WO2024196955A2 - Thérapie génique spécifique de la microglie ciblant l'apoe contre maladies neurologiques - Google Patents

Thérapie génique spécifique de la microglie ciblant l'apoe contre maladies neurologiques Download PDF

Info

Publication number
WO2024196955A2
WO2024196955A2 PCT/US2024/020591 US2024020591W WO2024196955A2 WO 2024196955 A2 WO2024196955 A2 WO 2024196955A2 US 2024020591 W US2024020591 W US 2024020591W WO 2024196955 A2 WO2024196955 A2 WO 2024196955A2
Authority
WO
WIPO (PCT)
Prior art keywords
disclosed
nucleic acid
seq
sequence
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/020591
Other languages
English (en)
Other versions
WO2024196955A3 (fr
Inventor
Ornit CHIBA-FALEK
Boris Kantor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Duke University
Original Assignee
Duke University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Publication of WO2024196955A2 publication Critical patent/WO2024196955A2/fr
Publication of WO2024196955A3 publication Critical patent/WO2024196955A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • AD Alzheimer’s disease
  • AD Alzheimer’s disease
  • the cost of AD to the US was $301 billion, including $206 billion in Medicare and Medicaid payments, while the caregivers provided $244 billion worth of care (Alzheimer’s Association, Alzheimer’s Impact Movement: Factsheet 2020).
  • AD disease modifying therapies
  • Late onset AD is a heterogenous disease with various genetic etiologies (Lo MT, et al. (2019). Neurobiol Aging. 84:243 el-243. e9; Nacmias B, et al. (2016) J Alzheimers Dis. 62:903-911). A major reason for the failure to identify an effective treatment is likely the inaccurate consideration of LOAD as a homogeneous disease. In this respect, increasing evidence demonstrate the heterogeneity in the underlying pathophysiologic processes of LOAD and show variability in the genetic risk and molecular profiles amongst AD patients (Reitz C. (2016) Ann Transl Med. 4: 107; Chiba-Falek O, et al. (2017) Expert Rev Precis Med Drug Dev. 2:47-55). Thus, AD remains an unmet medical need underscoring the urgent need for a paradigm shift in AD clinical research.
  • ApoE is a protein involved in metabolism of fat and is a major lipid and cholesterol carrier in the CNS.
  • apoE2 apoE3, and apoE4
  • APOE4 the genetic expression of APOE4 is considered to be a risk factor for the development of late-onset Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • mutations and abnormal expression of APOE has also been implicated in other neurological diseases like Parkinson’s disease and other neurodegenerative diseases including Age-Related Macular Degeneration AMD, and glaucoma.
  • AD disease modifying therapy for AD, particularly LOAD, that target specific genes like APOE in an allele specific manner.
  • FIG. 1A provides a UMAP plot showing separation of cell type-specific clusters in single nucleus (sn)RNA-seq data from 12 AD and 12 control temporal cortex. Microglial cluster 1 is circled.
  • FIG. IB provides UMAP plot showing differential expression analysis of APOE in AD vs control mice, with identified APOE as differentially expressed in microglia cluster 1.
  • FIG. 1C is a violin plot representing APOE gene expression in LOAD compared to control for each cell cluster.
  • FIG. ID shows single nucleus (sn)ATAC-seq data from the same 12 AD and 12 control temporal cortex for APOE gene.
  • the POE gene is located at chromosome 19ql3.2.
  • FIG. IE further shows LOAD Microl APOE DAP and co-accessible peaks within the range of gene range +/- Ik bp.
  • FIG. 1G depicts snRNA-seq of 12 LOAD and 12 aged and sex matched normal controls.
  • LEFT dotplots representing APOE expression in all brain cell clusters LOAD and control.
  • FIG. 1H depicts snATAC-seq of 12 LOAD and 12 aged and sex matched normal controls. Chromatin accessibility analysis across APOE genomic locus. UPPER PANELS: coverage plots for all 6 cell-types and microl cluster. Micro 1 differential accessible peak (DAP ) in the promoter region of APOE is labeled in a red box. MIDDLE PANEL: APOE genomic region and gene structure. LOWER PANEL: Open chromatin peaks are denoted. APOE promoter peak: chrl9 44905270-44906059 (790bp), APOE exon 2 peak, Microl specific : chrl9 44906364-44906667 (304bp).
  • FIG. II is a bar graph showing differential expression results for all clusters in which APOE was tested. Illustrated a bar plot of the logFCs (coef), where Microl is clearly the most robust.
  • FIG. 1J provides a scatter plot showing the adjusted p-value (-loglO(fdr)) vs. the logFC (coef) for snRNA-seq of 12 LOAD and 12 aged and sex matched normal controls.
  • APOE as DEG was tested in 5 clusters in which sufficient number of nuclei expressed APOE.
  • Microl is the only datapoint that passes both thresholds.
  • FIG. IK provides a schematic of chromosome 19 with individual gRNAs (SEQ ID NO: 30-33) sequences in the ApoE promoter and intron 1 listed.
  • FIG. IL shows clustering for snRNA-seq of 24 brain samples: 12 LOAD and 12 aged and sex matched normal controls. Analysis of genes expressed specifically and exclusively in Microl identified FCGR2A as a marker for Micro 1 cluster.
  • UPPER PANEL UMAP plot showing separation of cell type-specific clusters. Microl cluster circled in red.
  • LOWER PANEL FCGR2A expression is detected specifically in Microl cluster.
  • FIG. IM shows snATAC-seq of 24 brain samples: 12 LOAD and 12 aged and sex matched normal controls. Chromatin accessibility analysis across the genomic region of FCGR2A - the Micro 1 marker .
  • UPPER PANELS coverage plots for all cell-types and microl cluster.
  • MIDDLE PANEL FCGR2A genomic region and gene structure.
  • LOWER PANEL Open chromatin peaks are denoted.
  • the Micro 1 specific open chromatin/regulatory element overlapping FCGR2A promoter region is marked in Red box. 1904bp, coordinates chrl : 161505031-161506934 (hg38).
  • FIG. 2 shows measurement of repression capacity of developed AAV Cas repressor constructs.
  • Left panel depicts a schematic of the screening cascade, middle panel depicts the in vitro luciferase assay to characterize repression effects on gene expression and right panel shows that Cas-repressor 6 showed the most robust reduction in in luciferase expression. Normalized luciferase was measured on day 6 after AAV transduction.
  • FIG. 3A shows qRT-PCR data for expression of five microglial specific markers.
  • Human induced pluripotent stem cells harboring APOE3/3 (E3) and APOE4/4 (E4) were differentiated to microglia.
  • RNA was isolated, and qRT-PCR was performed to evaluate the expression of the five microglia markers: IBA1, CD68, CD11B, P2RY12 and TMEM119.
  • the results are displayed in the order of gene expression, with IBA1 having the highest expression in our cells. The results were normalized to GAPDH and PPIA1 internal controls.
  • FIG. 3B is the UCSC genome browser view of the AIF1 (IBA1) gene locus showing DAPS region as a red bar.
  • FIG. 3C shows representative immunofluorescence images testing IB Al promoter activity. The numbers correspond to three iterations of IBA promoter as indicated below. Immortalized microglia HMC3 in 0%FBS EMEM or 0.5% FBS EMEM on matrigel plates were transduced with different GFP reporter AAV vectors (serotype 6) driven by synthetic human IB Al promoters of 3 lengths.
  • FIG. 3D is a bar graph showing GFP expression for each of the IBA promoters tested.
  • the medium length promoter (1986: SEQ ID NO: 6) showed the strongest GFP expression.
  • FIG. 4A is a schematic representation of lentiviral vector system carrying a repressor domain to target promoter and exon 4 regions of APOE gene.
  • Microl specific regulatory element replace the constitutive promoter.
  • Promoter sequence of F4/80 or CD68 may be used for microglial specific expression OR the promoter region of FCGR2A: (1904bp) coordinates chrl : 161505031-161506934 (hg38).
  • Repressors DNMT3 A or KRAB-MeCP2 can be used.
  • FIG. 4B shows representative immunofluorescence images showing expression of GFP in organiods transformed with lentiviral or AAV viral vectors.
  • Human induced pluripotent stem cell derived cerebral organoids were transduced with either Lentivirus or AAV serotype (phpeb) expressing GFP.
  • Transduced organoids were spun down for 45 minutes at 300 g, and the media was changed the next day.
  • GFP fluorescence was monitored daily. Representative images show a higher efficiency of transduction with the AAV serotype (phpeb).
  • FIG. 4C are representative immunofluorescence images showing infection efficiency of different AAV serotypes.
  • Human induced pluripotent stem cell derived cerebral organoids were transduced with various AAV serotypes expressing GFP. Transduced organoids were spun down for 45 minutes at 300 g, and the media was changed the next day. GFP fluorescence was monitored daily. Images show infection efficiency at different time points.
  • FIG. 4D are representative immunofluorescence showing microglial infection efficiency of different AAV serotypes. Immortalized microglia HMC3 in 0% FBS EMEM or 0.5% FBS EMEM on matrigel plates were transduced with 2uL of different serotypes. Media was changed the next day. Images were taken at day 3. Serotype 6 showed the strongest GFP expression.
  • FIG. 4E are representative immunofluorescence showing microglial infection efficiency of different AAV serotypes. Immortalized microglia HMC3 in 0% FBS EMEM or 0.5% FBS EMEM on matrigel plates were transduced with 2uL of different serotypes. Media was changed the next day. Images were taken at day 6. Serotype 6 showed the strongest GFP expression.
  • FIG. 4F is a schematic showing in vivo proof-of-concept of the platform efficacy in the hippocampus.
  • Epigenome editing viral vector (AAV) with GFP as reporter were stereotaxically injected bilaterally into the left hemisphere of the hippocampus and a control vector with no repressor into the right hippocampus to provide within animal control.
  • FIG. 4G are representative immunofluorescence showing a robust and precise reduction in the expression of the reporter gene in the left side.
  • FIG. 4H is a box plot with statistical analysis of 11 mice, demonstrating stable and significant suppression over a period of 4 and 8 months.
  • FIG. 41 shows that was no significant change is weight in the injected animals over the duration they were monitored.
  • FIG. 5A is a schematic showing the in vivo validation experiment for the all-in-one AAV/dSaCas9-KRAB-MECP2(TRD) repressor platform targeting mouse endogenous APOE in the hippocampus.
  • 4-month old C57B/L6 mice were used to perform a bilateral stereotaxic inj ection directly into the mice’ s dorsal hippocampus (DH), the right DH hemisphere was inj ected with the AAV-gRNA/dCas9-repressor vectors with gRNA 1 or gRNA 2 or the left DH hemisphere with the control vector with no repressor (AAV-dCas-no repressor).
  • FIG. 5B shows representative images (2X magnification) of brain slices 6 weeks postinjection, showing expression of mouse APOE in the dorsal hippocampus. A 20X magnification of the image is provide below.
  • FIG. 5D shows that there was no significant change is weight in the injected animals over the duration they were monitored.
  • Daily weight post-injection (day 0) through when the mice were sacrificed were monitored (day 42).
  • FIG. 5E is a schematic showing the in vivo validation experiment for the all-in-one AAV/dSaCas9-KRAB-MECP2(TRD) repressor platform targeting human APOE in the hippocampus of APOE humanized mice model.
  • 4-month old mice were used to perform a bilateral stereotaxic injection directly into the mice’s dorsal hippocampus (DH), the right DH hemisphere was injected with the AAV-gRNA/dCas9-repressor vectors with gRNA 1 or gRNA 2 or the left DH hemisphere with the control vector with no repressor (AAV-dCas-no repressor).
  • FIG. 5F shows representative images (2X magnification) of brain slices 6 weeks postinjection, showing expression of human APOE in the dorsal hippocampus. A 20X magnification of the image is provide below.
  • FIG. 5G is a box plot showing that APOE was significantly repressed in the left dorsal hippocampus relative to the right dorsal hippocampus. Repression was significantly greater in males than females.
  • FIG. 6A is a bar graph quantifying the results of APOE mRNA expression in 4 month old organoids.
  • Human induced pluripotent stem cells harboring APOE3/3 (E3) and APOE4/4 (E4) were differentiated to cerebral organoids.
  • 4 months old organoids were transduced with gRNA/dCas-repressor (gRNA) virus harboring gRNA to target promoter region of APOE or no- gRNA (Control) virus.
  • gRNA gRNA/dCas-repressor
  • RNA was then isolated, and qRT-PCR was performed to evaluate the expression of APOE.
  • Results show a significant downregulation of APOE expression in both E3 and E4 organoids. The results were normalized to GAPDH, PPIA1 and the controls.
  • FIG. 6B is a bar graph showing beneficial effect of reduced APOE leading to a decrease in pTAU expression as evidenced by the western blot analysis.
  • the graph represents pTAU band intensity, normalized to total TAU. quantifying the results of APOE mRNA expression in 4 month-old organoids.
  • FIG. 7A shows in vitro validation studies of the microglia-specific APOEe4-targeted dCas9-repressor platform using hiPSC-derived microglia. Immunofluorescence image of hiPSC- derived microglia were stained with a microglia specific marker IBA1.
  • FIG. 7B is a bar graph showing a significant decrease in APOE-mRNA expression levels.
  • APOEe4 microglia were transduced with gRNA/dCas-repressor or control (no-gRNA) viruses using a vector driven by a synthetic human CD68 regulatory sequence harboring gRNA to target the APOE locus.
  • FIG. 7C is a bar graph showing that repressing APOEe4 expression in the microglia led to an increase in A ? uptake, comparable to that of APOEe3 microglia, ** p ⁇ 0.01, **** p ⁇ 0.0001
  • FIG. 7D are bar graphs showing reduction of the APOEe4 expression in the microglia led to an increase in TMEM119 level and decrease CD74, demonstrating rescue of neuroinflammation.
  • an isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding: (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting an APOE gene sequence or a regulatory element thereof; and (b) one or more microglia specific regulatory elements operably linked to (a)(i), (a)(ii), (a)(iii), or any combination thereof.
  • dCas Clustered Regularly Interspaced Short Palindromic Repeats associated
  • the one or more microglia specific regulatory element may comprise a promoter, wherein the promoter comprises a wild-type promoter, a synthetic promoter, or a minimal promoter.
  • the one or more microglia specific regulatory elements are operably linked to the nucleic acid sequence encoding the dCas endonuclease.
  • the one or more microglia specific regulatory elements are operably linked to the nucleic acid encoding the at least one polypeptide having enzymatic activity.
  • the one or more microglia specific regulatory elements are operably linked to the nucleic acid encoding the dCas endonuclease and the nucleic acid encoding the at least one polypeptide having enzymatic activity.
  • the one or more microglia specific regulatory elements is operably linked to the at least one guide RNA.
  • Non-limiting examples of micro-glia specific promoters include F4/80, CD68, FCGR2A, HexA, FCGR2A or IBA1 promoter, or a derivative thereof, or any combination thereof.
  • the promoter drives the expression of the at least one gRNA, the dCas endonuclease, the at least one polypeptide, or any combination thereof.
  • the promoter is a CD68 promoter, or a derivative thereof. In an aspect, the promoter is a IBA1 promoter, or a derivative thereof. In an aspect, the promoter is a F4/80 promoter, or a derivative thereof. In an aspect, the promoter comprises a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1-7, or a fragment or a derivative thereof.
  • the disclosed isolated nucleic acid may comprise a nucleic acid sequence encoding at least one gRNA which targets the APOE gene, or promoter region thereof, or an untranslated region (UTR) thereof, or any combination thereof.
  • the at least one gRNA targets the promoter region, or intron 1, or a flanking sequence thereof, of APOE.
  • the at least one gRNA may target the nucleic acid sequence set forth in SEQ ID NO: 15-33, or a sequence at least about 90% identical thereto.
  • the disclosed isolated nucleic acid may comprise a nucleic acid sequence encoding a Cas endonuclease comprising a deactivated Cas9 (dCas9) endonuclease.
  • the dCas endonuclease comprises a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • the dCas endonuclease may comprise a variant dCas9 endonuclease selected from a VRER, EQR or VQR variant of dCas9.
  • the dCas9 endonuclease comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 44- 47, or a fragment thereof.
  • the disclosed isolated nucleic acid may comprise a nucleic acid sequence encoding at least one polypeptide with an enzymatic activity, for example a transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nucleic acid association activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, or any combination thereof.
  • polypeptide having enzymatic activity comprises HP la, HP lb, MBD1, MBD2, Kriippel-Associated Box (KRAB), NIPP1, Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the at least one polypeptide having an enzymatic activity comprises Kriippel-associated box (KRAB), the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A or any fusion polypeptide thereof.
  • the fusion polypeptide comprises KRAB-MeCP2 (KRAB-MeCP2).
  • At least one polypeptide having enzymatic activity comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 36-39, or a derivative thereof.
  • the dCas endonuclease comprises dCas9 VRER and the polypeptide having enzymatic activity comprises Krtippel-associated box (KRAB), the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), or a fusion of KRAB-MeCP2 (KRAB-MeCP2).
  • KRAB Krtippel-associated box
  • TRD transcription repression domain
  • KRAB-MeCP2 Krtippel-associated box
  • the nucleic acid sequence encodes a fusion protein comprising the dCas endonuclease and the at least one polypeptide having an enzymatic activity.
  • the isolated nucleic acid molecule may comprise one of more of a P2A, T2A, E2A, F2A, and IRES sequence.
  • the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 36-39, or a sequence at least 80% identical thereto.
  • the isolated nucleic acid molecule may further comprising one or more additional regulatory elements, non-limiting examples of which include primer binding sites (PBS), one or more splice donor (SD) sites, one or more splice acceptor (SA) sites, one or more central polypurine tracts (cPPT), one or more polypurine tracts (PPT), one or more Rev Response elements (RRE), one or more Woodchuck Hepatitis Virus Posttranscriptional Regulatory Elements (WPRE), Spl responsive element, a p2A signal, a woodchuck hepatitis virus post-transcriptional regulatory element, a Phi signal-packaging signal, a rev responsive element, a 5’-LTR, and a 3’-LTR one or more retroviral vector packaging elements, or any combination thereof.
  • PBS primer binding sites
  • SD splice donor
  • SA splic
  • a viral vector comprising the isolated nucleic acid molecule as provided herein.
  • the vector is a recombinant adenovirus-associated viral vector (rAAV).
  • the viral vector comprises a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in the viral vector may a sequence as set forth in SEQ ID NO: 112-128.
  • the current disclosure also encompasses a pharmaceutical formulation comprising the isolated nucleic acid molecule as disclosed herein or the rAAV vector as disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation is for the treatment of a neurological disease associated with APOE overexpression.
  • the neurodegenerative disease is Alzheimer’s disease (AD) or Parkinson’s disease (PD).
  • a method of treating and/or preventing a neurological disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition.
  • the subject is a human.
  • the neurological disease is Alzheimer’s disease, Parkinson’s disease, or a neuroinflammatory disease.
  • the subject is suspected of having or has been diagnosed with having Alzheimer’s disease.
  • the method comprises reducing the pathological phenotype associated with Alzheimer’s disease.
  • administering the pharmaceutical composition comprises intravenous administration, intracerebral administration, intra-CSF administration, intracerebroventricular (ICV) administration, intraventricular administration, intra-cistema magna (ICM) administration, intraparenchymal administration, intrathecal (lumbar, cisternal, or both) administration, or any combination thereof.
  • the method further comprises administering to the subject a therapeutically effective amount of a therapeutic agent.
  • the administering to the subject a therapeutically effective amount of an immune modulator.
  • a method of treating and/or preventing Alzheimer’s disease progression in a subject comprising administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition, wherein the administering of the pharmaceutical composition reduces the expression of APOE at least one microglial cell of the subject, thereby reducing the pathological phenotype associated with Alzheimer’s disease.
  • the subject is a human.
  • administering the vector comprises intravenous administration, intracerebral administration, intra-CSF administration, intracerebroventricular (ICV) administration, intraventricular administration, intra-ci sterna magna (ICM) administration, intraparenchymal administration, intrathecal (lumbar, cisternal, or both) administration, or any combination thereof.
  • the method further comprising administering to the subject at least one additional therapeutic effective agent.
  • the therapeutically effective agent is an immune modulator.
  • the current disclosure also encompasses a method of controlling expression of APOE in a target cell comprising, contacting the target cell with the disclosed isolated nucleic acid molecule, or the viral vector, or the pharmaceutical composition, wherein the APOE expression in the target cell is reduced after contacting the cell.
  • the target cell comprises a microglial cell.
  • the target cell is located in vivo, ex vivo, or in vitro.
  • the target cell is located in a brain tissue.
  • the target cell is located in a human brain.
  • compositions compounds, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
  • AD Alzheimer’s disease
  • AD Alzheimer’s disease
  • the cost of AD to the US is $301 billion, including $206 billion in Medicare and Medicaid payments. It is estimated that caregivers provide 244 billion dollars’ worth of care (Costs of Alzheimer’s to Medicare and Medicaid. (2020); Alzheimer’s Association, Alzheimer’s Impact Movement: Fact Sheet 2020).
  • AD Alzheimer Res 9:746-758
  • Late onset AD is a heterogenous disease with various genetic etiologies. A major reason for the failure to identify an effective treatment is likely the inaccurate consideration of LOAD as a homogeneous disease. In this respect, mounting evidence demonstrates the heterogeneity in the underlying pathophysiologic processes of LOAD and shows variability in the genetic risk and molecular profiles amongst AD patients. Advancement in LOAD therapy requires the development and validation of new therapeutic targets including those tailored to a specific sub-group of patients with a specific set of risk factors. APOE has emerged as potential promising targets for LOAD treatment.
  • Apolipoprotein E (ApoE)
  • Apolipoprotein E is encoded by the APOE gene positioned on chromosome 19ql3.32 (GRCh 38: chrl9:44, 905, 795-44, 909, 392).
  • Two common coding SNPs in exon 4 give rise to three allelic variants (APOE e2, APOE e3, and APOE e4), encoding three corresponding protein isoforms that differ at two amino acid positions 112 and 158.
  • the e4 allele of the apolipoprotein E gene (APOE e4) is the first, strongest, and most firmly established genetic risk factor for LOAD.
  • the initial discovery was made nearly 30 years ago by linkage analysis of pedigrees.
  • APOE e4 has become the most highly replicated genetic risk factor for LOAD.
  • GWAS genome-wide association studies
  • APOE e3 is natural and APOE e2 confers a protective effect.
  • compositions and methods for decreasing the levels of ApoE are effective in decreasing the expression of APOE e4.
  • the allelic discrimination approach is innovative as it allows a precise and fine-tuned downregulation of APOEe4 allele expression.
  • compositions and methods for epigenetic editing at specifically targeted APOE loci representing appealing approach for gene therapy mediated by altering the expression of a specific gene. Allele specific modulation combined with epigenetic gene editing described herein provides a precise and targeted approach to treat diseases like Alzheimer’s and Parkinson’s. This technology offers an unprecedented opportunity to modify epigenetic markers to control the expression of disease-associated genes and variants.
  • the allelic discrimination approach is innovative as it allows a precise and fine-tuned downregulation of APOEe4 allele expression.
  • Also disclosed herein is a method of cell-type specific targeting of APOE in the microglia. This method has several benefits, for example minimal therapeutic side effects, often a concern with CRISPR based technologies.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • references in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.
  • a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included.
  • the present disclosure may include a variety of combinations and/or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues See, e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991), the disclosure of which is incorporated in its entirety herein.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • a protein is represented by an amino acid sequence and correspondingly a nucleic acid molecule or a polynucleotide represented by a nucleic acid sequence.
  • Identity and similarity between sequences throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%.
  • Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%.
  • Each amino acid sequence described herein by virtue of its identity or similarity percentage with a given amino acid sequence respectively has in a further preferred aspect an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given nucleotide or amino acid sequence, respectively
  • sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred aspect, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • the degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms.
  • Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm.
  • a Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps.
  • MAFFT for multiple sequence alignment
  • MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned, Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2, Perform FFTS: none is used).
  • Similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity may be used for determination of sequence similarity. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or He; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Vai to He or Leu.
  • “operably linked” means that expression of a gene is under the control of a regulatory element with which it is spatially connected.
  • the regulatory element is a promoter and when used in this context, the expression of a gene is under control of the promoter with which it is spatially connected.
  • a regulatory element e.g., a promoter
  • a regulatory element can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control.
  • the distance between the regulatory element (e.g., promoter) and a gene can be approximately the same as the distance between that regulatory element (promoter) and the gene it controls in the gene from which the regulatory element (e.g., promoter) is derived. As is known in the art, variation in this distance can be accommodated without loss of function (z.e., without loss of promoter function).
  • regulatory elements refer to any sequence elements that regulate, positively or negatively, the expression of an operably linked sequence. “Regulatory elements” include, without being limiting, a promoter, an enhancer, a leader, a transcription start site (TSS), a linker, 5' and 3' untranslated regions (UTRs), an intron, a polyadenylation signal, and a termination region or sequence, etc., that are suitable, necessary, or preferred for regulating or allowing expression of the gene or transcribable DNA sequence in a cell. Such additional regulatory element(s) can be optional and used to enhance or optimize expression of the gene or transcribable DNA sequence.
  • a regulatory sequence can, for example, be inducible, noninducible, constitutive, cell-cycle regulated, metabolically regulated, and the like.
  • a regulatory sequence may be a promoter.
  • promoter refers to a DNA sequence that comprises an RNA polymerase binding site, a transcription start site, and/or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene).
  • a promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence.
  • a promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences.
  • a promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein.
  • the promoter sequence is adapted to enable expression of a nucleic acid in the microglial cells.
  • the promoter is a microglia specific promoter or a derivative thereof, non-limiting examples of which include F4/80, CD68, FCGR2A, HexB or IBA I .
  • microglia specific regulatory element encompasses a sequence which regulates the expression of a gene-of-interest to which it is operably linked, such that the gene product exhibits enhanced and/or preferential expression in the microglia when compared to other cell-types.
  • the enhanced expression may result in at least about 1.1-fold to about 100-fold or more, expression of the gene-of-interest.
  • a microglia specific regulatory sequence may comprise a microglia specific wild-type promoter, a microglia specific enhancer, a synthetic promoter, a synthetic enhancer, a transcriptional or translational regulatory sequence or any combination thereof.
  • the microglia specific regulatory sequence is a promoter, an enhancer or a combination thereof.
  • microglia specific promoter includes wild-type promoter, or derivatives thereof, synthetic promoter, minimal promoter, and any combination of promoter and enhancers sequence, that drives enhanced expression of a gene- of-interest to which it is operably linked in the microglial cells.
  • nucleic acid sequence refers to a nucleic acid sequence related to a wild type sequence, for example either by the nucleic acid sequence or encoded amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function.
  • Derivatives, variants and fragments of a nucleic acid sequence can comprise one or more nucleotide variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type nucleic acid sequence.
  • derivative when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure e.g., secondary and/or tertiary), activity (e.g, enzymatic activity) and/or function.
  • Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g, mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide.
  • a “fragment” or “portion” of a nucleotide sequence can be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence.
  • nucleic acid fragment or portion can be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations.
  • a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise a target of interest.
  • a “fragment” or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more amino acids) to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference amino acid sequence.
  • Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent.
  • an “isolated” refers to a biological component (such as a nucleic acid molecule, nucleic acid sequence, protein, or virus) that has been substantially separated or purified away from other biological components (e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and/or organelles).
  • Nucleic acids, proteins, and/or viruses that have been “isolated” include nucleic acids, proteins, and viruses purified by standard purification methods. The term also embraces nucleic acids, proteins, and viruses prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins.
  • an isolated or purified nucleic acid, protein, virus, or other active compound is one that is isolated in whole or in part from associated nucleic acids, proteins, and other contaminants.
  • substantially purified refers to a nucleic acid, protein, virus or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.
  • isolated proteins or nucleic acids, or cells containing such are at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.
  • Isolated nucleic acid molecules may comprise one or more naturally occurring sequences, recombinant sequences, or combinations thereof.
  • An isolated nucleic acid molecule may comprise any one or more of a gene, a transcription regulatory sequence, a translation regulatory sequence, coding sequence, noncoding sequence, plasmids, vector, or viral vector.
  • the isolated nucleic acid molecule may also comprise one or more modified nucleotides.
  • a “heterologous” or a “recombinant” nucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide or amino acid sequence.
  • Different nucleic acids or proteins having homology can be referred to as “homologues.”
  • the term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • “Homology” refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (z.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • the disclosed compositions and disclosed methods can comprise homologues to the disclosed nucleotide sequences and/or disclosed polypeptide sequences.
  • Orthologous can refer to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation.
  • a homologue of a disclosed nucleotide sequence or a disclosed polypeptide can have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to a disclosed nucleotide sequence or a disclosed polypeptide.
  • “Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.
  • APOE associated diseases or disorders refers to any disease of disorder that results from an abnormal APOE gene or abnormal APOE gene expression/activity, or both.
  • Non-limiting examples of APOE associated diseases and disorders include Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative diseases including Age-Related Macular Degeneration (AMD), and glaucoma.
  • AMD Age-Related Macular Degeneration
  • the term “subject” refers to the target of administration, e.g., a human being.
  • the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent.
  • the term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered.
  • a subject can be a human patient.
  • a subject can have or is suspected of having Alzheimer’s disease (e.g., LOAD), be suspected of having Alzheimer’s disease, or be at risk of developing and/or acquiring Alzheimer’s disease.
  • Alzheimer’s disease e.g., LOAD
  • a subject can have or is suspected of having Parkinson’s disease (PD), or other neurodegenerative diseases associated with abnormal APOE gene, expression and/or activity, non-limiting examples of which include Age-Related Macular Degeneration (AMD), and glaucoma.
  • PD Parkinson’s disease
  • AMD Age-Related Macular Degeneration
  • diagnosisd means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed agents, disclosed therapeutic agents, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods.
  • “diagnosed with Alzheimer’s disease or LOAD” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be treated by one or more of the disclosed isolated nucleic acid molecules, disclosed viral vectors, disclosed lentiviral vectors, disclosed pharmaceutical formulations, disclosed host cells, disclosed gRNAs, disclosed plasmids, or any combination thereof, or by one or more of the disclosed methods.
  • “suspected of having Alzheimer’s disease” can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can likely be treated by one or more of the disclosed isolated nucleic acid molecules, disclosed viral vectors, disclosed lentiviral vectors, disclosed pharmaceutical formulations, disclosed host cells, disclosed gRNAs, disclosed plasmids, or any combination thereof, or by one or more of the disclosed methods.
  • an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzymatic assay), or a combination thereof.
  • a “patient” can refer to a subject that has been diagnosed with or is suspected of having an APOE associated disease or condition.
  • a patient can refer to a subject that has been diagnosed with or is suspected of having an APOE associated disease or condition, and is seeking treatment or receiving treatment for APOE associated disease or condition.
  • a “patient” can refer to a subject that has been diagnosed with or is suspected of having Alzheimer’s disease (AD) or late-onset Alzheimer’s disease (LOAD).
  • a patient can refer to a subject that has been diagnosed with or is suspected of having AD such as for example, LOAD, and is seeking treatment or receiving treatment for AD or LOAD.
  • the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder.
  • a subject can be identified as having a need for treatment of a disorder (e.g., such as APOE associated disease or disorder) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder (e.g., AD, LOAD, PD, AMD).
  • a subject can be identified as having a need for treatment of a disorder (e.g., such as AD) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder e.g., AD or LOAD).
  • the identification can be performed by a person different from the person making the diagnosis.
  • the administration can be performed by one who performed the diagnosis.
  • inhibit means to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not having Alzheimer’s disease). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels.
  • the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or predisorder level.
  • treat or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder (such as Alzheimer’s disease or APOE associated diseases).
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder (such as Alzheimer’s disease or APOE associated diseases).
  • the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease.
  • a mammal e.g., a human
  • treating AD or LOAD can reduce the severity of an established disease in a subject by 1 %- 100% as compared to a control (such as, for example, an individual not having AD or LOAD).
  • treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of AD or LOAD.
  • treating Alzheimer’s disease can reduce one or more symptoms of AD or LOAD in a subject by 1 %- 100% as compared to a control (such as, for example, an individual not having AD or LOAD).
  • treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established AD (such as LOAD).
  • AD such as LOAD
  • treatment does not necessarily refer to a cure or complete ablation or eradication of AD.
  • treatment can refer to a cure or complete ablation or eradication of AD or LOAD.
  • the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing AD or LOAD and/or AD or LOAD progression is intended. The words “prevent” and “preventing” and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having AD or LOAD or an AD or LOAD complication from progressing to that complication. In an aspect, preventing or reducing APOE expression and/or activity is intended.
  • administering refers to any method of providing one or more of the disclosed isolated nucleic acid molecules, disclosed pharmaceutical formulations, disclosed vectors, or any combination thereof to a subject.
  • Such methods are well known to those skilled in the art and include, but are not limited to, the following routes: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration.
  • Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV).
  • Administration of a disclosed therapeutic agent, a disclosed pharmaceutical composition, or a combination thereof can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventriular, inthrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS.
  • Administration can be continuous or intermittent.
  • a “therapeutic agent” can be a “biologically active agent” or “biologic active agent” or “bioactive agent,” which refers to an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied.
  • the bioactive agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions.
  • bioactive agents can include anti-viral agents, vaccines, hormones, antibodies (including active antibody fragments sFv, Fv, and Fab fragments), aptamers, peptide mimetics, functional nucleic acids, therapeutic proteins, peptides, or nucleic acids.
  • bioactive agents include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to bioactive agents through metabolism or some other mechanism.
  • any of the compositions of the invention can contain combinations of two or more bioactive agents. It is understood that a biologically active agent can be used in connection with administration to various subjects, for example, to humans (z.e., medical administration) or to animals (z.e., veterinary administration). As used herein, the recitation of a biologically active agent inherently encompasses the pharmaceutically acceptable salts thereof.
  • a “therapeutic agent” can be any agent that effects a desired clinical outcome in a subject having AD or LOAD, suspected of having AD or LOAD, and/or likely to develop or acquire AD or LOAD.
  • a disclosed therapeutic agent can be an oligonucleotide therapeutic agent.
  • a disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, gRNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof.
  • a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi.
  • a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable.
  • a therapeutic agent can be a “drug” or a “vaccine” and means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes.
  • This term includes externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like.
  • This term may also be used in reference to agricultural, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans.
  • Examples include but are not limited to a radiosensitizer, the combination of a radiosensitizer and a chemotherapeutic, a steroid, a xanthine, a beta-2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha-agonist, an alpha- 1 -antagonist, carbonic anhydrase inhibitors, prostaglandin analogs, a combination of an alpha agonist and a beta blocker, a combination of a carbonic anhydrase inhibitor and a beta blocker, an anticholinergic/antispasmodic agent, a vasopressin analogue, an anti arrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an ansiolytic agent, a
  • the pharmaceutically active agent can be coumarin, albumin, bromolidine, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2-agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory agents, including antiasthmatic antiinflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal antiinflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxene, acetominophen,
  • steroids such as
  • acetylcholine receptor agonists such as pilocarpine hydrochloride and carbachol
  • glutamate receptor agonists such as the N-methyl D-aspartate receptor agonist memantine
  • anti-Vascular endothelial growth factor (VEGF) aptamers such as pegaptanib
  • anti-VEGF antibodies including but not limited to anti-VEGF-A antibodies
  • carbonic anhydrase inhibitors such as methazolamide, brinzolamide, dorzolamide hydrochloride, and acetazolamide
  • anti arrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecaimide acetate, procainamide hydrochloride, moricizine hydrochloride, and diisopyramide phosphate; antiparkinsonian
  • a pharmaceutically active agent can be used in connection with administration to various subjects, for example, to humans (i.e., medical administration) or to animals (i.e., veterinary administration).
  • a pharmaceutically active agent inherently encompasses the pharmaceutically acceptable salts thereof.
  • AAV is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise.
  • the abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”).
  • AAV vector can include naturally isolated serotypes including, but not limited to, AAV 1 , AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, nonprimate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV.
  • ICTV International Committee on Taxonomy of Viruses
  • an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape.
  • Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV- HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81.
  • a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1).
  • an “rAAV vector” as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (z.e. a polynucleotide heterologous to AAV), typically a sequence of interest for introducing into a target cell.
  • the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • the term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.
  • An “AAV virus” or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated nucleic acid molecule AAV vector. If the particle comprises a heterologous nucleic acid molecule (z.e. a nucleic acid molecule other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector.”.” Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.
  • Packaging refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.
  • AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”
  • a “helper virus” for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell.
  • a variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used.
  • Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC.
  • Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • Helper virus function(s) refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.
  • an “infectious” virus or viral particle is one that comprises a nucleic acid molecule component which it is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus.
  • an “infectious” virus or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell.
  • “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity.
  • Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles.
  • Total viral particles can be expressed as the number of viral genome (vg) copies.
  • the ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as “transduction.”
  • the ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g.
  • GFP green fluorescent protein
  • virus comprises a nucleotide sequence encoding GFP
  • GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art.
  • a “replication-competent” virus refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e., in the presence of a helper virus or helper virus functions).
  • replication competence generally requires the presence of functional AAV packaging genes.
  • rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes.
  • rAAV vectors typically lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector.
  • rAAV vector preparations as described herein are those which contain few if any replication competent AAV (rcAAV, also referred to as RCA).
  • tropism refers to the specificity of an AAV capsid protein present in an AAV viral particle, for infecting a particular type of cell or tissue.
  • the tropism of an AAV capsid for a particular type of cell or tissue may be determined by measuring the ability of AAV vector particles comprising the hybrid AAV capsid protein to infect or to transduce a particular type of cell or tissue, using standard assays that are well-known in the art such as those disclosed in the examples of the present application.
  • liver tropism or “hepatic tropism” refers to the tropism for liver or hepatic tissue and cells, including hepatocytes.
  • codon optimization can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Various species exhibit particular bias for certain codons of a particular amino acid.
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes.urv.es/OPTIMIZER/).
  • expression cassette or “transgene cassette” can refer to a distinct component of vector DNA comprising a transgene and one or more regulatory sequences to be expressed by a transfected cell.
  • an expression cassette or transgene cassette can comprise a promoter sequence, an open reading frame (z.e., the transgene), and a 3’ untranslated region (e.g., in eukaryotes a polyadenylation site).
  • the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more of the disclosed isolated nucleic acid molecules, disclosed pharmaceutical formulations, disclosed vectors, or any combination thereof so as to treat or prevent AD or LOAD.
  • the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more of the disclosed isolated nucleic acid molecules, disclosed pharmaceutical formulations, disclosed vectors, or any combination thereof.
  • the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for any disclosed isolated nucleic acid molecule, disclosed pharmaceutical formulation, disclosed vector, disclosed therapeutic agent, or any combination thereof.
  • modifying the method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • a method can be altered by changing the amount of one or more of the disclosed isolated nucleic acid molecules, disclosed viral vectors, disclosed lentiviral vectors, disclosed pharmaceutical formulations, disclosed host cells, disclosed gRNAs, disclosed plasmids, or any combination thereof, or administered to a subject, or by changing the frequency of administration of one or more of the disclosed isolated nucleic acid molecules, disclosed viral vectors, disclosed lentiviral vectors, disclosed pharmaceutical formulations, disclosed host cells, disclosed gRNAs, disclosed plasmids, or any combination thereof, or by changing the duration of time that the one or more of the disclosed isolated nucleic acid molecules, disclosed viral vectors, disclosed lentiviral vectors, disclosed pharmaceutical formulations, disclosed host cells, disclosed gRNAs, disclosed plasmids, or any combination thereof, or are administered to a subject.
  • a target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, kidney, brain, etc.).
  • a target area or intended target area can be any cell or any organ infected by AD or LOAD (such as cholinergic neurons).
  • a target area or intended target area can be the brain or various neuronal populations.
  • determining can refer to measuring or ascertaining the presence and severity of AD such as, for example, LOAD.
  • Methods and techniques used to determine the presence and/or severity of AD are typically known to the medical arts.
  • the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of AD (such as, for example, a LOAD.
  • “determining” can also refer to measuring or ascertaining the level of one or more proteins or peptides in a biosample or measuring or ascertaining the level or one or more RNAs or miRNAs in a biosample. Methods and techniques for determining the level of proteins/peptides and RNAs/miRNAs are known to the art and are disclosed herein.
  • “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of AD or LOAD.
  • the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., an AD or LOAD).
  • a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.
  • “therapeutically effective amount” means an amount of a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed vector, or any combination thereof that (i) treats the particular disease, condition, or disorder (e.g., AD or LOAD), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder (e.g., AD or LOAD), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., AD or LOAD).
  • the particular disease, condition, or disorder e.g., AD or LOAD
  • attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., AD or LOAD
  • delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein e.g., AD or LOAD
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the isolated nucleic acid molecules, disclosed pharmaceutical formulations, disclosed vectors, or any combination thereof employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed isolated nucleic acid molecules, disclosed pharmaceutical formulations, disclosed vectors, or any combination thereof employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed isolated nucleic acid molecules, disclosed pharmaceutical formulations, disclosed vectors, or any combination thereof employed, and other like factors well known in the medical arts.
  • the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed isolated nucleic acid molecules, disclosed pharmaceutical formulations, disclosed vectors, or any combination thereof can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, AD or LOAD.
  • a pharmaceutical carrier refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • a pharmaceutical carrier employed can be a solid, liquid, or gas.
  • examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water.
  • examples of gaseous carriers can include carbon dioxide and nitrogen.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like
  • oral solid preparations such as powders, capsules and tablets.
  • tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
  • Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
  • the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the term “in combination” in the context of the administration of one or more of the disclosed agents, disclosed therapeutic agents, disclosed pharmaceutical formulations or a combination thereof includes the use of more than one therapy (e.g., additional therapeutic agents).
  • Administration “in combination with” one or more additional therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order.
  • the use of the term “in combination” does not restrict the order in which therapies are administered to a subject.
  • CRISPR-based endonucleases include RNA-guided endonucleases that comprise at least one nuclease domain and at least one domain that interacts with a guide RNA.
  • a guide RNA directs the CRISPR-based endonucleases to a targeted site in a nucleic acid at which site the CRISPR-based endonucleases cleaves at least one strand of the targeted nucleic acid sequence.
  • the CRISPR-based endonuclease is universal and can be used with different guide RNAs to cleave different target nucleic acid sequences.
  • CRISPR-based endonucleases are RNA-guided endonucleases derived from CRISPR/Cas systems.
  • a disclosed CRISPR-based endonuclease can be derived from a CRISPR/Cas type I, type II, or type III system.
  • suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cm
  • a disclosed CRISPR-based endonuclease can be derived from a type II CRISPR/Cas system.
  • a CRISPR-based endonuclease can be derived from a Cas9 protein.
  • the Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp, Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp
  • the CRISPR-based nuclease can be derived from a Cas9 protein from Streptococcus pyogenes.
  • the CRISPR-based nuclease can comprise the sequence set forth in any one of SEQ ID NO: 36-40, or a derivative thereof, or a sequence at least about 80% identical thereto.
  • CRISPRa refers to CRISPR Activation, which is using a dCas9 or dCas9-activator with a gRNA to increase transcription of a target gene.
  • CRISPRi refers to CRISPR Interference, which is using a dCas9 or dCas9-repressor with a gRNA to repress/decrease transcription of a target gene.
  • dCas9 refers to enzymatically inactive form of Cas9, which can bind, but cannot cleave, DNA.
  • Protospacer Adjacent Motif or “PAM” refers to a sequence adjacent to the target sequence that is necessary for Cas enzymes to bind target DNA.
  • these and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein.
  • an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting an APOE gene sequence or a regulatory element thereof; and (b) one or more microglia specific regulatory elements operably linked to (a)(i), (a)(ii), (a)(iii), or any combination thereof.
  • dCas Clustered Regularly Interspaced Short Palindromic Repeats associated
  • the microglia specific regulatory sequence may comprise a wild-type promoter, a microglia specific enhancer, a synthetic promoter, a synthetic enhancer, a transcriptional or translational regulatory sequence or any combination thereof.
  • the microglia specific regulatory sequence is a microglia specific promoter.
  • the term “microglia specific promoter” includes wild-type promoter, or derivatives thereof, synthetic promoter, minimal promoter, and any combination of promoter and enhancers sequence, that drives enhanced expression of a gene-of-interest to which it is operably linked in the microglial cells.
  • the microglia specific promoter may drive enhanced expression of the gene-of-interest to which it is operably linked, in the microglia compared to other brain cells, for example neurons.
  • the microglia specific promoter may be functional in enhancing expression in the microglia, of gene-of-interest to which it is operably linked, by about 1.1 fold to about 100-fold or more.
  • the microglia specific promoter may be functional in enhancing expression in the microglia, of gene-of-interest to which it is operably linked, by about 1.1 -fold to about 2-fold, about 2-fold to about 10-fold, about 10-fold to about 20-fold, about 20- fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50- fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80- fold to about 90-fold, about 90-fold to about 100-fold or more compared to other brain cells.
  • the microglia specific promoter can be any promoter known in the art, that exhibits enhanced activity in the microglia.
  • Non-limiting examples of microglia specific promoters include F4/80, CD68, FCGR2A, HexB or IBA1 promoter, or a derivative thereof.
  • the promoter is a CD68 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one or more of the sequences set forth in SEQ ID NO: 1-3, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 1-3.
  • the promoter is a F4/80 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 4.
  • the promoter is a IBA1 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5-7, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 5-7.
  • the promoter is a FCGR2A promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 8, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 8.
  • a disclosed microglia specific regulatory element ie., a microglia specific promoter
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, or a derivative thereof.
  • the isolated nucleic acid molecule disclosed herein may comprising a nucleic acid sequence encoding a deactivated Cas (dCas) endonuclease.
  • the nucleic acid sequence encoding the dCas may be operably linked to the microglia specific regulatory element disclosed herein above.
  • the nucleic acid sequence encoding the dCas may be operably linked to the microglia specific promoter disclosed herein above.
  • the microglia specific promoter may comprise the nucleic acid sequence as set forth in any one of SEQ ID NOS: 1-8, or a sequence at least about 60% identical thereto.
  • a disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • the nucleic acid sequence encoding the disclosed dSpCas9 may comprise a sequence as set forth in any one of SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSpCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof.
  • a disclosed dSpCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment, or a derivative thereof.
  • a disclosed dSpCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dSaCas9 may comprise a sequence as set forth in SEQ ID NO: 42, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSaCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 42, or a fragment thereof.
  • a disclosed dSaCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 38 or a fragment, or a derivative thereof.
  • a disclosed dSaCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 38, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCjCas9 may comprise a sequence as set forth in SEQ ID NO: 43, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dCjCas9 endonuclease may comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 43, or a fragment thereof.
  • a disclosed dCjCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 39, or a fragment, or a derivative thereof.
  • a disclosed dCjCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 39 or a fragment thereof.
  • a disclosed variant of dCas9 dVQR can comprise DI 135V, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dEQR can comprise D1135E, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dVRER can comprise DI 135V, G1218R, R1335E, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed dVRER can comprise the sequence set forth in SEQ ID NO: 37 or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCas9 endonuclease may comprise a sequence as set forth in any one of SEQ ID NO: 40-43.
  • the sequence encoding the disclosed dCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40-43, or a fragment, or a derivative thereof.
  • the isolated nucleic acid molecule may comprise a nucleic acid sequence encoding at least one polypeptide having an enzymatic activity.
  • a disclosed encoded polypeptide can comprise transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nucleic acid association activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise HP la, HP lb, MBD1, MBD2, Kriippel-Associated Box (KRAB), NIPP1, the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • KRAB Kriippel-Associated Box
  • NIPP1 the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed fusion can comprise HPla-HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb- HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb- DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1- NIPP1, MBDl-MeCP2, MBD1-DNMT3A, MBD2-MBD2, MBD2-HPla, MBD2-HPlb, MBD2- MBD1, MBD2-KRAB, MBD2-NIPP1, MBD2-MeCP2, MBD2-DNMT3A, KRAB-KRAB, KRA
  • the nucleic acid sequence encoding the disclosed polypeptide HP la may comprise a sequence as set forth in any one of SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP la may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment thereof.
  • a disclosed polypeptide HPla can comprise the amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPla can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide HP lb may comprise a sequence as set forth in any one of SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP lb may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment thereof.
  • a disclosed polypeptide HPlb can comprise the amino acid sequence as set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPlb can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD1 may comprise a sequence as set forth in any one of SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment thereof.
  • a disclosed polypeptide MBD1 can comprise the amino acid sequence as set forth in any one of SEQ ID NO: 48-50, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MDB1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 48-50, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD2 may comprise a sequence as set forth in any one of SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment thereof.
  • a disclosed polypeptide MBD2 can comprise the amino acid sequence as set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MBD2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide NIPP1 may comprise a sequence as set forth in any one of SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide NIPP1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment thereof.
  • a disclosed polypeptide NIPP1 can comprise the amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment, or a derivative thereof.
  • a disclosed polypeptide NIPP1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide DNMT3A may comprise a sequence as set forth in SEQ ID NO: 69, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide DNMT3 A may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 69, or a fragment thereof.
  • a disclosed polypeptide DNMT3A can comprise the amino acid sequence as set forth in SEQ ID NO: 55, or a fragment, or a derivative thereof.
  • a disclosed polypeptide DNMT3A can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 72, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 72, or a fragment thereof.
  • a disclosed polypeptide MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 57, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 57, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed fusion polypeptide KRAB- MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB-MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 58, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 58, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity.
  • a disclosed encoded polypeptide comprising transcription activation activity can comprise a transactivation domain.
  • a disclosed transactivation domain can comprise a VP16 protein, a series of linked VP16 proteins, a p65 domain of NFKB, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide VP 16 may comprise a sequence as set forth in any one of SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide VP 16 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment thereof.
  • a disclosed polypeptide VP16 can comprise the amino acid sequence as set forth in SEQ ID NO: 131, or a fragment, or a derivative thereof.
  • a disclosed polypeptide VP 16 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131, or a fragment thereof.
  • nucleic acid sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a sequence as set forth in any one of SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise the amino acid sequence as set forth in SEQ ID NO: 134, or a fragment, or a derivative thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription repression activity.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise a Kriippel associated box domain, an ERF repressor domain, a MXI1 repressor domain, a SID4x repressor domain, a fused KRAB-MeCP2 domain, a MeCP2 TRD domain, a MAS-SID repressor domain, a TATA box binding protein activity, or any combination thereof.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise HP1 repressor activity, MeCP2 repressor activity, MBD1 repressor activity, MBD2 repressor activity, MBD3 repressor activity, MBD4 repressor activity, KRAB repressor activity, SUV39H1 repressor activity, SUV39H2 repressor activity, CTCF insulator-repressor activity, LSD-1 hi stone-dem ethyl ase repressor activity, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide comprising Eukaryotic Release Factor 1 (ERF1) activity or Eukaryotic Release Factor 3 (ERF3) activity may comprise a sequence as set forth in any one of SEQ ID NO: 138, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide comprising ERF1 activity or ERF3 activity may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 138, or a fragment thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise the amino acid sequence as set forth in SEQ ID NO: 137, or a fragment, or a derivative thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 137, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise histone modification activity.
  • a disclosed encoded polypeptide comprising histone modification activity can comprise histone acetyltransferase, histone deacetylase, histone demethylase, histone methyltransferase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise methyltransferase activity.
  • a disclosed encoded polypeptide comprising methyltransferase activity can comprise DNA (cytosine-5)-methyltransferase 3a (DNMT3A).
  • DNMT3A DNA
  • a disclosed encoded polypeptide can comprise demethylase activity.
  • a disclosed encoded polypeptide comprising demethylase activity can comprise ten-eleven translocation methylcytosine dioxygenase 1 (TET1) or lysine-specific histone demethylase 1 (LSD1).
  • a disclosed encoded polypeptide can comprise acetyltransferase activity.
  • a disclosed encoded polypeptide comprising acetyltransferase activity can comprise histone acetyltransferase.
  • a disclosed encoded polypeptide can comprise deacetylase activity.
  • a disclosed encoded polypeptide comprising deacetylase activity can comprise histone deacetylase.
  • a disclosed dCas endonuclease can comprise dSaCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Krtippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Krtippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Krtippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dVQR, dEQR, or dVRER and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Krtippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER and a disclosed fusion can comprise HPla- HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb- NIPP1, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9 and a disclosed encoded polypeptide can comprise KRAB-MeCP2.
  • a disclosed dCas endonuclease can comprise dSaCas9, and a disclosed encoded polypeptide (z.e., a disclosed fusion) can comprise KRAB-MeCP2.
  • the encoded fusion protein can comprise a dSpCas9 and a polypeptide having enzymatic activity comprising HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dCjCas9 and a polypeptide having enzymatic activity comprising HP la, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HP la, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dVQR, dEQR, or dVRER and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel- associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel- associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER) and one or more of HPla-HPla, HPla-HPlb, HP 1 a-MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP 1 a-DNMT3 A, HP 1b- HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dSaCas9 and KRAB-MeCP2.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76 respectively, or a derivative thereof.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 78 respectively, or a derivative thereof.
  • the nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A may comprise a sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the sequences set forth in SEQ ID NO: 77 or SEQ ID NO: 78, or a fragment thereof.
  • a disclosed polypeptide dSpCas9 can comprise the amino acid sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment, or a derivative thereof.
  • the isolated nucleic acid molecule comprises a nucleic acid sequence encoding at least one guide RNA targeting a gene of interest.
  • a gene of interest can be any gene which has a disease linked mutation and/or exhibits abnormal expression or activity in the microglia, linked to a disease or disorder. Examples of such genes are known in the art.
  • a disclosed gene of interest or portion thereof can demonstrate a reduced expression and/or activity level when compared to wild-type or control expression level.
  • a disclosed gRNA can target a gene of interest or portion thereof that demonstrates a reduced expression and/or activity level when compared to wild-type or control expression level.
  • a disclosed gene of interest or portion thereof can demonstrate an increased expression and/or activity level when compared to wild-type or control expression level.
  • the gene of interest can be any gene which exhibits unusual expression in microglia in a diseased condition.
  • the gene-of-interest is the APOE gene.
  • a disclosed isolated nucleic acid can comprise at least one gRNA that can target a gene of interest or portion thereof (e.g., an APOE gene).
  • a disclosed gRNA targeting a gene of interest or portion thereof can comprise at least two gRNAs.
  • disclosed gRNAs can target the same gene of interest or portion thereof.
  • disclosed gRNAs can target different genes of interest or portions thereof.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a loss of function.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a gain of function.
  • a suitable gRNA that “targets” a nucleic acid is designed to hybridize to a sequence that is complementary to the “target nucleic acid.”.”
  • the gRNA can comprise an RNA sequence corresponding to the “target nucleic acid.”.”
  • a disclosed gRNA can target the APOE gene sequence or a regulatory element thereof.
  • the at least one gRNA can target the gene, or promoter region thereof, or an untranslated region (UTR) thereof, or any combination thereof.
  • the at least one gRNA may target the promoter region, or intron 1, or a flanking sequence thereof, of APOE gene.
  • a disclosed gRNA can target exon 4 of the APOE gene.
  • a disclosed gRNA can target a protospacer-adjacent motif (PAM) created by a SNP rs429358 in exon 4 of the APOE gene.
  • PAM protospacer-adjacent motif
  • the at least one gRNA may target a region in the APOE gene comprising a nucleic acid sequence as set forth in any one of SEQ ID NO: 11-14, or a complement thereof. In an aspect, the at least one gRNA targets a nucleic acid sequence comprising at least 10 consecutive nucleotides as set forth in SEQ ID NO: 11-14, or a complement thereof. In an aspect, the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33. In an aspect, the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • the nucleic acid molecule can further comprise a gRNA scaffold.
  • a disclosed gRNA scaffold can comprise the sequence set forth in SEQ ID NO: 129, SEQ ID NO: 130, or a fragment thereof.
  • a disclosed isolated nucleic acid molecule can further comprise one or more regulatory elements (z.e., promoters) in addition to the microglia specific regulatory element (e.g., microglia specific promoter).
  • a disclosed additional promoter can comprise a U6 promoter, a chicken P-actin promoter, an EF-la, a CMV promoter, a CMV promoter/enhancer, a fragment thereof, a derivative thereof, or any combination thereof.
  • a disclosed U6 promoter can comprise the sequence set forth in SEQ ID NO: 81, or a fragment thereof.
  • a disclosed U6 promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 81 or a fragment thereof.
  • a disclosed EF-la promoter can comprise the sequence set forth in SEQ ID NO: 79, SEQ ID NO: 80, or a fragment thereof.
  • a disclosed EF-la promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 79, SEQ ID NO:80, or a fragment thereof.
  • a promoter can be a short EFlalpha (EFS-NF) promoter.
  • a disclosed promoter operably can be linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed promoter operably linked to the at least one guide RNA can comprise a U6 promoter.
  • a disclosed promoter operably linked to the at least one guide RNA targeting an APOE gene can comprise a U6 promoter.
  • a disclosed isolated nucleic acid molecule can further comprise two or more promoters, wherein a first promoter can be operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed isolated nucleic acid molecule can further comprise one or more promoters, wherein a first promoter comprises a microglia specific promoter that is operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest thereof.
  • the second promoter operably linked to the gRNA may not be a microglia specific promoter.
  • the isolated nucleic acid can comprise a first promoter selected from a F4/80 promoter, a CD68 promoter, a IBA1 promoter, or a FCGR2A promoter or derivatives thereof, and a second promoter comprising a U6 promoter, or a derivative thereof.
  • a disclosed isolated nucleic acid molecule can further comprise a nucleic acid sequencing encoding one or more regulatory elements.
  • a disclosed regulatory element can comprise a promoter, an enhancer, a promoter/enhancer, an internal ribosomal entry site, a transcription terminal signal, a polyadenylation signal, a Spl and/or NF-kB transcriptional factor binding site, a p2A signal, a woodchuck hepatitis virus post-transcriptional regulatory element, a Phi signal -packaging signal, a rev responsive element, a 5’-ITR, a 3’-ITR, an inverted terminal repeat, a nuclear localization signal (NLS), or any combination thereof.
  • NLS nuclear localization signal
  • a disclosed 5’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 82 or SEQ ID NO: 83.
  • a disclosed 3’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 84 or SEQ ID NO: 85.
  • a disclosed NLS can comprise the sequence set forth in any one of SEQ ID NO: 88 - SEQ ID NO: 94, or a fragment thereof.
  • a disclosed PolyA sequence can comprise the sequence set forth in SEQ ID NO: 86, SEQ ID NO: 87, or a fragment thereof.
  • a disclosed nucleic acid sequence can be CpG depleted and codon-optimized for expression in a human cell.
  • CpG-free can mean completely free of CpGs or partially free of CpGs.
  • CpG-free can mean “CpG-depleted.”.”
  • CpG- depleted can mean “CpG-free.”
  • CpG-depleted can mean completely depleted of CpGs or partially depleted of CpGs.
  • CpG-free can mean “CpG-optimized” for a desired and/or ideal expression level.
  • any disclosed Cas9 endonuclease, a disclosed polypeptide having enzymatic activities, a disclosed fusion product or a disclosed fusion protein, or any combination thereof can be codon-optimized.
  • a disclosed isolated nucleic acid molecule can comprise a sequence that is about 4.5 kilobases or less than about 4.5 kilobases.
  • an isolated nucleic acid molecule comprising: a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof.
  • dCas deactivated Cas
  • an isolated nucleic acid molecule comprising: a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, (iii) at least one guide RNA targeting a gene of interest or portion thereof, (iv) a promoter operably linked to the dCas endonuclease and the fusion protein, and (v) a promoter operably linked to the at least one guide RNA.
  • dCas deactivated Cas
  • an isolated nucleic acid molecule comprising: a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion protein comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting APOE or portion thereof.
  • dCas deactivated Cas
  • an isolated nucleic acid molecule comprising: a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion protein comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, (iii) at least one guide RNA targeting APOE or portion thereof, (iv) a microglia specific promoter (e.g.
  • a disclosed isolated nucleic acid molecule can treat and/or prevent Alzheimer’s disease progression. In an aspect, a disclosed isolated nucleic acid molecule can treat and/or prevent Parkinson’s disease progression.
  • a viral vector comprising a disclosed isolated nucleic acid molecule.
  • a viral vector comprising a disclosed nucleic acid molecule.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picornavirus vector.
  • a disclosed viral vector can be a lentiviral vector.
  • a recombinant AAV vector comprising a disclosed isolated nucleic acid molecule.
  • a recombinant AAV vector comprising a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting an APOE gene sequence or a regulatory element thereof; and (b) one or more microglia specific regulatory elements operably linked to (a)(i), (a)(ii), (a)(iii), or any combination thereof.
  • dCas Clustered Regularly Interspaced Short Palindromic Repeats associated
  • a recombinant AAV vector comprising a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a recombinant AAV vector comprising a disclosed isolated nucleic acid molecule comprising (a) (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a recombinant AAV vector comprising a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • the microglia specific regulatory sequence may comprise a wild-type promoter, a microglia specific enhancer, a synthetic promoter, a synthetic enhancer, a transcriptional or translational regulatory sequence or any combination thereof.
  • the microglia specific regulatory sequence is a microglia specific promoter.
  • the microglia specific promoter can be any promoter known in the art, that exhibits enhanced activity in the microglia.
  • Non-limiting examples of microglia specific promoters include F4/80, CD68, FCGR2A, HexB or IBA1 promoter, or a derivative thereof.
  • the promoter is a CD68 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one or more of the sequences set forth in SEQ ID NO: 1-3, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 1-3.
  • the promoter is a F4/80 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 4.
  • the promoter is a IBA1 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5-7, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 5-7.
  • the promoter is a FCGR2A promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 8, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 8.
  • the disclosed viral vector or disclosed recombinant AAV vector may comprise at least a microglia specific regulatory element (z.e., a microglia specific promoter) which may be operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, or a derivative thereof.
  • the disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • the disclosed dSpCas9 may comprise a sequence as set forth in any one of SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSpCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof.
  • a disclosed dSpCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment, or a derivative thereof.
  • a disclosed dSpCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment thereof.
  • the disclosed dSaCas9 may comprise a sequence as set forth in SEQ ID NO: 42, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSaCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 42, or a fragment thereof.
  • a disclosed dSaCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 38 or a fragment, or a derivative thereof.
  • a disclosed dSaCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 38, or a fragment thereof.
  • the disclosed dCjCas9 may comprise a sequence as set forth in SEQ ID NO: 43, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dCjCas9 endonuclease may comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 43, or a fragment thereof.
  • a disclosed dCjCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 39, or a fragment, or a derivative thereof.
  • a disclosed dCjCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 39 or a fragment thereof.
  • a disclosed variant of dCas9 dVQR can comprise DI 135V, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dEQR can comprise D1135E, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dVRER can comprise DI 135V, G1218R, R1335E, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed dVRER can comprise the sequence set forth in SEQ ID NO: 37 or a fragment thereof.
  • the disclosed dCas9 endonuclease may comprise a sequence as set forth in any one of SEQ ID NO: 40-43.
  • the sequence encoding the disclosed dCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40-43, or a fragment, or a derivative thereof.
  • the disclosed viral vector or disclosed recombinant AAV vector may comprise a nucleic acid sequence encoding at least one polypeptide having an enzymatic activity.
  • a disclosed encoded polypeptide can comprise transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nucleic acid association activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, or any combination thereof.
  • the disclosed encoded polypeptide can comprise HP la, HP lb, MBD1, MBD2, Kriippel-Associated Box (KRAB), NIPP1, the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • KRAB Kriippel-Associated Box
  • NIPP1 the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed fusion can comprise HPla-HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb- HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb- DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1- NIPP1, MBDl-MeCP2, MBD1-DNMT3A, MBD2-MBD2, MBD2-HPla, MBD2-HPlb, MBD2- MBD1, MBD2-KRAB, MBD2-NIPP1, MBD2-MeCP2, MBD2-DNMT3A, KRAB-KRAB, KRA
  • the disclosed viral vector or disclosed recombinant AAV vector encoding the polypeptide HP la may comprise a sequence as set forth in any one of SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP la may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment thereof.
  • a disclosed polypeptide HPla can comprise the amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPla can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide HP lb may comprise a sequence as set forth in any one of SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP lb may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment thereof.
  • a disclosed polypeptide HPlb can comprise the amino acid sequence as set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPlb can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD1 may comprise a sequence as set forth in any one of SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment thereof.
  • a disclosed polypeptide MBD1 can comprise the amino acid sequence as set forth in any one of SEQ ID NO: 48-50, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MDB1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 48-50, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD2 may comprise a sequence as set forth in any one of SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment thereof.
  • a disclosed polypeptide MBD2 can comprise the amino acid sequence as set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MBD2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide NIPP1 may comprise a sequence as set forth in any one of SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide NIPP1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment thereof.
  • a disclosed polypeptide NIPP1 can comprise the amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment, or a derivative thereof.
  • a disclosed polypeptide NIPP1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide DNMT3 A may comprise a sequence as set forth in SEQ ID NO: 69, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide DNMT3 A may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 69, or a fragment thereof.
  • a disclosed polypeptide DNMT3A can comprise the amino acid sequence as set forth in SEQ ID NO: 55, or a fragment, or a derivative thereof.
  • a disclosed polypeptide DNMT3A can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 72, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 72, or a fragment thereof.
  • a disclosed polypeptide MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 57, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 57, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed fusion polypeptide KRAB- MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB-MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 58, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 58, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity.
  • a disclosed encoded polypeptide comprising transcription activation activity can comprise a transactivation domain.
  • a disclosed transactivation domain can comprise a VP16 protein, a series of linked VP16 proteins, a p65 domain of NFKB, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide VP 16 may comprise a sequence as set forth in any one of SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide VP 16 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment thereof.
  • a disclosed polypeptide VP16 can comprise the amino acid sequence as set forth in SEQ ID NO: 131, or a fragment, or a derivative thereof.
  • a disclosed polypeptide VP 16 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription repression activity.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise a Kriippel associated box domain, an ERF repressor domain, a MXI1 repressor domain, a SID4x repressor domain, a fused KRAB-MeCP2 domain, a MeCP2 TRD domain, a MAS-SID repressor domain, a TATA box binding protein activity, or any combination thereof.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise HP1 repressor activity, MeCP2 repressor activity, MBD1 repressor activity, MBD2 repressor activity, MBD3 repressor activity, MBD4 repressor activity, KRAB repressor activity, SUV39H1 repressor activity, SUV39H2 repressor activity, CTCF insulator-repressor activity, LSD-1 hi stone-dem ethyl ase repressor activity, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide comprising Eukaryotic Release Factor 1 (ERF1) activity or Eukaryotic Release Factor 3 (ERF3) activity may comprise a sequence as set forth in any one of SEQ ID NO: 138, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide comprising ERF1 activity or ERF3 activity may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 138, or a fragment thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise the amino acid sequence as set forth in SEQ ID NO: 137, or a fragment, or a derivative thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 137, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise histone modification activity.
  • a disclosed encoded polypeptide comprising histone modification activity can comprise histone acetyltransferase, histone deacetylase, histone demethylase, histone methyltransferase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise nucleic acid association activity.
  • a disclosed encoded polypeptide comprising nucleic acid association activity can comprise a helix-turn-helix region, a leucine zipper region, a winged helix region, a winged helix-turn-helix region, a helix-loop-helix region, an immunoglobulin fold, a B3 domain, a zinc finger, a HMG-box, a Wor3 domain, a TAL effector DNA-binding domain, or any combination thereof.
  • a disclosed encoded polypeptide can comprise methyltransferase activity.
  • a disclosed encoded polypeptide comprising methyltransferase activity can comprise DNA (cytosine-5)-methyltransferase 3a (DNMT3A).
  • DNMT3A DNA
  • a disclosed encoded polypeptide can comprise demethylase activity.
  • a disclosed encoded polypeptide comprising demethylase activity can comprise ten-eleven translocation methylcytosine dioxygenase 1 (TET1) or lysine-specific histone demethylase 1 (LSD1).
  • a disclosed encoded polypeptide can comprise acetyltransferase activity.
  • a disclosed encoded polypeptide comprising acetyltransferase activity can comprise histone acetyltransferase.
  • a disclosed encoded polypeptide can comprise deacetylase activity.
  • a disclosed encoded polypeptide comprising deacetylase activity can comprise histone deacetylase.
  • the vector may comprise a nucleic acid sequence encoding a disclosed dCas endonuclease, comprising dSpCas9 and a disclosed encoded polypeptide comprising any one or more of HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease comprising dSpCas9 and a disclosed encoded polypeptide comprising any one or more of HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSaCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dVQR, dEQR, or dVRER and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel- associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER and a disclosed fusion can comprise HPla- HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb- NIPP1, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9 and a disclosed encoded polypeptide can comprise KRAB-MeCP2.
  • a disclosed dCas endonuclease can comprise dSaCas9, and a disclosed encoded polypeptide (z.e., a disclosed fusion) can comprise KRAB-MeCP2.
  • the disclosed viral vector or disclosed recombinant AAV vector may comprise a nucleic acid sequence encoding a dCas endonuclease fused to the at least one encoded polypeptide having an enzymatic activity.
  • the isolated nucleic acid can encode for a “fusion protein” wherein the fusion protein comprises a first component comprising a dCas endonuclease and a second component comprising at least one polypeptide having an enzymatic activity.
  • the encoded fusion protein can comprise a dSpCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dSaCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dCjCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dVQR, dEQR, or dVRER and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER) and one or more of HPla-HPla, HPla-HPlb, HP 1 a-MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP 1 a-DNMT3 A, HP 1b- HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dSaCas9 and KRAB-MeCP2.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 78 respectively, or a derivative thereof.
  • the nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3 A may comprise a sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the sequences set forth in SEQ ID NO: 77 or SEQ ID NO: 78, or a fragment thereof.
  • a disclosed polypeptide dSpCas9 can comprise the amino acid sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment thereof.
  • the disclosed viral vector or disclosed recombinant AAV vector may further encode at least one guide RNA targeting a gene of interest.
  • a disclosed gene of interest or portion thereof can demonstrate a reduced expression and/or activity level when compared to wild-type or control expression level.
  • a disclosed gRNA can target a gene of interest or portion thereof that demonstrates a reduced expression and/or activity level when compared to wild-type or control expression level.
  • a disclosed gene of interest or portion thereof can demonstrate an increased expression and/or activity level when compared to wild-type or control expression level.
  • the gene of interest can be any gene which exhibits unusual expression in microglia in a diseased condition.
  • the gene-of-interest is the APOE gene.
  • the at least one gRNA can target a gene of interest or portion thereof (e.g., an APOE gene).
  • the viral vector encoding gRNAs targeting a gene of interest or portion thereof can comprise at least two gRNAs.
  • disclosed gRNAs can target the same gene of interest or portion thereof.
  • disclosed gRNAs can target different genes of interest or portions thereof.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a loss of function.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a gain of function.
  • a suitable gRNA that “targets” a nucleic acid is designed to hybridize to a sequence that is complementary to the “target nucleic acid.” This, in effect, results in a gRNA that comprises an RNA sequence having a certain percent identity to the target nucleic acid (DNA), with the understanding that uracil in the RNA sequence is equivalent to thymidine in the DNA target sequence.
  • the gRNA can comprise an RNA sequence corresponding to the “target nucleic acid.”
  • a disclosed gRNA scaffold [0249]
  • a disclosed gRNA can target the APOE gene sequence or a regulatory element thereof.
  • the at least one gRNA can target xe. APOE gene, or promoter region thereof, or an untranslated region (UTR) thereof, or any combination thereof.
  • the at least one gRNA may target the promoter region, or intron 1, or a flanking sequence thereof, of APOE gene.
  • a disclosed gRNA can target exon 4 of the APOE gene.
  • a disclosed gRNA can target a protospacer-adjacent motif (PAM) created by a SNP rs429358 in exon 4 of the APOE gene.
  • PAM protospacer-adjacent motif
  • the at least one gRNA may target a region in the APOE gene comprising a nucleic acid sequence as set forth in any one of SEQ ID NO: 11-14, or a complement thereof. In an aspect, the at least one gRNA targets a nucleic acid sequence comprising at least 10 consecutive nucleotides as set forth in SEQ ID NO: 11-14, or a complement thereof. . In an aspect, the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof. In an aspect, the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • the disclosed viral vector or disclosed recombinant AAV vector can further comprise one or more regulatory elements (z.e., promoters) in addition to the microglia specific regulatory element (e.g., microglia specific promoter).
  • a disclosed additional promoter can comprise a U6 promoter, a chicken [3-actin promoter, an EF-la, a CMV promoter, a CMV promoter/enhancer, a fragment thereof, a derivative thereof, or any combination thereof.
  • a disclosed U6 promoter can comprise the sequence set forth in SEQ ID NO: 81, or a fragment thereof.
  • a disclosed U6 promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 81 or a fragment thereof.
  • a disclosed EF-la promoter can comprise the sequence set forth in SEQ ID NO: 79, SEQ ID NO: 80, or a fragment thereof.
  • a disclosed EF-la promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 79, SEQ ID NO:80, or a fragment thereof.
  • a promoter can be a short EFlalpha (EFS-NF) promoter.
  • a disclosed promoter operably can be linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed promoter operably linked to the at least one guide RNA can comprise a U6 promoter.
  • a disclosed promoter operably linked to the at least one guide RNA targeting an APOE gene can comprise a U6 promoter.
  • the disclosed viral vector or disclosed recombinant AAV vector can further comprise two or more promoters, wherein a first promoter can be operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed isolated nucleic acid molecule can further comprise one or more promoters, wherein a first promoter comprises a microglia specific promoter that is operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest thereof.
  • the second promoter operably linked to the gRNA may not be a microglia specific promoter.
  • the isolated nucleic acid can comprise a first promoter selected from a F4/80 promoter, a CD68 promoter, a IBA1 promoter, or a FCGR2A promoter or derivatives thereof, and a second promoter comprising a U6 promoter, or a derivative thereof.
  • the disclosed viral vector or disclosed recombinant AAV vector can further comprise one or more regulatory elements.
  • a disclosed regulatory element can comprise a promoter, an enhancer, a promoter/enhancer, an internal ribosomal entry site, a transcription terminal signal, a polyadenylation signal, a Spl and/or NF-kB transcriptional factor binding site, a p2A signal, a woodchuck hepatitis virus post-transcriptional regulatory element, a Phi signal-packaging signal, a rev responsive element, a 5’-ITR, a 3’-ITR, an inverted terminal repeat, a nuclear localization signal (NLS), or any combination thereof.
  • NLS nuclear localization signal
  • a disclosed 5’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 82 or SEQ ID NO: 83.
  • a disclosed 3’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 84 or SEQ ID NO: 85.
  • a disclosed NLS can comprise the sequence set forth in any one of SEQ ID NO: 88 - SEQ ID NO: 94, or a fragment thereof.
  • a disclosed PolyA sequence can comprise the sequence set forth in SEQ ID NO: 86, SEQ ID NO: 87, or a fragment thereof.
  • the disclosed viral vector or disclosed recombinant AAV vector sequence can be CpG depleted and codon- optimized for expression in a human cell.
  • “CpG-free” can mean completely free of CpGs or partially free of CpGs.
  • CpG-free can mean “CpG-depleted.” In an aspect, “CpG-depleted” can mean “CpG-free.” In an aspect, “CpG-depleted” can mean completely depleted of CpGs or partially depleted of CpGs. In an aspect, “CpG-free” can mean “CpG- optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art. In an aspect, any disclosed Cas9 endonuclease, a disclosed polypeptide having enzymatic activities, a disclosed fusion product or a disclosed fusion protein, or any combination thereof can be codon-optimized.
  • the disclosed viral vector or disclosed recombinant AAV vector can comprise a sequence that is about 4.5 kilobases or less than about 4.5 kilobases.
  • a viral vector or a recombinant AAV vector comprising a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, KRAB-MeCP2 or DNMT3A, and (iii) at least one guide RNA targeting APOE (e.g. APOE e4 allele) or portion thereof.
  • APOE e.g. APOE e4 allele
  • a viral vector or a recombinant AAV vector comprising a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, KRAB-MeCP2 or DNMT3 A, (iii) at least one guide RNA targeting APOE (e.g.
  • APOE e4 allele or portion thereof, (iv) a microglia specific regulatory sequence (e. g. promoter) operably linked to the dCas endonuclease and the fusion protein, and (v) a promoter operably linked to the at least one guide RNA.
  • a microglia specific regulatory sequence e. g. promoter
  • a promoter operably linked to the at least one guide RNA.
  • a disclosed recombinant AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV.
  • AAV1, AAV2, AAV3 including 3a and 3b
  • AAV4 AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, A
  • an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape.
  • Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A- String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV- PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81.
  • a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1).
  • a disclosed AAV vector can be AAV8.
  • a disclosed AAV vector can be AAVhum.8.
  • a disclosed AAV vector can be AAV9.
  • a disclosed viral vector or disclosed recombinant AAV vector can comprise the sequence set forth in any of SEQ ID NO: 112 - SEQ ID NO: 128, or a variant thereof, or a fragment thereof, or a portion thereof.
  • a disclosed viral vector or disclosed recombinant AAV vector can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 112 - SEQ ID NO: 128, or a variant thereof, or a fragment thereof, or a portion thereof.
  • a lentiviral vector comprising the disclosed isolated nucleic acid molecule.
  • a lentiviral vector comprising the disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest sequence or a regulatory element thereof; and (b) one or more microglia specific regulatory elements operably linked to (a)(i), (a)(ii), (a)(iii), or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a lentiviral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest (e.g. APOE) or portion thereof.
  • a deactivated Cas dCas
  • APOE guide RNA targeting a gene of interest
  • a lentiviral vector comprising the nucleic acid sequence encoding (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene-of-interest or a regulatory element thereof; and (b) one or more promoters elements operably linked to (a)(i), (a)(ii), (a)(iii), or any combination thereof, wherein at least one of the promoter is a microglia specific promoter.
  • the gene of interest is d/YV/ or an allele thereof.
  • a lentiviral vector comprising (a) a nucleic acid sequence encoding (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a lentiviral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • the lentiviral vector may comprise a sequence encoding any one of the disclosed dCas9, any one or more of the disclosed polypeptides having an enzymatic activity, any one or more of the disclosed gRNA.
  • the lentiviral vector may further comprise one or more of the disclosed microglia specific regulatory elements (e. g. microglia specific promoter) and optionally additional disclosed promoters.
  • a lentiviral vector comprising the disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease comprising an amino acid sequence as set forth in any one of SEQ ID NO: 36- SEQ ID NO: 38, or a fragment, or derivative thereof, or a sequence at least about 60% identical thereto (ii) at least one polypeptide having an enzymatic activity comprising an amino acid sequence as set forth in any one or more of SEQ ID NO: 44- SEQ ID NO: 58, or a sequence at least about 60% identical thereto and (iii) at least one guide RNA targeting an APOE gene sequence or a regulatory element thereof comprising, wherein the targeted sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 15 - SEQ ID NO: 33, or a complement thereof, or a sequence at least about 60%
  • a disclosed lentiviral vector can comprise the sequence set forth in any of SEQ ID NO: 105 - SEQ ID NO: 111, or a variant thereof, or a fragment thereof, or a portion thereof.
  • a disclosed viral vector or disclosed recombinant AAV vector can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 105 - SEQ ID NO: 111, or a variant thereof, or a fragment thereof, or a portion thereof.
  • a disclosed viral vector or a disclosed recombinant AAV vector of lentiviral vector can treat and/or prevent Alzheimer’s disease progression.
  • a disclosed viral vector or a disclosed recombinant AAV vector can treat and/or prevent Parkinson’s disease progression.
  • a plasmid vector comprising a disclosed isolated nucleic acid molecule.
  • a plasmid vector comprising a disclosed nucleic acid molecule.
  • a plasmid vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is d/YV/ or an allele thereof.
  • a plasmid vector comprising a disclosed isolated nucleic acid molecule.
  • a plasmid vector comprising a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a plasmid vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a plasmid vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • the microglia specific regulatory sequence may comprise a wild-type promoter, a microglia specific enhancer, a synthetic promoter, a synthetic enhancer, a transcriptional or translational regulatory sequence or any combination thereof.
  • the microglia specific regulatory sequence is a microglia specific promoter.
  • the microglia specific promoter can be any promoter known in the art, that exhibits enhanced activity in the microglia.
  • Non-limiting examples of microglia specific promoters include F4/80, CD68, FCGR2A, HexB or IBA1 promoter, or a derivative thereof.
  • the promoter is a CD68 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one or more of the sequences set forth in SEQ ID NO: 1-3, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 1-3.
  • the promoter is a F4/80 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 4.
  • the promoter is a IBA1 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5-7, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 5-7.
  • the promoter is a FCGR2A promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 8, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 8.
  • the disclosed plasmid vector may comprise at least a microglia specific regulatory element (z.e., a microglia specific promoter) which may be operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, or a derivative thereof.
  • the disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • the disclosed dSpCas9 may comprise a sequence as set forth in any one of SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSpCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof.
  • a disclosed dSpCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment, or a derivative thereof.
  • a disclosed dSpCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment thereof.
  • the disclosed dSaCas9 may comprise a sequence as set forth in SEQ ID NO: 42, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSaCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 42, or a fragment thereof.
  • a disclosed dSaCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 38 or a fragment, or a derivative thereof.
  • a disclosed dSaCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 38, or a fragment thereof.
  • the disclosed dCjCas9 may comprise a sequence as set forth in SEQ ID NO: 43, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dCjCas9 endonuclease may comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 43, or a fragment thereof.
  • a disclosed dCjCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 39, or a fragment, or a derivative thereof.
  • a disclosed dCjCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 39 or a fragment thereof.
  • a disclosed variant of dCas9 dVQR can comprise DI 135V, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dEQR can comprise D1135E, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dVRER can comprise DI 135V, G1218R, R1335E, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed dVRER can comprise the sequence set forth in SEQ ID NO: 37 or a fragment thereof.
  • the disclosed dCas9 endonuclease may comprise a sequence as set forth in any one of SEQ ID NO: 40-43.
  • the sequence encoding the disclosed dCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40-43, or a fragment, or a derivative thereof.
  • the disclosed plasmid vector may comprise a nucleic acid sequence encoding at least one polypeptide having an enzymatic activity.
  • a disclosed encoded polypeptide can comprise transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nucleic acid association activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, or any combination thereof.
  • the disclosed encoded polypeptide can comprise HP la, HP lb, MBD1, MBD2, Kriippel-Associated Box (KRAB), NIPP1, the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • KRAB Kriippel-Associated Box
  • NIPP1 the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed fusion can comprise HPla-HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb- HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb- DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1- NIPP1, MBDl-MeCP2, MBD1-DNMT3A, MBD2-MBD2, MBD2-HPla, MBD2-HPlb, MBD2- MBD1, MBD2-KRAB, MBD2-NIPP1, MBD2-MeCP2, MBD2-DNMT3A, KRAB-KRAB, KRA
  • the polypeptide HP la may comprise a sequence as set forth in any one of SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP la may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment thereof.
  • a disclosed polypeptide HPla can comprise the amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPla can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide HP lb may comprise a sequence as set forth in any one of SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP lb may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment thereof.
  • a disclosed polypeptide HPlb can comprise the amino acid sequence as set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPlb can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD1 may comprise a sequence as set forth in any one of SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment thereof.
  • a disclosed polypeptide MBD1 can comprise the amino acid sequence as set forth in any one of SEQ ID NO: 48-50, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MDB1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 48-50, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD2 may comprise a sequence as set forth in any one of SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment thereof.
  • a disclosed polypeptide MBD2 can comprise the amino acid sequence as set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MBD2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide NIPP1 may comprise a sequence as set forth in any one of SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide NIPP1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment thereof.
  • a disclosed polypeptide NIPP1 can comprise the amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment, or a derivative thereof.
  • a disclosed polypeptide NIPP1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide DNMT3 A may comprise a sequence as set forth in SEQ ID NO: 69, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide DNMT3 A may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 69, or a fragment thereof.
  • a disclosed polypeptide DNMT3A can comprise the amino acid sequence as set forth in SEQ ID NO: 55, or a fragment, or a derivative thereof.
  • a disclosed polypeptide DNMT3A can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 72, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 72, or a fragment thereof.
  • a disclosed polypeptide MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 57, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 57, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed fusion polypeptide KRAB- MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB-MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 58, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 58, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity.
  • a disclosed encoded polypeptide comprising transcription activation activity can comprise a transactivation domain.
  • a disclosed transactivation domain can comprise a VP16 protein, a series of linked VP16 proteins, a p65 domain of NFKB, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide VP 16 may comprise a sequence as set forth in any one of SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide VP 16 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment thereof.
  • a disclosed polypeptide VP16 can comprise the amino acid sequence as set forth in SEQ ID NO: 131, or a fragment, or a derivative thereof.
  • a disclosed polypeptide VP 16 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131, or a fragment thereof.
  • nucleic acid sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a sequence as set forth in any one of SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise the amino acid sequence as set forth in SEQ ID NO: 134, or a fragment, or a derivative thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription repression activity.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise a Kriippel associated box domain, an ERF repressor domain, a MXI1 repressor domain, a SID4x repressor domain, a fused KRAB-MeCP2 domain, a MeCP2 TRD domain, a MAS-SID repressor domain, a TATA box binding protein activity, or any combination thereof.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise HP1 repressor activity, MeCP2 repressor activity, MBD1 repressor activity, MBD2 repressor activity, MBD3 repressor activity, MBD4 repressor activity, KRAB repressor activity, SUV39H1 repressor activity, SUV39H2 repressor activity, CTCF insulator-repressor activity, LSD-1 hi stone-dem ethyl ase repressor activity, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide comprising Eukaryotic Release Factor 1 (ERF1) activity or Eukaryotic Release Factor 3 (ERF3) activity may comprise a sequence as set forth in any one of SEQ ID NO: 138, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide comprising ERF1 activity or ERF3 activity may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 138, or a fragment thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise the amino acid sequence as set forth in SEQ ID NO: 137, or a fragment, or a derivative thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 137, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise histone modification activity.
  • a disclosed encoded polypeptide comprising histone modification activity can comprise histone acetyltransferase, histone deacetylase, histone demethylase, histone methyltransferase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise nucleic acid association activity.
  • a disclosed encoded polypeptide comprising nucleic acid association activity can comprise a helix-turn-helix region, a leucine zipper region, a winged helix region, a winged helix-turn-helix region, a helix-loop-helix region, an immunoglobulin fold, a B3 domain, a zinc finger, a HMG-box, a Wor3 domain, a TAL effector DNA-binding domain, or any combination thereof.
  • a disclosed encoded polypeptide can comprise methyltransferase activity.
  • a disclosed encoded polypeptide comprising methyltransferase activity can comprise DNA (cytosine-5)-methyltransferase 3a (DNMT3A).
  • DNMT3A DNA
  • a disclosed encoded polypeptide can comprise demethylase activity.
  • a disclosed encoded polypeptide comprising demethylase activity can comprise ten-eleven translocation methylcytosine dioxygenase 1 (TET1) or lysine-specific histone demethylase 1 (LSD1).
  • a disclosed encoded polypeptide can comprise acetyltransferase activity.
  • a disclosed encoded polypeptide comprising acetyltransferase activity can comprise histone acetyltransferase.
  • a disclosed encoded polypeptide can comprise deacetylase activity.
  • a disclosed encoded polypeptide comprising deacetylase activity can comprise histone deacetylase.
  • a disclosed plasmid vector may comprise a nucleic acid sequence encoding a disclosed dCas endonuclease, comprising dSpCas9 and a disclosed encoded polypeptide comprising any one or more of HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease comprising dSpCas9 and a disclosed encoded polypeptide comprising any one or more of HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof
  • a disclosed dCas endonuclease can comprise dSaCas9 and a disclosed encoded polypeptide can comprise HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl -CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dVQR, dEQR, or dVRER and a disclosed encoded polypeptide can comprise HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER and a disclosed fusion can comprise HPla- HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb- NIPP1, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9 and a disclosed encoded polypeptide can comprise KRAB-MeCP2.
  • a disclosed dCas endonuclease can comprise dSaCas9, and a disclosed encoded polypeptide (z.e., a disclosed fusion) can comprise KRAB-MeCP2.
  • a disclosed plasmid vector may comprise a nucleic acid sequence encoding a dCas endonuclease fused to the at least one encoded polypeptide having an enzymatic activity.
  • the isolated nucleic acid can encode for a “fusion protein” wherein the fusion protein comprises a first component comprising a dCas endonuclease and a second component comprising at least one polypeptide having an enzymatic activity.
  • the encoded fusion protein can comprise a dSpCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dSaCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dCjCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dVQR, dEQR, or dVRER and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER) and one or more of HPla-HPla, HPla-HPlb, HP 1 a-MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP 1 a-DNMT3 A, HP 1b- HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dSaCas9 and KRAB-MeCP2.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 78 respectively, or a derivative thereof.
  • the nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3 A may comprise a sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the sequences set forth in SEQ ID NO: 77 or SEQ ID NO: 78, or a fragment thereof.
  • a disclosed polypeptide dSpCas9 can comprise the amino acid sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment thereof.
  • a disclosed plasmid vector may further encode at least one guide RNA targeting a gene of interest.
  • a disclosed gene of interest or portion thereof can demonstrate a reduced expression and/or activity level when compared to wild-type or control expression level.
  • a disclosed gRNA can target a gene of interest or portion thereof that demonstrates a reduced expression and/or activity level when compared to wild-type or control expression level.
  • a disclosed gene of interest or portion thereof can demonstrate an increased expression and/or activity level when compared to wild-type or control expression level.
  • the gene of interest can be any gene which exhibits unusual expression in microglia in a diseased condition.
  • the gene-of-interest is the APOE gene.
  • the at least one gRNA can target a gene of interest or portion thereof (e.g., an APOE gene).
  • the plasmid vector encoding gRNAs targeting a gene of interest or portion thereof can comprise at least two gRNAs.
  • disclosed gRNAs can target the same gene of interest or portion thereof.
  • disclosed gRNAs can target different genes of interest or portions thereof.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a loss of function.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a gain of function.
  • a suitable gRNA that “targets” a nucleic acid is designed to hybridize to a sequence that is complementary to the “target nucleic acid.”
  • the gRNA can comprise an RNA sequence corresponding to the “target nucleic acid.”
  • a disclosed gRNA scaffold is designed to hybridize to a sequence that is complementary to the “target nucleic acid.”
  • a disclosed gRNA can target the APOE gene sequence or a regulatory element thereof.
  • the at least one gRNA can target xe. APOE gene, or promoter region thereof, or an untranslated region (UTR) thereof, or any combination thereof.
  • the at least one gRNA may target the promoter region, or intron 1, or a flanking sequence thereof, of APOE gene.
  • a disclosed gRNA can target exon 4 of the APOE gene.
  • a disclosed gRNA can target a protospacer-adjacent motif (PAM) created by a SNP rs429358 in exon 4 of the APOE gene.
  • PAM protospacer-adjacent motif
  • the at least one gRNA may target a region in the APOE gene comprising a nucleic acid sequence as set forth in any one of SEQ ID NO: 11-14, or a complement thereof. In an aspect, the at least one gRNA targets a nucleic acid sequence comprising at least 10 consecutive nucleotides as set forth in SEQ ID NO: 11-14, or a complement thereof. . In an aspect, the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof. In an aspect, the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • a disclosed plasmid vector can further comprise one or more regulatory elements (z.e., promoters) in addition to the microglia specific regulatory element (e.g., microglia specific promoter).
  • a disclosed additional promoter can comprise a U6 promoter, a chicken [3-actin promoter, an EF-la, a CMV promoter, a CMV promoter/enhancer, a fragment thereof, a derivative thereof, or any combination thereof.
  • a disclosed U6 promoter can comprise the sequence set forth in SEQ ID NO: 81, or a fragment thereof.
  • a disclosed U6 promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 81 or a fragment thereof.
  • a disclosed EF-la promoter can comprise the sequence set forth in SEQ ID NO: 79, SEQ ID NO: 80, or a fragment thereof.
  • a disclosed EF-la promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 79, SEQ ID NO: 80, or a fragment thereof.
  • a promoter can be a short EFlalpha (EFS-NF) promoter.
  • a disclosed promoter operably can be linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed promoter operably linked to the at least one guide RNA can comprise a U6 promoter.
  • a disclosed promoter operably linked to the at least one guide RNA targeting an APOE gene can comprise a U6 promoter.
  • a disclosed plasmid vector can further comprise two or more promoters, wherein a first promoter can be operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed isolated nucleic acid molecule can further comprise one or more promoters, wherein a first promoter comprises a microglia specific promoter that is operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest thereof.
  • the second promoter operably linked to the gRNA may not be a microglia specific promoter.
  • the isolated nucleic acid can comprise a first promoter selected from a F4/80 promoter, a CD68 promoter, a IBA1 promoter, or a FCGR2A promoter or derivatives thereof, and a second promoter comprising a U6 promoter, or a derivative thereof.
  • a disclosed plasmid vector can further comprise one or more regulatory elements.
  • a disclosed regulatory element can comprise a promoter, an enhancer, a promoter/enhancer, an internal ribosomal entry site, a transcription terminal signal, a polyadenylation signal, a Spl and/or NF-kB transcriptional factor binding site, a p2A signal, a woodchuck hepatitis virus post-transcriptional regulatory element, a Phi signal-packaging signal, a rev responsive element, a nuclear localization signal (NLS), or any combination thereof.
  • a disclosed NLS can comprise the sequence set forth in any one of SEQ ID NO: 88 - SEQ ID NO: 94, or a fragment thereof.
  • a disclosed PolyA sequence can comprise the sequence set forth in SEQ ID NO: 86, SEQ ID NO: 87, or a fragment thereof.
  • a disclosed plasmid vector can be CpG depleted and codon-optimized for expression in a human cell.
  • CpG-free can mean completely free of CpGs or partially free of CpGs.
  • CpG-free can mean “CpG-depleted.”
  • CpG- depleted can mean “CpG-free.”
  • CpG-depleted can mean completely depleted of CpGs or partially depleted of CpGs.
  • CpG-free can mean “CpG-optimized” for a desired and/or ideal expression level.
  • any disclosed Cas9 endonuclease, a disclosed polypeptide having enzymatic activities, a disclosed fusion product or a disclosed fusion protein, or any combination thereof can be codon-optimized.
  • a plasmid vector comprising a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, KRAB-MeCP2 or DNMT3A, and (iii) at least one guide RNA targeting APOE (e.g. APOE e4 allele) or portion thereof.
  • APOE e.g. APOE e4 allele
  • a plasmid vector comprising a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion protein having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, KRAB-MeCP2 or DNMT3 A, (iii) at least one guide RNA targeting APOE (e.g.
  • APOE e4 allele or portion thereof, (iv) a microglia specific regulatory sequence (e. g. promoter) operably linked to the dCas endonuclease and the fusion protein, and (v) a promoter operably linked to the at least one guide RNA.
  • a microglia specific regulatory sequence e. g. promoter
  • a promoter operably linked to the at least one guide RNA.
  • a disclosed plasmid vector can comprise the sequence set forth in any of SEQ ID NO: 95 - SEQ ID NO: 104, or a variant thereof, or a fragment thereof, or a portion thereof.
  • a disclosed plasmid vector can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 95 - SEQ ID NO: 104, or a variant thereof, or a fragment thereof, or a portion thereof.
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) one or more microglia specific regulatory elements operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • dCas deactivated Cas
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting APOE or portion thereof and (b) one or more microglia specific promoters operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • dCas deactivated Cas
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting APOE or portion thereof, (b) one or more microglia specific regulatory elements operably linked to (a)(i) and/or (a)(ii), and (c) a promoter operably linked to (a)(iii).
  • dCas deactivated Cas
  • a host cell or a host cell line comprising an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting APOE or portion thereof, (b) one or more neuron specific promoters operably linked to (a)(i) and/or (a)(ii), and (c) a promoter operably linked to (a)(iii).
  • dCas deactivated Cas
  • the isolated nucleic acid molecule may comprise a sequence encoding any one of the disclosed dCas9, any one or more of the disclosed polypeptides having an enzymatic activity, any one or more of the disclosed gRNA.
  • the isolated nucleic acid molecule may further comprise one or more of the disclosed microglia specific regulatory elements (e. g. microglia specific promoter) and optionally additional disclosed promoters.
  • a host cell or a host cell line comprising the disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease comprising an amino acid sequence as set forth in any one of SEQ ID NO: 36- SEQ ID NO: 38, or a fragment, or derivative thereof, or a sequence at least about 60% identical thereto (ii) at least one polypeptide having an enzymatic activity comprising an amino acid sequence as set forth in any one or more of SEQ ID NO: 44- SEQ ID NO: 58, or a sequence at least about 60% identical thereto and (iii) at least one guide RNA targeting an APOE gene sequence or a regulatory element thereof comprising, wherein the targeted sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 15 - SEQ ID NO: 33, or a complement thereof, or a sequence at
  • a host cell or a host cell line transduced by a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a host cell or a host cell line transduced by a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a host cell or a host cell line transduced by a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • a host cell or a host cell line transduced by a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof.
  • the gene of interest is APOE, or an allele thereof.
  • the disclosed host cell or a host cell line transduced by a viral vector comprises a nucleic acid sequence encoding any one of the disclosed dCas9, any one or more of the disclosed polypeptides having an enzymatic activity, and any one or more of the disclosed gRNA.
  • a nucleic acid sequence further comprising one or more of the disclosed microglia specific regulatory elements (e. g. microglia specific promoter) and optionally additional disclosed promoters.
  • a host cell or a host cell line transduced by a viral vector comprising comprising (a) a nucleic acid sequence encoding (i) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) endonuclease comprising an amino acid sequence as set forth in any one of SEQ ID NO: 36- SEQ ID NO: 38, or a fragment, or derivative thereof, or a sequence at least about 60% identical thereto (ii) at least one polypeptide having an enzymatic activity comprising an amino acid sequence as set forth in any one or more of SEQ ID NO: 44- SEQ ID NO: 58, or a sequence at least about 60% identical thereto and (iii) at least one guide RNA targeting an APOE gene sequence or a regulatory element thereof comprising, wherein the targeted sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 15 - SEQ ID NO: 33, or a complement thereof, or a sequence at least
  • a host cell or a host cell line transduced by a AAV viral vector comprising a sequence as set forth in any one of SEQ ID NO: 112-SEQ ID NO: 128, or a sequence at least about 60% identical thereto.
  • a host cell or a host cell line transduced by a lentiviral vector comprising a sequence as set forth in any one of SEQ ID NO: 105-SEQ ID NO: 111, or a sequence at least about 60% identical thereto.
  • a host cell or a host cell line transfected by a plasmid comprising the sequence set forth in any one of SEQ ID NO: 95 - SEQ ID NO: 104, or a sequence at least about 60% identical thereto.
  • a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • pharmaceutical formulation comprising (1) an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof; and (2) a pharmaceutically acceptable carrier.
  • the gene of interest is APOE, or an allele thereof.
  • a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • pharmaceutical formulation comprising (1) an isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof; and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof; and (2) a pharmaceutically acceptable carrier.
  • the gene of interest is APOE, or an allele thereof.
  • a pharmaceutical formulation comprising an isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising (1) an isolated nucleic acid comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof; and (2) a pharmaceutically acceptable carrier.
  • the gene of interest is APOE, or an allele thereof.
  • a pharmaceutical formulation comprising an isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising (1) an isolated nucleic acid comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof ; and (2) a pharmaceutically acceptable carrier.
  • the gene of interest is APOE, or an allele thereof.
  • a pharmaceutical formulation comprising an isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising (1) an isolated nucleic acid comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof;
  • a pharmaceutical formulation comprising an isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising (1) an isolated nucleic acid comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof;
  • a pharmaceutical formulation comprising an isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising (1) an isolated nucleic acid comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) a microglia specific regulatory element operably linked to (a)(i) and/or (a)(ii) and (c) a promoter operably linked to
  • a pharmaceutical formulation comprising an isolated nucleic acid molecule and a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising (1) an isolated nucleic acid comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) a microglia specific promoter operably linked to (a)(i) and/or (a)(ii) and (c) a promoter operably linked to
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof; and (2) a pharmaceutically acceptable carrier.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) one or more microglia specific regulatory element operably linked to (a)(i), (a)(iii), (a)(i
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof; and (2) a pharmaceutically acceptable carrier.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) one or more microglia specific promoter operably linked to (a)(i), (a)(iii), (a)(i
  • compositions comprising a (1) viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific regulatory elements operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, and (2) a pharmaceutically acceptable carrier.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (d
  • compositions comprising a (1) viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) one or more microglia specific promoters operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, and (2) a pharmaceutically acceptable carrier.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (
  • a pharmaceutical formulation comprising 1) viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) one or more microglia specific regulatory elements operably linked to (a)(i) and/or (a)(ii), and (c) a promoter operably linked to (a)(iii), and (2) a pharmaceutically acceptable carrier.
  • dCas deactivated Cas
  • a pharmaceutical formulation comprising 1) viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) one or more microglia specific promoters operably linked to (a)(i) and/or (a)(ii), and (c) a promoter operably linked to (a)(iii), and (2) a pharmaceutically acceptable carrier.
  • dCas deactivated Cas
  • a disclosed formulation can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof.
  • a disclosed composition can comprise one or more proteasome inhibitors.
  • a disclosed composition can comprise one or more immunosuppressives or immunosuppressive agents.
  • an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof.
  • a disclosed formulation can comprise a RNA therapeutic.
  • An RNA therapeutic can comprise RNA-mediated interference (RNAi) and/or antisense oligonucleotides (ASO).
  • a disclosed formulation can comprise a disclosed small molecule.
  • a disclosed pharmaceutical formulation can treat and/or prevent Alzheimer’s disease progression. In an aspect, a disclosed pharmaceutical formulation can treat and/or prevent Parkinson’s disease progression.
  • Disclosed herein is a method of effecting precision epigenetic modulation of microglial cells, the method comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is modulated.
  • a method of effecting precision epigenetic modulation the method comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is reduced or decreased.
  • Disclosed herein is a method of effecting precision epigenetic modulation, the method comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is augmented or increased.
  • Disclosed herein is a method of effecting precision epigenetic modulation, the method comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, wherein the expression and/or activity of APOE in the one or more microglial cells is reduced or decreased.
  • a method of effecting precision epigenetic modulation in microglial cells comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, wherein the expression and/or activity the gene of interest in the one or more microglial cells is modulated.
  • a disclosed isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptid
  • a method of effecting precision epigenetic modulation in microglial cells comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, wherein the expression and/or activity the gene of interest in the one or more microglial cells is modulated.
  • a disclosed isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide
  • a method of effecting precision epigenetic modulation in microglial cells comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(dCas) endonuclease
  • microglia specific promoters include F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), or a derivative thereof, a derivative thereof, a fragment thereof, or any combination thereof.
  • F4/80 promoter SEQ ID NO: 4
  • CD68 promoter SEQ ID NO: 1-3
  • FCGR2A promoter SEQ ID NO: 8
  • IBA1 promoter SEQ ID NO: 5-7
  • a method of effecting precision epigenetic modulation in microglial cells comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(dCas) endonuclease
  • the microglia specific promoter of b) may comprise F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), or a derivative thereof, a derivative thereof, a fragment thereof, or any combination thereof.
  • the gene of interest in the APOE gene may comprise F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), or a derivative thereof, a derivative thereof, a fragment thereof, or any combination thereof.
  • the gene of interest in the APOE gene may comprise F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), or a
  • a method of effecting precision epigenetic modulation in microglial cells comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) a microglia specific regulatory element operably linked (a)(i) and/or (a)(ii), and (c
  • the microglia specific regulatory element of (b) may comprise F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1- 3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), any derivative thereof, any fragment thereof, or any combination thereof.
  • the promoter of (c) comprises a U6 promoter, a derivative thereof, or a fragment thereof.
  • the gene of interest in the APOE gene may comprise F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1- 3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), any derivative thereof, any fragment thereof, or any combination thereof.
  • the promoter of (c) comprises a U6 promoter, a derivative thereof, or a fragment thereof.
  • the gene of interest in the APOE gene comprises a U6 promoter, a derivative thereof, or a fragment thereof.
  • a method of effecting precision epigenetic modulation in microglial cells comprising contacting one or more microglial cells with a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) a microglia specific promoter operably linked to (a)(i) and/or (a)(ii), and (c
  • the microglia specific promoter of (b) may comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or any combination thereof.
  • the promoter of (v) comprises a U6 promoter a derivative thereof or a fragment thereof.
  • the gene of interest in the APOE gene may comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or any combination thereof.
  • the promoter of (v) comprises a U6 promoter a derivative thereof or a fragment thereof.
  • Disclosed herein is a method of effecting precision epigenetic modulation in microglial cells in a subject, the method comprising contacting one or more microglial cells in a subject with a therapeutically effective amount of a disclosed viral vector, wherein the expression and/or activity of one or more genes of interest in the one or more cells is modulated.
  • a method of effecting precision epigenetic modulation in microglial cells the method comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed viral vector, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is reduced or decreased.
  • Disclosed herein is a method of effecting precision epigenetic modulation in microglial cells, the method comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed viral vector, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is augmented or increased.
  • a method of effecting precision epigenetic modulation the method comprising contacting one or more cells in a subj ect with a therapeutically effective amount of a disclosed viral vector, wherein the expression and/or activity of APOE in the one or more microglial cells is reduced or decreased.
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising contacting one or more microglial cells in a subject with a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or a combination thereof, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is modulated.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having
  • the at least one guide RNA of (a)(iii) targets the d/Y/A gene or a portion thereof.
  • the microglia specific regulatory element of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising contacting one or more microglial cells in a subject with a therapeutically effective amount of a viral vector comprising a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or a combination thereof, wherein the expression and/or activity of one or more genes of interest in the one or more cells is modulated.
  • a viral vector comprising a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, (i
  • the at least one guide RNA of (a)(iii) targets the APOE gene or a portion thereof.
  • the microglia specific promoter of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii), or a combination thereof, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is modulated.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii), or a combination thereof, wherein the expression and/or activity of one or more genes of interest in the one or more microglial cells is modulated.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising contacting one or more microglial cells in a subject with a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(
  • the dCas of (a)(i) is dSaCas9.
  • the fusion product of (a)(ii) can comprise KRAB-MeCP2.
  • the at least one guide RNA of (a)(iii) targets the d/Y/A’ gene or a portion thereof.
  • the microglia specific regulatory element of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising contacting one or more microglial cells in a subject with a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(
  • the dCas of (a)(i) is dSaCas9.
  • the fusion product of (a)(ii) can comprise KRAB-MeCP2.
  • the at least one guide RNA of (a)(iii) targets the APOE gene or a portion thereof.
  • the microglia specific promoter of (b) can comprise F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7) a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising contacting one or more cells in a subject with a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding
  • dCas deactivated Cas endonuclease
  • dCas deactivated Cas endonuclease
  • the dCas is a dSpCas9, dSaCas9, or dCjCas9
  • a fusion product having one or more enzymatic activities comprising any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) a microglia specific regulatory element operably linked to (a)(i) and/or (a)(ii), and (c) a promoter operably linked to (a)(iii), wherein the expression and/or activity of one or more genes of interest in the one or more cells is modulated.
  • the dCas of (a)(i) is dSaCas9.
  • the fusion product of (a)(ii) can comprise KRAB-MeCP2.
  • the at least one guide RNA of (a)(iii) targets the APOE gene or a portion thereof.
  • the microglia specific regulatory element of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1- 3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or any combination thereof.
  • the promoter of (c) can comprise any promoter known in the art.
  • a method of effecting precision epigenetic modulation in microglial cells in a subject comprising contacting one or more cells in a subject with a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding
  • dCas deactivated Cas endonuclease
  • dCas deactivated Cas endonuclease
  • the dCas is a dSpCas9, dSaCas9, or dCjCas9
  • a fusion product having one or more enzymatic activities comprising any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting a gene of interest or portion thereof, (b) a microglia specific promoter element operably linked to (a)(i) and/or (a)(ii) and (c) a promoter operably linked to (a)(iii), wherein the expression and/or activity of one or more genes of interest in the one or more cells is modulated.
  • the dCas of (a)(i) is dSaCas9.
  • the fusion product of (a)(ii) can comprise KRAB-MeCP2.
  • the at least one guide RNA of (a)(iii) targets the APOE gene or a portion thereof.
  • the microglia specific promoter of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof.
  • the promoter of (c) can comprise a U6 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed gene of interest or portion thereof can demonstrate a reduced expression and/or activity level when compared to wild-type or control expression level. In an aspect, a disclosed gene of interest or portion thereof can demonstrate an increased expression and/or activity level when compared to wild-type or control expression level. In an aspect, a disclosed gRNA can target a gene of interest or portion thereof that demonstrates an increased expression level when compared to wild-type or control expression level. In an aspect, a disclosed gRNA can target a gene of interest or portion thereof that demonstrates a reduced expression and/or activity level when compared to wild-type or control expression level.
  • a disclosed method of effecting precision epigenetic modulation comprises measuring the level of expression and/or the level of activity of the disclosed gene of interest. In an aspect, a disclosed method can comprise repeating the measuring step one or more times.
  • a disclosed gRNA targeting a gene of interest or portion thereof can comprise at least two gRNAs.
  • disclosed gRNAs can target the same gene of interest or portion thereof.
  • disclosed gRNAs can target different genes of interest or portions thereof.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a loss of function.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a gain of function.
  • a disclosed gRNA can target the APOE gene sequence or a regulatory element thereof.
  • the at least one gRNA can target the APOE gene, or promoter region thereof, or an untranslated region (UTR) thereof, or any combination thereof.
  • the at least one gRNA may target the promoter region, or intron 1, or a flanking sequence thereof, of APOE gene.
  • a disclosed gRNA can target exon 4 of the APOE gene.
  • a disclosed gRNA can target a protospacer-adjacent motif (PAM) created by a SNP rs429358 in exon 4 of the POE gene.
  • PAM protospacer-adjacent motif
  • a disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • a nucleic acid sequence the disclosed dSpCas9 may comprise a sequence as set forth in any one of SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSpCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof.
  • a disclosed dSpCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment, or a derivative thereof.
  • a disclosed dSpCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dSaCas9 may comprise a sequence as set forth in SEQ ID NO: 42, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSaCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 42, or a fragment thereof.
  • a disclosed dSaCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 38 or a fragment, or a derivative thereof.
  • a disclosed dSaCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 38, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCjCas9 may comprise a sequence as set forth in SEQ ID NO: 43, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dCjCas9 endonuclease may comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 43, or a fragment thereof.
  • a disclosed dCjCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 39, or a fragment, or a derivative thereof.
  • a disclosed dCjCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 39 or a fragment thereof.
  • a disclosed variant of dCas9 dVQR can comprise DI 135V, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dEQR can comprise D1135E, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dVRER can comprise DI 135V, G1218R, R1335E, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed dVRER can comprise the sequence set forth in SEQ ID NO: 37 or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCas9 endonuclease may comprise a sequence as set forth in any one of SEQ ID NO: 40-43.
  • the sequence encoding the disclosed dCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40-43, or a fragment, or a derivative thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity, transcription repression activity, transcription release factor activity, histone modulation activity, nucleic acid association activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise HPla, HP lb, MBD1, MBD2, Kriippel-Associated Box (KRAB), NIPP1, the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed fusion or disclosed fusion product can comprise HPla-HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb- KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBD1- HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP1, MBDl-MeCP2, MBD1-DNMT3A, MBD2-MBD2, MBD2-HPla, MBD2-HPlb, MBD2-MBD1, MBD2-KRAB, MBD2-NIPP1, MBD2-MeCP2, MBD2-DNMT3A, MBD2-MBD2, MBD2-HP
  • the nucleic acid sequence encoding the disclosed polypeptide HPla may comprise a sequence as set forth in any one of SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HPla may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment thereof.
  • a disclosed polypeptide HPla can comprise the amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPla can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide HP lb may comprise a sequence as set forth in any one of SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP lb may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment thereof.
  • a disclosed polypeptide HPlb can comprise the amino acid sequence as set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPlb can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD1 may comprise a sequence as set forth in any one of SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment thereof.
  • a disclosed polypeptide MBD1 can comprise the amino acid sequence as set forth in any one of SEQ ID NO: 48-50, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MDB1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 48-50, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD2 may comprise a sequence as set forth in any one of SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment thereof.
  • a disclosed polypeptide MBD2 can comprise the amino acid sequence as set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MBD2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide NIPP1 may comprise a sequence as set forth in any one of SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide NIPP1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment thereof.
  • a disclosed polypeptide NIPP1 can comprise the amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment, or a derivative thereof.
  • a disclosed polypeptide NIPP1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide DNMT3 A may comprise a sequence as set forth in SEQ ID NO: 69, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide DNMT3 A may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 69, or a fragment thereof.
  • a disclosed polypeptide DNMT3A can comprise the amino acid sequence as set forth in SEQ ID NO: 55, or a fragment, or a derivative thereof.
  • a disclosed polypeptide DNMT3A can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 72, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 72, or a fragment thereof.
  • a disclosed polypeptide MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 57, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 57, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed fusion polypeptide KRAB- MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB-MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 58, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 58, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity.
  • a disclosed encoded polypeptide comprising transcription activation activity can comprise a transactivation domain.
  • a disclosed transactivation domain can comprise a VP16 protein, a series of linked VP16 proteins, a p65 domain of NFKB, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide VP16 may comprise a sequence as set forth in any one of SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide VP 16 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 132 or SEQ NO: 133, or a fragment thereof.
  • a disclosed polypeptide VP16 can comprise the amino acid sequence as set forth in SEQ ID NO: 131, or a fragment, or a derivative thereof.
  • a disclosed polypeptide VP 16 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131, or a fragment thereof.
  • nucleic acid sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a sequence as set forth in any one of SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise the amino acid sequence as set forth in SEQ ID NO: 134, or a fragment, or a derivative thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription repression activity.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise a Kriippel associated box domain, an ERF repressor domain, a MXI1 repressor domain, a SID4x repressor domain, a fused KRAB-MeCP2 domain, a MeCP2 TRD domain, a MAS-SID repressor domain, a TATA box binding protein activity, or any combination thereof.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise HP1 repressor activity, MeCP2 repressor activity, MBD1 repressor activity, MBD2 repressor activity, MBD3 repressor activity, MBD4 repressor activity, KRAB repressor activity, SUV39H1 repressor activity, SUV39H2 repressor activity, CTCF insulator-repressor activity, LSD-1 hi stone-dem ethyl ase repressor activity, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide comprising Eukaryotic Release Factor 1 (ERF1) activity or Eukaryotic Release Factor 3 (ERF3) activity may comprise a sequence as set forth in any one of SEQ ID NO: 138, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide comprising ERF1 activity or ERF3 activity may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 138, or a fragment thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise the amino acid sequence as set forth in SEQ ID NO: 137, or a fragment, or a derivative thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 137, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise histone modulation activity.
  • a disclosed encoded polypeptide comprising histone modulation activity can comprise histone acetyltransferase, histone deacetylase, histone demethylase, histone methyltransferase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise nucleic acid association activity.
  • a disclosed encoded polypeptide comprising nucleic acid association activity can comprise a helix-tum-helix region, a leucine zipper region, a winged helix region, a winged helix-tum-helix region, a helix-loop-helix region, an immunoglobulin fold, a B3 domain, a zinc finger, a HMG-box, a Wor3 domain, a TAL effector DNA-binding domain, or any combination thereof.
  • a disclosed encoded polypeptide can comprise methyltransferase activity.
  • a disclosed encoded polypeptide comprising methyltransferase activity can comprise DNA (cytosine-5)-methyltransferase 3a (DNMT3 A).
  • a disclosed encoded polypeptide can comprise demethylase activity.
  • a disclosed encoded polypeptide comprising demethylase activity can comprise ten-eleven translocation methylcytosine dioxygenase 1 (TET1) or lysine-specific histone demethylase 1 (LSD1).
  • a disclosed encoded polypeptide can comprise acetyltransferase activity.
  • a disclosed encoded polypeptide comprising acetyltransferase activity can comprise histone acetyltransferase.
  • a disclosed encoded polypeptide can comprise deacetylase activity.
  • a disclosed encoded polypeptide comprising deacetylase activity can comprise histone deacetylase.
  • a disclosed dCas endonuclease can comprise dSpCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel- associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSaCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dVQR, dEQR, or dVRER and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9 and a disclosed encoded polypeptide can comprise KRAB-MeCP2.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9
  • a disclosed fusion can comprise HPla-HPla, HPla-HPlb, HPla- MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP1 a-DNMT3 A, HP Ib-HP lb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb- DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1- NIPP1, MBDl-MeCP2, MBD
  • an isolated nucleic acid or viral vector can encode for a dCas endonuclease fused to the at least one encoded polypeptide having an enzymatic activity.
  • the isolated nucleic acid can encode for a “fusion protein” wherein the fusion protein comprises a first component comprising a dCas endonuclease and a second component comprising at least one polypeptide having an enzymatic activity.
  • the encoded fusion protein can comprise a dSpCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dSaCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dCjCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dVQR, dEQR, or dVRER and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER) and one or more of HPla-HPla, HPla-HPlb, HP 1 a-MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP 1 a-DNMT3 A, HP 1b- HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP
  • the encoded fusion protein can comprise a variant dCas9 selected from dVQR, dEQR, or dVRER and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dSaCas9 and KRAB-MeCP2. In an aspect, the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and DNMTA. In an aspect, the encoded fusion protein can comprise a dSaCas9 and DNMTA.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76 respectively, or a derivative thereof.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 78 respectively, or a derivative thereof.
  • the nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A may comprise a sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the sequences set forth in SEQ ID NO: 77 or SEQ ID NO: 78, or a fragment thereof.
  • a disclosed polypeptide dSpCas9 can comprise the amino acid sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment, or a derivative thereof.
  • a disclosed gene of interest or portion thereof can demonstrate a reduced expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can targes a gene of interest or portion thereof that demonstrates a reduced expression level when compared to wild-type or control expression level.
  • a disclosed gene of interest or portion thereof can demonstrate an increased expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can target a gene of interest or portion thereof that demonstrates an increased expression level when compared to wild-type or control expression level.
  • a disclosed gRNA targeting a gene of interest or portion thereof can comprise at least two gRNAs.
  • disclosed gRNAs can target the same gene of interest or portion thereof.
  • disclosed gRNAs can target different genes of interest or portions thereof.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a loss of function.
  • a disclosed gRNA can target a gene or portion thereof that demonstrates a gain of function. Disclosed target genes and disclosed genes of interest are discussed supra.
  • a disclosed gRNA can target the APOE gene.
  • the at least one gRNA may target a region in the APOE gene comprising a nucleic acid sequence as set forth in any one of SEQ ID NO: 11-14, or a complement thereof.
  • the at least one gRNA targets a nucleic acid sequence comprising at least 10 consecutive nucleotides as set forth in SEQ ID NO: 11-14, or a complement thereof.
  • a disclosed gRNA can target the promoter of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 4 of the APOE gene. In an aspect, a disclosed gRNA can target a CpG island region of intron 1 of the APOE gene.
  • the CpG island region can include CpGl, CpG2, CpG3, CpG4, CpG5, CpG6, CpG7, CpG8, CpG9, CpGlO, CpGl l, CpG12, CpG13, CpG14, CpG15, CpG16, CpG17, CpG18, CpG19, CpG20, CpG21, CpG22, CpG23, or a combination thereof.
  • the CpG island region can include CpGl, CpG3, CpG6, CpG7, CpG8, CpG9, CpG18, CpG19, CpG20, CpG21, CpG22, or a combination thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • a disclosed viral vector or disclosed recombinant AAV vector can further comprise a gRNA scaffold.
  • a disclosed gRNA scaffold can comprise the sequence set forth in SEQ ID NO: 129, SEQ ID NO: 130, or a fragment thereof.
  • a disclosed isolated nucleic acid or disclosed viral vector comprises one or more microglia specific regulatory elements.
  • the one or more microglia specific regulatory elements can comprise a microglia specific wild-type promoter, a microglia specific enhancer, a synthetic promoter, a synthetic enhancer, a transcriptional or translational regulatory sequence or any combination thereof.
  • the one or more microglia specific regulatory elements comprise at least one microglia specific promoter (e.g., a microglia specific wild-type promoter or a microglia specific synthetic promoter).
  • the microglia specific regulatory element e.g., promoter
  • the microglia specific regulatory element may be specific for a certain type of microglial cell (e.g., a dopaminergic or a cholinergic microglial cell).
  • the microglia specific regulatory element may include a dopaminergic specific regulatory element (z.e., a dopaminergic promoter) and/or a cholinergic microglia specific regulatory element (z.e., a cholinergic promoter).
  • the microglia specific regulatory element can comprise a microglia specific promoter.
  • a disclosed microglia specific promoter can comprise a F4/80 promoter, CD68 promoter, FCGR2A promoter, HexB promoter or IBA1 promoter, a derivative thereof, a fragment thereof, or any combination thereof.
  • the promoter is a CD68 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one or more of the sequences set forth in SEQ ID NO: 1-3, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 1-3.
  • the promoter is a F4/80 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 4.
  • the promoter is a IB Al promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5-7, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 5-7.
  • the promoter is a FCGR2A promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 8, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 8.
  • a disclosed microglia specific regulatory element ie., a microglia specific promoter
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at least one polypeptide having enzymatic activity can comprise a F4/80promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at least one polypeptide having enzymatic activity can comprise a CD68 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at least one polypeptide having enzymatic activity can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • at least one polypeptide having enzymatic activity can comprise a FCGR2A promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the at least one polypeptide having enzymatic activity
  • a FGCR2A promoter a derivative thereof, or a fragment thereof
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at dCas endonuclease and the at least one polypeptide having enzymatic activity can comprise a F4/80 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the at least one polypeptide having enzymatic activity
  • a CD68 promoter a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the at least one polypeptide having enzymatic activity can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, a derivative thereof, or a fragment thereof.
  • a disclosed isolated nucleic acid or disclosed viral vector can further comprise one or more regulatory elements (i.e., promoters) in addition to the microglia specific regulatory element (e.g., microglia specific promoter).
  • a disclosed additional promoter can comprise a U6 promoter, a chicken P-actin promoter, an EF-la, a CMV promoter, a CMV promoter/enhancer, a fragment thereof, a derivative thereof, or any combination thereof.
  • a disclosed U6 promoter can comprise the sequence set forth in SEQ ID NO: 81, or a fragment thereof.
  • a disclosed U6 promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 81 or a fragment thereof.
  • a disclosed EF-la promoter can comprise the sequence set forth in SEQ ID NO: 79, SEQ ID NO: 80, or a fragment thereof.
  • a disclosed EF-la promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 79, SEQ ID NO:80, or a fragment thereof.
  • a promoter can be a short EFl alpha (EFS-NF) promoter.
  • a disclosed promoter operably can be linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed promoter operably linked to the at least one guide RNA can comprise a U6 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed promoter operably linked to the at least one guide RNA targeting an APOE gene can comprise a U6 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed isolated nucleic acid molecule or viral vector can further two or more promoters, wherein a first promoter can be operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed isolated nucleic acid molecule can further comprise one or more promoters, wherein a first promoter comprises a microglia specific promoter that is operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest thereof.
  • the second promoter operably linked to the gRNA may not be a microglia specific promoter.
  • the isolated nucleic acid can comprise a first promoter selected from a F4/80 promoter, a CD68 promoter, a IBA1 promoter, or a FCGR2A promoter, a fragment thereof, a derivative thereof, or any combination thereof, and a second promoter comprising a U6 promoter.
  • a disclosed isolated nucleic acid molecule or viral vector can further comprise one or more additional regulatory elements.
  • a disclosed additional regulatory element can comprise a promoter (described supra), an enhancer, a promoter/enhancer, an internal ribosomal entry site, a transcription terminal signal, a polyadenylation signal, a Spl and/or NF-kB transcriptional factor binding site, a p2A signal, a woodchuck hepatitis virus post-transcriptional regulatory element, a Phi signal-packaging signal, a rev responsive element, a 5’-ITR, a 3’-ITR, an inverted terminal repeat, a nuclear localization signal (NLS), or any combination thereof.
  • a disclosed 5’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 82 or SEQ ID NO: 83.
  • a disclosed 3’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 84 or SEQ ID NO: 85.
  • a disclosed NLS can comprise the sequence set forth in any one of SEQ ID NO: 88 - SEQ ID NO: 94, or a fragment thereof.
  • a disclosed PolyA sequence can comprise the sequence set forth in SEQ ID NO: 86, SEQ ID NO: 87, or a fragment thereof.
  • a disclosed viral vector or isolated nucleic acid can comprise a nucleic acid that is CpG depleted and codon-optimized for expression in a human cell.
  • CpG-free can mean completely free of CpGs or partially free of CpGs.
  • CpG-free can mean “CpG-depleted.”
  • CpG-depleted can mean “CpG-free.”
  • CpG-depleted can mean completely depleted of CpGs or partially depleted of CpGs.
  • CpG-free can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art.
  • any disclosed Cas9 endonuclease, a disclosed polypeptide having enzymatic activities, a disclosed fusion product or a disclosed fusion protein, or any combination thereof can be codon-optimized.
  • a disclosed viral vector or disclosed recombinant AAV vector can comprise a sequence that is about 4.5 kilobases or less than about 4.5 kilobases.
  • modulating can comprise increasing expression and/or activity of one or more genes of interest. In an aspect of a disclosed method of effecting precision epigenetic modulation, modulating can comprise decreasing expression and/or activity of one or more genes of interest.
  • a disclosed method of effecting precision epigenetic modulation can comprise reducing expression and/or activity of APOE.
  • the disclosed cells can be microglial cells.
  • the disclosed cells can be cells affected by an overexpression level and/or activity level of one or more genes of interest or one or more target genes.
  • the disclosed cells can be in a subject.
  • a subject can be a human.
  • a subject can be suspected of having or can be diagnosed with having Alzheimer’s disease.
  • a subject can be suspected of having or can be diagnosed with having Parkinson’s disease.
  • a disclosed subject can be symptomatic or asymptomatic.
  • a disclosed method of effecting precision epigenetic modulation can comprise reducing the pathological phenotype associated with Alzheimer’s disease.
  • reducing the pathological phenotype associated with Alzheimer’s disease can comprise reducing the number and/or amount of Tau aggregates.
  • a disclosed method can comprise diagnosing the subject with Alzheimer’s disease.
  • a disclosed method of effecting precision epigenetic modulation can comprise reducing the pathological phenotype associated with Parkinson’s disease.
  • reducing the pathological phenotype associated with Parkinson’s disease can comprise reducing the number and/or amount of Tau aggregates.
  • a disclosed method can comprise diagnosing the subject with Parkinson’s disease.
  • a subject can be a subject having a disease or disorder.
  • a disease or disorder can be any disease or disorder disclosed herein, for example, characterized by an overexpression and/or over-activity or characterized by a loss of function.
  • a subject can be a subject in need of treatment of a disclosed disease or disorder (e.g., a genetic disease or disorder).
  • a disclosed disease or disorder e.g., a genetic disease or disorder.
  • a disclosed method of effecting precision epigenetic modulation can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.
  • a disclosed method of effecting precision epigenetic modulation can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation can comprise reducing the expression and/or activity level of at least one gene of interest or at least one target gene.
  • a gene of interest can be a gene that demonstrates over expression and/or over activity.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation can comprise increasing the expression and/or activity level of at least one gene of interest or at least one target gene.
  • a gene of interest can be a gene that demonstrates a loss of function or reduced expression and/or activity level.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi -systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof.
  • restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.
  • restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level.
  • the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level.
  • restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme).
  • restoration can be a partial or incomplete restoration.
  • restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is like that of a wild-type or control level.
  • restoring the activity and/or functionality of a gene of interest can comprise increasing or enhancing the expression and/or activity level of that gene.
  • increasing or enhancing can comprise an elevation of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more as compared to a control (such as a pre-treatment level).
  • restoring the activity and/or functionality of a gene of interest can comprise decreasing or reducing the expression and/or activity level of that gene.
  • decreasing or reducing can comprise a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more as compared to a control (such as a pre-treatment level).
  • techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.
  • administering can comprise intravenous administration, intracerebral administration, intra-CSF administration, intracerebroventricular (ICV) administration, intraventricular administration, intra-ci sterna magna (ICM) administration, intraparenchymal administration, intrathecal (lumbar, cisternal, or both) administration, intrahepatic administration, hepatic intra-arterial administration, hepatic portal vein (HPV) administration, or any combination thereof.
  • a disclosed vector can be administered via LNP administration.
  • administering can comprise use of non-viral vectors to deliver a disclosed isolated nucleic acid.
  • these non-viral vectors can include, but are not limited to, nanoparticles or liposomes.
  • the disclosed isolated nucleic acids may be delivered via viral vectors (such as the disclosed viral vectors provided herein).
  • Exemplary viral vectors are described supra and include adenovirus, adeno-associated virus (AAV), and lentivirus (LV).
  • AAV adeno-associated virus
  • LV lentivirus
  • Viral vectors are used more commonly due to their robust delivery, long-lasting expression, low immunogenicity and toxicity, and compatibility with the advanced manufacturing techniques. Most research in gene therapy has utilized AAV or LV due to their lowered immunogenicity compared to adenovirus.
  • LV integrates its viral genome into the host gene to create permanent change in gene expression.
  • a disclosed method of effecting precision epigenetic modulation can employ multiple routes of administration to the subject.
  • a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.
  • a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation can be concurrently and/or serially administered to a subject via multiple routes of administration.
  • administering a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistem magna (ICM) administration.
  • ICM intra-cistem magna
  • administering a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise IV administration and intrathecal (ITH) administration.
  • a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x IO 10 vg/kg to about 2 x 10 14 vg/kg.
  • a disclosed vector can be administered at a dose of about 1 x 10 11 vg/kg to about 8 x 10 13 vg/kg or about 1 x 10 12 vg/kg to about 8 x 10 13 vg/kg.
  • a disclosed vector can be administered at a dose of about 1 x 10 13 vg/kg to about 6 x 10 13 vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x IO 10 vg/kg, at least about 5 x IO 10 vg/kg, at least about 1 x 10 11 vg/kg, at least about 5 x 10 11 vg/kg, at least about 1 x 10 12 vg/kg, at least about 5 x 10 12 vg/kg, at least about 1 x 10 13 vg/kg, at least about 5 x 10 13 vg/kg, or at least about 1 x 10 14 vg/kg.
  • a disclosed vector can be administered at a dose of no more than about 1 x IO 10 vg/kg, no more than about 5 x IO 10 vg/kg, no more than about 1 x 10 11 vg/kg, no more than about 5 x 10 11 vg/kg, no more than about 1 x 10 12 vg/kg, no more than about 5 x 10 12 vg/kg, no more than about 1 x 10 13 vg/kg, no more than about 5 x 10 13 , or no more than about 1 x 10 14 vg/kg.
  • a disclosed vector can be administered at a dose of about 1 x 10 12 vg/kg.
  • a disclosed vector can be administered at a dose of about 1 x 10 11 vg/kg. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.
  • a disclosed method of effecting precision epigenetic modulation can further comprise monitoring the subject for adverse effects.
  • the method in the absence of adverse effects, can further comprise continuing to treat the subject.
  • the method in the presence of adverse effects, can further comprise modifying the treating step.
  • Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.
  • a disclosed method of effecting precision epigenetic modulation can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent.
  • a therapeutic agent can be any disclosed agent that effects a desired clinical outcome.
  • a disclosed method of effecting precision epigenetic modulation can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway.
  • such an agent can comprise an enzyme for enzyme replacement therapy.
  • a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway.
  • a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.
  • a disclosed method of effecting precision epigenetic modulation can further comprise administering one or more immune modulators.
  • a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof.
  • a disclosed immune modulator can be bortezomib or SVP- Rapamycin.
  • a disclosed immune modulator can be Tacrolimus.
  • a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose.
  • a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.
  • a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.
  • a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.
  • a disclosed method of effecting precision epigenetic modulation can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib).
  • a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab).
  • an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledron
  • a disclosed method of effecting precision epigenetic modulation can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation.
  • a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation.
  • a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation.
  • a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time.
  • a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.
  • a disclosed method of effecting precision epigenetic modulation can further comprise administering one or more immunosuppressive agents.
  • an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, antithymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof.
  • a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time.
  • a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time.
  • a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.
  • a disclosed method of effecting precision epigenetic modulation can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response.
  • a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD 19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof.
  • a subject can be a human.
  • a subject can be suspected of having or can be diagnosed with having Alzheimer’ s disease (such as, for example, PD).
  • a disclosed subject can be symptomatic or asymptomatic.
  • a disclosed method can comprise repeating one or more steps of the method and/or modifying one or more steps of the method (such as, for example, an administering step).
  • a disclosed method of effecting precision epigenetic modulation can comprise modifying one or more of the disclosed steps.
  • modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • a method can be altered by changing the amount of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject, or by changing the duration of time one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination are administered to a subject.
  • a disclosed method of effecting precision epigenetic modulation can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.
  • a disclosed method of effecting precision epigenetic modulation can further comprise generating and/or validating one or more of the disclosed isolated nucleic acid molecules, one or more of the disclosed vectors, one or more of the disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method of effecting precision epigenetic modulation can further comprise contacting the cells with a second disclosed isolated nucleic acid molecule, a second disclosed vector, a second disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method of effecting precision epigenetic modulation can further comprise contacting the cells with additional disclosed isolated nucleic acid molecules, additional disclosed vectors, additional disclosed pharmaceutical formulations, or any combination thereof.
  • Disclosed herein is a method of treating and/or preventing progression of an APOE associated disease or disorder, the method comprising administering to a subject a therapeutically effective amount of a disclosed isolated nucleic acid molecule, thereby reducing the pathological phenotype associated with the APOE associated disease or disorder.
  • a method of treating and/or preventing an APOE associated disease or disorder comprises administering to a subject a therapeutically effective amount of a disclosed viral vector, thereby reducing the pathological phenotype associated with the APOE associated disease or disorder.
  • Disclosed herein is a method of treating and/or preventing a tauopathy.
  • the method comprises administering to a subject a therapeutically effective amount of a disclosed isolated nucleic acid molecule, thereby reducing the pathological phenotype associated with the tauopathy.
  • a method of treating and/or preventing a tauopathy comprises administering to a subject a therapeutically effective amount of a disclosed viral vector, thereby reducing the pathological phenotype associated with the tauopathy.
  • a method of treating and/or preventing progression of a tauopathy or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a APOE gene or portion thereof and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a APOE gene or portion thereof and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonucleas
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • an isolated nucleic acid comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease,
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP 1 , MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting an APOE gene or portion thereof and b) at least one microglia specific regulatory element operably linked to (a)(i), (a
  • the microglia specific regulatory element of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof, a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific promoter operably linked to (a)
  • the microglia specific promoter of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IB Al promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof, a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific regulatory element operably linked to (a)(dCas) endonuclease
  • the microglia specific regulatory element of (iv) may comprise a (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IB Al promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof.
  • the promoter of (c) comprises a U6 promoter, a derivative thereof or a fragment thereof.
  • Disclosed herein is a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject, the method comprising administering to a subject a therapeutically effective amount of a disclosed viral vector, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting the APOE gene or a portion of WIQ APOE gene and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or a combination thereof, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a disclosed viral vector, comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a APOE gene or portion thereof and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • a disclosed viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one poly
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii), or a combination thereof, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii)
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii), or a combination thereof, thereby reducing the pathological phenotype associated with the Alzheimer’s or other APOE associated disease or disorder.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii)
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific regulatory element operably linked to the (a)(i) and/or (
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific promoter operably linked to the (a)(i) and/or (a)(i
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific regulatory element operably linked to (a)(i) and/or (
  • a method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific promoter operably linked to (a)(i) and/or (a)(i
  • the microglia specific regulatory element or microglia specific promoter of (b) can comprise a (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof.
  • the fusion product of (a)(ii) can comprise KRAB-MeCP2.
  • the promoter of (c) comprises a U6 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed method of treating and/or preventing progression of Alzheimer’s or other APOE associated disease or disorder can reduce and/or decrease the expression and/or activity of APOE. In an aspect of a disclosed method of treating and/or preventing progression of Alzheimer’ s or other APOE associated disease or disorder, the expression and/or activity of APOE is reduced and/or decreased. In an aspect, a disclosed method can comprise diagnosing the subject with Alzheimer’s or other APOE associated disease or disorder. [0482] In an aspect, a disclosed method of treating and/or preventing Alzheimer’s or other APOE associated disease or disorder comprises measuring the level of expression and/or the level of activity of the APOE gene. In an aspect, a disclosed method can comprise repeating the measuring step one or more times.
  • the expression and/or activity level of APOE prior to the administering step, can be elevated and/or increased when compared to wild-type or control expression level. In an aspect, following the administering step, the expression and/or activity level of APOE can be reduced and/or decreased when compared to the pre-administering step level.
  • a disclosed gRNA targeting a portion of the APOE gene can comprise at least two gRNAs. In an aspect, disclosed gRNAs can target the same portion of the APOE gene or different portions of the APOE gene. In an aspect, a disclosed gRNA can target the promoter of the APOE gene. In an aspect, a disclosed gRNA can target exon 1 of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 4 of the APOE gene. In an aspect, a disclosed gRNA can target a CpG island region of intron 1 of the APOE gene.
  • the CpG island region can include CpGl, CpG2, CpG3, CpG4, CpG5, CpG6, CpG7, CpG8, CpG9, CpGlO, CpGl l, CpG12, CpG13, CpG14, CpG15, CpG16, CpG17, CpG18, CpG19, CpG20, CpG21, CpG22, CpG23, or a combination thereof.
  • the CpG island region can include CpGl, CpG3, CpG6, CpG7, CpG8, CpG9, CpGl 8, CpGl 9, CpG20, CpG21, CpG22, or a combination thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • a disclosed viral vector or disclosed recombinant AAV vector can further comprise a gRNA scaffold.
  • a disclosed gRNA scaffold can comprise the sequence set forth in SEQ ID NO: 129, SEQ ID NO: 130, or a fragment thereof.
  • a disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • a disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • a nucleic acid sequence the disclosed dSpCas9 may comprise a sequence as set forth in any one of SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSpCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof.
  • a disclosed dSpCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment, or a derivative thereof.
  • a disclosed dSpCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dSaCas9 may comprise a sequence as set forth in SEQ ID NO: 42, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSaCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 42, or a fragment thereof.
  • a disclosed dSaCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 38 or a fragment, or a derivative thereof.
  • a disclosed dSaCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 38, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCjCas9 may comprise a sequence as set forth in SEQ ID NO: 43, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dCjCas9 endonuclease may comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 43, or a fragment thereof.
  • a disclosed dCjCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 39, or a fragment, or a derivative thereof.
  • a disclosed dCjCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 39 or a fragment thereof.
  • a disclosed variant of dCas9 dVQR can comprise DI 135V, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dEQR can comprise D1135E, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dVRER can comprise DI 135V, G1218R, R1335E, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed dVRER can comprise the sequence set forth in SEQ ID NO: 37 or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCas9 endonuclease may comprise a sequence as set forth in any one of SEQ ID NO: 40-43.
  • the sequence encoding the disclosed dCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40-43, or a fragment, or a derivative thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity, transcription repression activity, transcription release factor activity, histone modulation activity, nucleic acid association activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise HPla, HP lb, MBD1, MBD2, Krtippel-Associated Box (KRAB), NIPP1, the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed fusion or disclosed fusion product can comprise HPla-HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb- KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBD1- HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP1, MBDl-MeCP2, MBD1-DNMT3A, MBD2-MBD2, MBD2-HPla, MBD2-HPlb, MBD2-MBD1, MBD2-KRAB, MBD2-NIPP1, MBD2-MeCP2, MBD2-DNMT3A, MBD2-MBD2, MBD2-HP
  • the nucleic acid sequence encoding the disclosed polypeptide HP la may comprise a sequence as set forth in any one of SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP la may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment thereof.
  • a disclosed polypeptide HPla can comprise the amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPla can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide HP lb may comprise a sequence as set forth in any one of SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP lb may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment thereof.
  • a disclosed polypeptide HPlb can comprise the amino acid sequence as set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPlb can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD1 may comprise a sequence as set forth in any one of SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment thereof.
  • a disclosed polypeptide MBD1 can comprise the amino acid sequence as set forth in any one of SEQ ID NO: 48-50, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MDB1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 48-50, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD2 may comprise a sequence as set forth in any one of SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment thereof.
  • a disclosed polypeptide MBD2 can comprise the amino acid sequence as set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MBD2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide NIPP1 may comprise a sequence as set forth in any one of SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide NIPP1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment thereof.
  • a disclosed polypeptide NIPP1 can comprise the amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment, or a derivative thereof.
  • a disclosed polypeptide NIPP1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide DNMT3 A may comprise a sequence as set forth in SEQ ID NO: 69, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide DNMT3 A may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 69, or a fragment thereof.
  • a disclosed polypeptide DNMT3A can comprise the amino acid sequence as set forth in SEQ ID NO: 55, or a fragment, or a derivative thereof.
  • a disclosed polypeptide DNMT3A can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 72, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 72, or a fragment thereof.
  • a disclosed polypeptide MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 57, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 57, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed fusion polypeptide KRAB- MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB-MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 58, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 58, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity.
  • a disclosed encoded polypeptide comprising transcription activation activity can comprise a transactivation domain.
  • a disclosed transactivation domain can comprise a VP16 protein, a series of linked VP16 proteins, a p65 domain of NFKB, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide VP 16 may comprise a sequence as set forth in any one of SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide VP 16 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment thereof.
  • a disclosed polypeptide VP16 can comprise the amino acid sequence as set forth in SEQ ID NO: 131, or a fragment, or a derivative thereof.
  • a disclosed polypeptide VP 16 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131, or a fragment thereof.
  • nucleic acid sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a sequence as set forth in any one of SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise the amino acid sequence as set forth in SEQ ID NO: 134, or a fragment, or a derivative thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription repression activity.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise a Kriippel associated box domain, an ERF repressor domain, a MXI1 repressor domain, a SID4x repressor domain, a fused KRAB-MeCP2 domain, a MeCP2 TRD domain, a MAS-SID repressor domain, a TATA box binding protein activity, or any combination thereof.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise HP1 repressor activity, MeCP2 repressor activity, MBD1 repressor activity, MBD2 repressor activity, MBD3 repressor activity, MBD4 repressor activity, KRAB repressor activity, SUV39H1 repressor activity, SUV39H2 repressor activity, CTCF insulator-repressor activity, LSD-1 hi stone-dem ethyl ase repressor activity, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide comprising Eukaryotic Release Factor 1 (ERF1) activity or Eukaryotic Release Factor 3 (ERF3) activity may comprise a sequence as set forth in any one of SEQ ID NO: 138, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide comprising ERF1 activity or ERF3 activity may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 138, or a fragment thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise the amino acid sequence as set forth in SEQ ID NO: 137, or a fragment, or a derivative thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 137, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise histone modulation activity.
  • a disclosed encoded polypeptide comprising histone modulation activity can comprise histone acetyltransferase, histone deacetylase, histone demethylase, histone methyltransferase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise nucleic acid association activity.
  • a disclosed encoded polypeptide comprising nucleic acid association activity can comprise a helix-tum-helix region, a leucine zipper region, a winged helix region, a winged helix-tum-helix region, a helix-loop-helix region, an immunoglobulin fold, a B3 domain, a zinc finger, a HMG-box, a Wor3 domain, a TAL effector DNA-binding domain, or any combination thereof.
  • a disclosed encoded polypeptide can comprise methyltransferase activity.
  • a disclosed encoded polypeptide comprising methyltransferase activity can comprise DNA (cytosine-5)-methyltransferase 3a (DNMT3 A).
  • a disclosed encoded polypeptide can comprise demethylase activity.
  • a disclosed encoded polypeptide comprising demethylase activity can comprise ten-eleven translocation methylcytosine dioxygenase 1 (TET1) or lysine-specific histone demethylase 1 (LSD1).
  • a disclosed encoded polypeptide can comprise acetyltransferase activity.
  • a disclosed encoded polypeptide comprising acetyltransferase activity can comprise histone acetyltransferase.
  • a disclosed encoded polypeptide can comprise deacetylase activity.
  • a disclosed encoded polypeptide comprising deacetylase activity can comprise histone deacetylase.
  • a disclosed dCas endonuclease can comprise dSpCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel- associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSaCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dVQR, dEQR, or dVRER and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9 and a disclosed encoded polypeptide can comprise KRAB-MeCP2.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9
  • a disclosed fusion can comprise HPla-HPla, HPla-HPlb, HPla- MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP1 a-DNMT3 A, HP Ib-HP lb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb- DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1- NIPP1, MBDl-MeCP2, MBD
  • an isolated nucleic acid or viral vector can encode for a dCas endonuclease fused to the at least one encoded polypeptide having an enzymatic activity.
  • the isolated nucleic acid can encode for a “fusion protein” wherein the fusion protein comprises a first component comprising a dCas endonuclease and a second component comprising at least one polypeptide having an enzymatic activity.
  • the encoded fusion protein can comprise a dSpCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dSaCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dCjCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dVQR, dEQR, or dVRER and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER) and one or more of HPla-HPla, HPla-HPlb, HP 1 a-MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP 1 a-DNMT3 A, HP 1b- HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP
  • the encoded fusion protein can comprise a variant dCas9 selected from dVQR, dEQR, or dVRER and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dSaCas9 and KRAB-MeCP2. In an aspect, the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and DNMTA. In an aspect, the encoded fusion protein can comprise a dSaCas9 and DNMTA.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76 respectively, or a derivative thereof.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 78 respectively, or a derivative thereof.
  • the nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A may comprise a sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the sequences set forth in SEQ ID NO: 77 or SEQ ID NO: 78, or a fragment thereof.
  • a disclosed polypeptide dSpCas9 can comprise the amino acid sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment, or a derivative thereof.
  • a disclosed gene of interest or portion thereof can demonstrate a reduced expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can targes a gene of interest or portion thereof that demonstrates a reduced expression level when compared to wild-type or control expression level.
  • a disclosed gene of interest or portion thereof can demonstrate an increased expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can target a gene of interest or portion thereof that demonstrates an increased expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can target the APOE gene.
  • the at least one gRNA may target a region in the APOE gene comprising a nucleic acid sequence as set forth in any one of SEQ ID NO: 11-14, or a complement thereof.
  • the at least one gRNA targets a nucleic acid sequence comprising at least 10 consecutive nucleotides as set forth in SEQ ID NO: 11-14, or a complement thereof.
  • a disclosed gRNA can target the promoter of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 4 of the APOE gene. In an aspect, a disclosed gRNA can target a CpG island region of intron 1 of the APOE gene.
  • the CpG island region can include CpGl, CpG2, CpG3, CpG4, CpG5, CpG6, CpG7, CpG8, CpG9, CpGlO, CpGl l, CpG12, CpG13, CpG14, CpG15, CpG16, CpG17, CpG18, CpG19, CpG20, CpG21, CpG22, CpG23, or a combination thereof.
  • the CpG island region can include CpGl, CpG3, CpG6, CpG7, CpG8, CpG9, CpG18, CpG19, CpG20, CpG21, CpG22, or a combination thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • a disclosed viral vector or disclosed recombinant AAV vector can further comprise a gRNA scaffold.
  • a disclosed gRNA scaffold can comprise the sequence set forth in SEQ ID NO: 129, SEQ ID NO: 130, or a fragment thereof.
  • a disclosed isolated nucleic acid or disclosed viral vector comprises one or more microglia specific regulatory elements.
  • the one or more microglia specific regulatory elements can comprise a microglia specific wild-type promoter, a microglia specific enhancer, a synthetic promoter, a synthetic enhancer, a transcriptional or translational regulatory sequence or any combination thereof.
  • the one or more microglia specific regulatory elements comprise at least one microglia specific promoter (e.g., a microglia specific wild-type promoter or a microglia specific synthetic promoter).
  • the microglia specific regulatory element may be specific for a certain type of microglial cell (e.g., a dopaminergic or a cholinergic microglial cell).
  • the microglia specific regulatory element may include a dopaminergic specific regulatory element (z.e., a dopaminergic promoter) and/or a cholinergic microglia specific regulatory element (z.e., a cholinergic promoter).
  • the microglia specific regulatory element can comprise a microglia specific promoter.
  • a disclosed microglia specific promoter can comprise a F4/80 promoter, CD68 promoter, FCGR2A promoter, HexB promoter or IBA1 promoter, a derivative thereof, a fragment thereof, or any combination thereof.
  • the promoter is a CD68 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one or more of the sequences set forth in SEQ ID NO: 1-3, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 1-3.
  • the promoter is a F4/80 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 4.
  • the promoter is a IBA1 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5-7, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 5-7.
  • the promoter is a FCGR2A promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 8, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 8.
  • a disclosed microglia specific regulatory element ie., a microglia specific promoter
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element z.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, or a derivative thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at least one polypeptide having enzymatic activity can comprise a F4/80promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at least one polypeptide having enzymatic activity can comprise a CD68 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at least one polypeptide having enzymatic activity can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to at least one polypeptide having enzymatic activity can comprise a FCGR2A promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the at least one polypeptide having enzymatic activity can comprise a FGCR2A promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the at dCas endonuclease and the at least one polypeptide having enzymatic activity can comprise a F4/80 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the at least one polypeptide having enzymatic activity can comprise a CD68 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the at least one polypeptide having enzymatic activity can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a F4/80 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • a disclosed microglia specific regulatory element operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a CD68 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a IBA1 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed microglia specific regulatory element i.e., a microglia specific promoter
  • operably linked to the dCas endonuclease and the fusion protein having one or more enzymatic activities can comprise a FCGR2A promoter, a derivative thereof, or a fragment thereof.
  • a disclosed isolated nucleic acid or disclosed viral vector can further comprise one or more regulatory elements (i.e., promoters) in addition to the microglia specific regulatory element (e.g., microglia specific promoter).
  • a disclosed additional promoter can comprise a U6 promoter, a chicken P-actin promoter, an EF-la, a CMV promoter, a CMV promoter/enhancer, a fragment thereof, a derivative thereof, or any combination thereof.
  • a disclosed U6 promoter can comprise the sequence set forth in SEQ ID NO: 81, or a fragment thereof.
  • a disclosed U6 promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 81 or a fragment thereof.
  • a disclosed EF-la promoter can comprise the sequence set forth in SEQ ID NO: 79, SEQ ID NO: 80, or a fragment thereof.
  • a disclosed EF-la promoter can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% identity to the sequence set forth in SEQ ID NO: 79, SEQ ID NO: 80, or a fragment thereof.
  • a promoter can be a short EFl alpha (EFS-NF) promoter.
  • a disclosed promoter operably can be linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed promoter operably linked to the at least one guide RNA can comprise a U6 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed promoter operably linked to the at least one guide RNA targeting an APOE gene can comprise a U6 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed isolated nucleic acid molecule or viral vector can further two or more promoters, wherein a first promoter can be operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest or portion thereof.
  • a disclosed isolated nucleic acid molecule can further comprise one or more promoters, wherein a first promoter comprises a microglia specific promoter that is operably linked to the dCas endonuclease, a polypeptide having enzymatic activity, a fusion of two or more polypeptides having enzymatic activities or any combination thereof, and wherein a second promoter can be operably linked to the at least one guide RNA targeting a gene of interest thereof.
  • the second promoter operably linked to the gRNA may not be a microglia specific promoter.
  • the isolated nucleic acid can comprise a first promoter selected from a F4/80 promoter, a CD68 promoter, a IBA1 promoter, or a FCGR2A promoter, a fragment thereof, a derivative thereof, or any combination thereof, and a second promoter comprising a U6 promoter.
  • a disclosed isolated nucleic acid molecule or viral vector can further comprise one or more additional regulatory elements.
  • a disclosed additional regulatory element can comprise a promoter (described supra), an enhancer, a promoter/enhancer, an internal ribosomal entry site, a transcription terminal signal, a polyadenylation signal, a Spl and/or NF-kB transcriptional factor binding site, a p2A signal, a woodchuck hepatitis virus post-transcriptional regulatory element, a Phi signal-packaging signal, a rev responsive element, a 5’-ITR, a 3’-ITR, an inverted terminal repeat, a nuclear localization signal (NLS), or any combination thereof.
  • a disclosed 5’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 82 or SEQ ID NO: 83.
  • a disclosed 3’-ITR may comprise the sequence as set forth in any one of SEQ ID NO: 84 or SEQ ID NO: 85.
  • a disclosed NLS can comprise the sequence set forth in any one of SEQ ID NO: 88 - SEQ ID NO: 94, or a fragment thereof.
  • a disclosed PolyA sequence can comprise the sequence set forth in SEQ ID NO: 86, SEQ ID NO: 87, or a fragment thereof.
  • a disclosed viral vector or isolated nucleic acid can comprise a nucleic acid that is CpG depleted and codon-optimized for expression in a human cell.
  • CpG-free can mean completely free of CpGs or partially free of CpGs.
  • CpG-free can mean “CpG-depleted.”
  • CpG-depleted can mean “CpG-free.”
  • CpG-depleted can mean completely depleted of CpGs or partially depleted of CpGs.
  • CpG-free can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art.
  • any disclosed Cas9 endonuclease, a disclosed polypeptide having enzymatic activities, a disclosed fusion product or a disclosed fusion protein, or any combination thereof can be codon-optimized.
  • a disclosed viral vector or disclosed recombinant AAV vector can comprise a sequence that is about 4.5 kilobases or less than about 4.5 kilobases.
  • a disclosed method of treating and/or preventing Alzheimer’s diseases or an APOE related disease can comprise increasing expression and/or activity of one or more genes of interest.
  • modulating can comprise decreasing expression and/or activity of one or more genes of interest.
  • a disclosed method of effecting precision epigenetic modulation can comprise reducing expression and/or activity of APOE.
  • the disclosed cells can be microglial cells.
  • the disclosed cells can be cells affected by an overexpression level and/or activity level of one or more genes of interest or one or more target genes.
  • the disclosed cells can be in a subject.
  • a subject can be a human.
  • a subject can be suspected of having or can be diagnosed with having Alzheimer’s disease.
  • a subject can be suspected of having or can be diagnosed with having Parkinson’s disease.
  • a disclosed subject can be symptomatic or asymptomatic.
  • techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.
  • administering can comprise intravenous administration, intracerebral administration, intra-CSF administration, intracerebroventricular (ICV) administration, intraventricular administration, intra-cistema magna (ICM) administration, intraparenchymal administration, intrathecal (lumbar, cisternal, or both) administration, intrahepatic administration, hepatic intra-arterial administration, hepatic portal vein (HPV) administration, or any combination thereof.
  • a disclosed vector can be administered via LNP administration.
  • administering can comprise use of non-viral vectors to deliver a disclosed isolated nucleic acid.
  • these non-viral vectors can include, but are not limited to, nanoparticles or liposomes.
  • the disclosed isolated nucleic acids may be delivered via viral vectors (such as the disclosed viral vectors provided herein).
  • Exemplary viral vectors are described supra and include adenovirus, adeno-associated virus (AAV), and lentivirus (LV).
  • AAV adeno-associated virus
  • LV lentivirus
  • Viral vectors are used more commonly due to their robust delivery, long-lasting expression, low immunogenicity and toxicity, and compatibility with the advanced manufacturing techniques. Most research in gene therapy has utilized AAV or LV due to their lowered immunogenicity compared to adenovirus.
  • LV integrates its viral genome into the host gene to create permanent change in gene expression.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can employ multiple routes of administration to the subject.
  • a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.
  • a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation can be concurrently and/or serially administered to a subject via multiple routes of administration.
  • administering a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistern magna (ICM) administration.
  • administering a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise IV administration and intrathecal (ITH) administration.
  • a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x IO 10 vg/kg to about 2 x 10 14 vg/kg.
  • IV intravenous
  • a disclosed vector can be administered at a dose of about 1 x 10 11 vg/kg to about 8 x 10 13 vg/kg or about 1 x 10 12 vg/kg to about 8 x 10 13 vg/kg.
  • a disclosed vector can be administered at a dose of about 1 x 10 13 vg/kg to about 6 x 10 13 vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x IO 10 vg/kg, at least about 5 x IO 10 vg/kg, at least about 1 x 10 11 vg/kg, at least about 5 x 10 11 vg/kg, at least about 1 x 10 12 vg/kg, at least about 5 x 10 12 vg/kg, at least about 1 x 10 13 vg/kg, at least about 5 x 10 13 vg/kg, or at least about 1 x 10 14 vg/kg.
  • a disclosed vector can be administered at a dose of no more than about 1 x IO 10 vg/kg, no more than about 5 x IO 10 vg/kg, no more than about 1 x 10 11 vg/kg, no more than about 5 x 10 11 vg/kg, no more than about 1 x 10 12 vg/kg, no more than about 5 x 10 12 vg/kg, no more than about 1 x 10 13 vg/kg, no more than about 5 x 10 13 , or no more than about 1 x 10 14 vg/kg.
  • a disclosed vector can be administered at a dose of about 1 x 10 12 vg/kg.
  • a disclosed vector can be administered at a dose of about 1 x 10 11 vg/kg. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise monitoring the subject for adverse effects.
  • the method in the absence of adverse effects, can further comprise continuing to treat the subject.
  • the method in the presence of adverse effects, can further comprise modifying the treating step.
  • Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent.
  • a therapeutic agent can be any disclosed agent that effects a desired clinical outcome.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway.
  • an agent can comprise an enzyme for enzyme replacement therapy.
  • a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway.
  • a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise administering one or more immune modulators.
  • a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof.
  • a disclosed immune modulator can be bortezomib or SVP-Rapamycin.
  • a disclosed immune modulator can be Tacrolimus.
  • a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose.
  • a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.
  • a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib).
  • a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab).
  • an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledron
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation.
  • a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation.
  • a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells after administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise administering one or more immunosuppressive agents.
  • an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof.
  • a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time.
  • a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time.
  • a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response.
  • a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD 19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof.
  • Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV).
  • a subject in an aspect of a disclosed method, can be a human. In an aspect, a subject can be suspected of having or can be diagnosed with having Alzheimer’s or other APOE associated disease or disorder (such as, for example, PD or DLB). In an aspect, a disclosed subject can be symptomatic or asymptomatic.
  • a disclosed method can comprise repeating one or more steps of the method and/or modifying one or more steps of the method (such as, for example, an administering step).
  • a disclosed method of treating and/or preventing progression of a Alzheimer’s or other APOE associated disease or disorder can comprise modifying one or more of the disclosed steps.
  • modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • a method can be altered by changing the amount of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject, or by changing the duration of time one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination are administered to a subject.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.
  • a disclosed method of treating and/or preventing progression of a Alzheimer’ s or other APOE associated disease or disorder can further comprise generating and/or validating one or more of the disclosed isolated nucleic acid molecules, one or more of the disclosed vectors, one or more of the disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method of treating and/or preventing progression of a synucleinopathy or other APOE associated disease or disorder can further comprise administering to the subject a second disclosed isolated nucleic acid molecule, a second disclosed vector, a second disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method of treating and/or preventing progression of a synucleinopathy or other APOE associated disease or disorder can further comprise administering to the subject additional disclosed isolated nucleic acid molecules, additional disclosed vectors, additional disclosed pharmaceutical formulations, or any combination thereof.
  • Disclosed herein is a method of treating and/or preventing Parkinson’s disease progression in a subject, the method comprising administering to a subject a therapeutically effective amount of a disclosed isolated nucleic acid molecule, thereby reducing the pathological phenotype associated with Parkinson’s disease.
  • a method of treating and/or preventing Parkinson’s disease progression in a subject comprising administering to a subject a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a APOE gene or portion thereof and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with Parkinson’s disease.
  • a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity,
  • a method of treating and/or preventing Parkinson’s disease progression in a subject comprising administering to a subject a therapeutically effective amount of a disclosed isolated nucleic acid molecule, comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a APOE gene or portion thereof and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with Parkinson’s disease.
  • a disclosed isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity,
  • a method of treating and/or preventing Parkinson’s disease progression in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with Parkinson’s disease.
  • an isolated nucleic acid comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (
  • a method of treating and/or preventing Parkinson’s disease progression in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting an APOE gene or portion thereof and b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or
  • the microglia specific regulatory element of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof, a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(dCas) endonuclease
  • the microglia specific promoter of (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IBA1 promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof, a derivative thereof, a fragment thereof, or any combination thereof.
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of an isolated nucleic acid molecule, comprising: (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific regulatory element operably linked to (a)(i) and/or (a
  • the microglia specific regulatory element of (iv) may comprise a (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IB Al promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof.
  • the promoter of (c) comprises a U6 promoter, a derivative thereof or a fragment thereof.
  • Disclosed herein is a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject, the method comprising administering to a subject a therapeutically effective amount of a disclosed viral vector, thereby reducing the pathological phenotype associated with the Parkinson’s disease or disorder.
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii) or a combination thereof, thereby reducing the pathological phenotype associated with the Parkinson’s disease or disorder.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a disclosed viral vector, comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (iii) at least one guide RNA targeting a APOE gene or portion thereof and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii) or any combination thereof, thereby reducing the pathological phenotype associated with the Parkinson’s disease or disorder.
  • a disclosed viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) at least one polypeptide having an enzymatic activity, and (
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)(iii), or a combination thereof, thereby reducing the pathological phenotype associated with the Parkinson’s disease or disorder.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzy
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzymatic activities, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii), or a combination thereof, thereby reducing the pathological phenotype associated with the Parkinson’s disease or disorder.
  • a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, (ii) a fusion product having one or more enzy
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, and (b) at least one microglia specific regulatory element operably linked to (a)(i), (a)(ii), (a)
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, and (b) at least one microglia specific promoter operably linked to (a)(i), (a)(ii), (a)(iii)
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific regulatory element operably linked to the (a)(i) and/or (a)(ii),
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding
  • dCas deactivated Cas endonuclease
  • dCas deactivated Cas endonuclease
  • the dCas is a dSpCas9, dSaCas9, or dCjCas9
  • a fusion product having one or more enzymatic activities comprising any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific promoter operably linked to the (a)(i) and/or (a)(ii), and (c) a promoter operably linked to (a)(iii), thereby reducing the pathological phenotype associated with the Parkinson’s disease or disorder.
  • the microglia specific regulatory element or microglia specific promoter of (b) can comprise Alzheimer’s.
  • the fusion product of (a)(ii) can comprise KRAB-MeCP2.
  • a method of treating and/or preventing progression of Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding (i) a deactivated Cas (dCas) endonuclease, wherein the dCas is a dSpCas9, dSaCas9, or dCjCas9, (ii) a fusion product having one or more enzymatic activities, wherein the fusion product comprises any combination of HP la, HP lb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific regulatory element operably linked to (a)(i) and/or (a)(ii), and (
  • a method of treating and/or preventing progression of a Parkinson’s disease or disorder in a subject comprising administering to a subject a therapeutically effective amount of a viral vector comprising (a) a nucleic acid sequence encoding
  • dCas deactivated Cas endonuclease
  • dCas deactivated Cas endonuclease
  • the dCas is a dSpCas9, dSaCas9, or dCjCas9
  • a fusion product having one or more enzymatic activities comprising any combination of HPla, HPlb, MBD1, MBD2, KRAB, NIPP1, MeCP2, or DNMT3 A, and (iii) at least one guide RNA targeting the APOE gene or a portion of the APOE gene, (b) a microglia specific promoter operably linked to (a)(i) and/or (a)(ii), and (c) a promoter operably linked to (a)(iii), thereby reducing the pathological phenotype associated with the Alzhiemer’s or other APOE associated disease or disorder.
  • the microglia specific regulatory element or microglia specific promoter of (b) can comprise a (b) can comprise a F4/80 promoter (SEQ ID NO: 4), CD68 promoter (SEQ ID NO: 1-3), FCGR2A promoter (SEQ ID NO: 8), or IB Al promoter (SEQ ID NO: 5-7), a derivative thereof, a fragment thereof, or a combination thereof.
  • the fusion product of (a)(ii) can comprise KRAB-MeCP2.
  • the promoter of (c) comprises a U6 promoter, a derivative thereof, or a fragment thereof.
  • a disclosed method of treating and/or preventing progression of Parkinson’s disease or disorder can reduce and/or decrease the expression and/or activity of APOE. In an aspect of a disclosed method of treating and/or preventing progression of Parkinson’s disease or disorder, the expression and/or activity of APOE is reduced and/or decreased. In an aspect, a disclosed method can comprise diagnosing the subject with Parkinson’s disease or disorder.
  • a disclosed method of treating and/or preventing Parkinson’s disease or disorder comprises measuring the level of expression and/or the level of activity of the APOE gene. In an aspect, a disclosed method can comprise repeating the measuring step one or more times.
  • the expression and/or activity level of APOE prior to the administering step, can be elevated and/or increased when compared to wild-type or control expression level. In an aspect, following the administering step, the expression and/or activity level of APOE can be reduced and/or decreased when compared to the pre-administering step level.
  • a disclosed gRNA targeting a portion of the APOE gene can comprise at least two gRNAs. In an aspect, disclosed gRNAs can target the same portion of the APOE gene or different portions of the APOE gene. In an aspect, a disclosed gRNA can target the promoter of the APOE gene. In an aspect, a disclosed gRNA can target exon 1 of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 4 of the APOE gene. In an aspect, a disclosed gRNA can target a CpG island region of intron 1 of the APOE gene.
  • the CpG island region can include CpGl, CpG2, CpG3, CpG4, CpG5, CpG6, CpG7, CpG8, CpG9, CpGlO, CpGl l, CpG12, CpG13, CpG14, CpG15, CpG16, CpG17, CpG18, CpG19, CpG20, CpG21, CpG22, CpG23, or a combination thereof.
  • the CpG island region can include CpGl, CpG3, CpG6, CpG7, CpG8, CpG9, CpGl 8, CpGl 9, CpG20, CpG21, CpG22, or a combination thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • a disclosed viral vector or disclosed recombinant AAV vector can further comprise a gRNA scaffold.
  • a disclosed gRNA scaffold can comprise the sequence set forth in SEQ ID NO: 129, SEQ ID NO: 130, or a fragment thereof.
  • a disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • a disclosed dCas endonuclease can comprise a dCas9 endonuclease.
  • a disclosed dCas9 endonuclease can comprise a deactivated Staphylococcus aureus Cas9 (dSaCas9), a deactivated Streptococcus pyogenes Cas9 (dSpCas9), a deactivated Campylobacter jejuni Cas9 (dCjCas9), or a variant dCas9 endonuclease.
  • a disclosed variant dCas9 can comprise a variant dSaCas9, a variant dSpCas9, or a variant dCjCa9.
  • a disclosed variant dSpCas9 can comprise dVQR, dEQR, or dVRER.
  • a nucleic acid sequence the disclosed dSpCas9 may comprise a sequence as set forth in any one of SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSpCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40 or SEQ ID NO: 41, or a fragment thereof.
  • a disclosed dSpCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment, or a derivative thereof.
  • a disclosed dSpCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dSaCas9 may comprise a sequence as set forth in SEQ ID NO: 42, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dSaCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 42, or a fragment thereof.
  • a disclosed dSaCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 38 or a fragment, or a derivative thereof.
  • a disclosed dSaCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 38, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCjCas9 may comprise a sequence as set forth in SEQ ID NO: 43, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed dCjCas9 endonuclease may comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 43, or a fragment thereof.
  • a disclosed dCjCas9 endonuclease can comprise the amino acid sequence as set forth in SEQ ID NO: 39, or a fragment, or a derivative thereof.
  • a disclosed dCjCas9 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 39 or a fragment thereof.
  • a disclosed variant of dCas9 dVQR can comprise DI 135V, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dEQR can comprise D1135E, R1335Q, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed variant of dCas9 dVRER can comprise DI 135V, G1218R, R1335E, and T1337R with reference to SEQ ID NO: 36.
  • a disclosed dVRER can comprise the sequence set forth in SEQ ID NO: 37 or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed dCas9 endonuclease may comprise a sequence as set forth in any one of SEQ ID NO: 40-43.
  • the sequence encoding the disclosed dCas9 endonuclease may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 40-43, or a fragment, or a derivative thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity, transcription repression activity, transcription release factor activity, histone modulation activity, nucleic acid association activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise HP la, HP lb, MBD1, MBD2, Krtippel-Associated Box (KRAB), NIPP1, the Transcription Repression Domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed fusion or disclosed fusion product can comprise HPla-HPla, HPla-HPlb, HPla-MBDl, HPla-MBD2, HPla-KRAB, HPla-NIPPl, HPla-MeCP2, HPla-DNMT3A, HPlb-HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb- KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBD1- HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP1, MBDl-MeCP2, MBD1-DNMT3A, MBD2-MBD2, MBD2-HPla, MBD2-HPlb, MBD2-MBD1, MBD2-KRAB, MBD2-NIPP1, MBD2-MeCP2, MBD2-DNMT3A, MBD2-MBD2, MBD2-HP
  • the nucleic acid sequence encoding the disclosed polypeptide HP la may comprise a sequence as set forth in any one of SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HP la may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 59 or SEQ ID NO: 60, or a fragment thereof.
  • a disclosed polypeptide HPla can comprise the amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPla can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 45, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide HPlb may comprise a sequence as set forth in any one of SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide HPlb may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 61 or SEQ ID NO: 62, or a fragment thereof.
  • a disclosed polypeptide HPlb can comprise the amino acid sequence as set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment, or a derivative thereof.
  • a disclosed polypeptide HPlb can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD1 may comprise a sequence as set forth in any one of SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 63 or SEQ ID NO: 64, or a fragment thereof.
  • a disclosed polypeptide MBD1 can comprise the amino acid sequence as set forth in any one of SEQ ID NO: 48-50, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MDB1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 48-50, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MBD2 may comprise a sequence as set forth in any one of SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MBD2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 65 or SEQ ID NO: 66, or a fragment thereof.
  • a disclosed polypeptide MBD2 can comprise the amino acid sequence as set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MBD2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide NIPP1 may comprise a sequence as set forth in any one of SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide NIPP1 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 67 or SEQ ID NO: 68, or a fragment thereof.
  • a disclosed polypeptide NIPP1 can comprise the amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment, or a derivative thereof.
  • a disclosed polypeptide NIPP1 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide DNMT3 A may comprise a sequence as set forth in SEQ ID NO: 69, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide DNMT3 A may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 69, or a fragment thereof.
  • a disclosed polypeptide DNMT3A can comprise the amino acid sequence as set forth in SEQ ID NO: 55, or a fragment, or a derivative thereof.
  • a disclosed polypeptide DNMT3A can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide KRAB may comprise a sequence as set forth in any one of SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of the sequences set forth in SEQ ID NO: 70 or SEQ ID NO: 71, or a fragment thereof.
  • a disclosed polypeptide KRAB can comprise the amino acid sequence as set forth in SEQ ID NO: 56, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 56, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 72, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 72, or a fragment thereof.
  • a disclosed polypeptide MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 57, or a fragment, or a derivative thereof.
  • a disclosed polypeptide MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 57, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed fusion polypeptide KRAB- MeCP2 may comprise a sequence as set forth in any one of SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide KRAB-MeCP2 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 73 or SEQ ID NO: 74, or a fragment thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise the amino acid sequence as set forth in SEQ ID NO: 58, or a fragment, or a derivative thereof.
  • a disclosed polypeptide KRAB-MeCP2 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 58, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription activation activity.
  • a disclosed encoded polypeptide comprising transcription activation activity can comprise a transactivation domain.
  • a disclosed transactivation domain can comprise a VP16 protein, a series of linked VP16 proteins, a p65 domain of NFKB, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide VP16 may comprise a sequence as set forth in any one of SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment, or a derivative thereof.
  • the sequence encoding the disclosed polypeptide VP 16 may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 132 or SEQ ID NO: 133, or a fragment thereof.
  • a disclosed polypeptide VP16 can comprise the amino acid sequence as set forth in SEQ ID NO: 131, or a fragment, or a derivative thereof.
  • a disclosed polypeptide VP 16 can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 131, or a fragment thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a sequence as set forth in any one of SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide p65 domain of NFKB or RELA may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 135 or SEQ ID NO: 136, or a fragment thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise the amino acid sequence as set forth in SEQ ID NO: 134, or a fragment, or a derivative thereof.
  • a disclosed polypeptide p65 domain of NFKB or RELA can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 134, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise transcription repression activity.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise a Kriippel associated box domain, an ERF repressor domain, a MXI1 repressor domain, a SID4x repressor domain, a fused KRAB-MeCP2 domain, a MeCP2 TRD domain, a MAS-SID repressor domain, a TATA box binding protein activity, or any combination thereof.
  • a disclosed encoded polypeptide comprising transcription repression activity can comprise HP1 repressor activity, MeCP2 repressor activity, MBD1 repressor activity, MBD2 repressor activity, MBD3 repressor activity, MBD4 repressor activity, KRAB repressor activity, SUV39H1 repressor activity, SUV39H2 repressor activity, CTCF insulator-repressor activity, LSD-1 hi stone-dem ethyl ase repressor activity, or any combination thereof.
  • the nucleic acid sequence encoding the disclosed polypeptide comprising Eukaryotic Release Factor 1 (ERF1) activity or Eukaryotic Release Factor 3 (ERF3) activity may comprise a sequence as set forth in any one of SEQ ID NO: 138, or a fragment, or a derivative thereof.
  • sequence encoding the disclosed polypeptide comprising ERF1 activity or ERF3 activity may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 138, or a fragment thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise the amino acid sequence as set forth in SEQ ID NO: 137, or a fragment, or a derivative thereof.
  • a disclosed polypeptide comprising ERF1 activity or ERF3 activity can comprise a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 137, or a fragment thereof.
  • a disclosed encoded polypeptide can comprise histone modulation activity.
  • a disclosed encoded polypeptide comprising histone modulation activity can comprise histone acetyltransferase, histone deacetylase, histone demethylase, histone methyltransferase activity, or any combination thereof.
  • a disclosed encoded polypeptide can comprise nucleic acid association activity.
  • a disclosed encoded polypeptide comprising nucleic acid association activity can comprise a helix-tum-helix region, a leucine zipper region, a winged helix region, a winged helix-tum-helix region, a helix-loop-helix region, an immunoglobulin fold, a B3 domain, a zinc finger, a HMG-box, a Wor3 domain, a TAL effector DNA-binding domain, or any combination thereof.
  • a disclosed encoded polypeptide can comprise methyltransferase activity.
  • a disclosed encoded polypeptide comprising methyltransferase activity can comprise DNA (cytosine-5)-methyltransferase 3a (DNMT3 A).
  • a disclosed encoded polypeptide can comprise demethylase activity.
  • a disclosed encoded polypeptide comprising demethylase activity can comprise ten-eleven translocation methylcytosine dioxygenase 1 (TET1) or lysine-specific histone demethylase 1 (LSD1).
  • a disclosed encoded polypeptide can comprise acetyltransferase activity.
  • a disclosed encoded polypeptide comprising acetyltransferase activity can comprise histone acetyltransferase.
  • a disclosed encoded polypeptide can comprise deacetylase activity.
  • a disclosed encoded polypeptide comprising deacetylase activity can comprise histone deacetylase.
  • a disclosed dCas endonuclease can comprise dSpCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel- associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSaCas9 and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dCjCas9 and a disclosed polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl- CpG Binding Protein 2 (MeCP2), DNMT3 A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dVQR, dEQR, or dVRER and a disclosed encoded polypeptide can comprise HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9 and a disclosed encoded polypeptide can comprise KRAB-MeCP2.
  • a disclosed dCas endonuclease can comprise dSpCas9, dSaCas9, or dCjCas9
  • a disclosed fusion can comprise HPla-HPla, HPla-HPlb, HPla- MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP1 a-DNMT3 A, HP Ib-HP lb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb- DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1- NIPP1, MBDl
  • an isolated nucleic acid or viral vector can encode for a dCas endonuclease fused to the at least one encoded polypeptide having an enzymatic activity.
  • the isolated nucleic acid can encode for a “fusion protein” wherein the fusion protein comprises a first component comprising a dCas endonuclease and a second component comprising at least one polypeptide having an enzymatic activity.
  • the encoded fusion protein can comprise a dSpCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dSaCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise a dCjCas9 and a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HPla, HPlb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dVQR, dEQR, or dVRER and a polypeptide having enzymatic activity comprising HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • a polypeptide having enzymatic activity comprising HP la, HP lb, MBD1, MBD2, Kriippel-associated box (KRAB), NIPP1, the transcription repression domain (TRD) of Methyl-CpG Binding Protein 2 (MeCP2), DNMT3A, any combination thereof, or any fusion thereof.
  • the encoded fusion protein can comprise dSpCas9, dSaCas9, dCjCas9, or a variant dCas9 (e.g., dVQR, dEQR or dVRER) and one or more of HPla-HPla, HPla-HPlb, HP 1 a-MBD 1 , HP 1 a-MBD2, HP 1 a-KRAB, HP 1 a-NIPP 1 , HP 1 a-MeCP2, HP 1 a-DNMT3 A, HP 1b- HPlb, HPlb-HPla, HPlb-MBDl, HPlb-MBD2, HPlb-KRAB, HPlb-NIPPl, HPlb-MeCP2, HPlb-DNMT3A, MBD1-MBD1, MBDl-HPla, MBDl-HPla, MBDl-HPlb, MBD1-MBD2, MBD1-KRAB, MBD1-NIPP
  • the encoded fusion protein can comprise a variant dCas9 selected from dVQR, dEQR, or dVRER and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and a fusion comprising KRAB-MeCP2.
  • the encoded fusion protein can comprise a dSaCas9 and KRAB-MeCP2. In an aspect, the encoded fusion protein can comprise a dCas9 selected from dSpCas9, dSaCas9, or dCjCas9 and DNMTA. In an aspect, the encoded fusion protein can comprise a dSaCas9 and DNMTA.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76 respectively, or a derivative thereof.
  • nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A or dVRER and DNMT3A may comprise a sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 78 respectively, or a derivative thereof.
  • the nucleic acid sequence encoding the fusion protein comprising dSpCas9 and DNMT3A may comprise a sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the sequences set forth in SEQ ID NO: 77 or SEQ ID NO: 78, or a fragment thereof.
  • a disclosed polypeptide dSpCas9 can comprise the amino acid sequence as set forth in SEQ ID NO: 75 or SEQ ID NO: 76, or a fragment, or a derivative thereof.
  • a disclosed gene of interest or portion thereof can demonstrate a reduced expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can targes a gene of interest or portion thereof that demonstrates a reduced expression level when compared to wild-type or control expression level.
  • a disclosed gene of interest or portion thereof can demonstrate an increased expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can target a gene of interest or portion thereof that demonstrates an increased expression level when compared to wild-type or control expression level.
  • a disclosed gRNA can target the APOE gene.
  • the at least one gRNA may target a region in the APOE gene comprising a nucleic acid sequence as set forth in any one of SEQ ID NO: 11-14, or a complement thereof.
  • the at least one gRNA targets a nucleic acid sequence comprising at least 10 consecutive nucleotides as set forth in SEQ ID NO: 11-14, or a complement thereof.
  • a disclosed gRNA can target the promoter of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene.
  • a disclosed gRNA can target exon 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 1 of the APOE gene. In an aspect, a disclosed gRNA can target intron 4 of the APOE gene. In an aspect, a disclosed gRNA can target a CpG island region of intron 1 of the APOE gene.
  • the CpG island region can include CpGl, CpG2, CpG3, CpG4, CpG5, CpG6, CpG7, CpG8, CpG9, CpGlO, CpGl l, CpG12, CpG13, CpG14, CpG15, CpG16, CpG17, CpG18, CpG19, CpG20, CpG21, CpG22, CpG23, or a combination thereof.
  • the CpG island region can include CpGl, CpG3, CpG6, CpG7, CpG8, CpG9, CpG18, CpG19, CpG20, CpG21, CpG22, or a combination thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33.
  • the disclosed gRNA may comprise a nucleic acid sequence as set forth in SEQ ID NO: 15-33, or a complement thereof.
  • the disclosed gRNA may comprise a nucleic acid sequence with at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or more sequence identity to any one of the sequences set forth in SEQ ID NO: 15-33, or a complement thereof, or a fragment thereof.
  • a disclosed viral vector or disclosed recombinant AAV vector can further comprise a gRNA scaffold.
  • a disclosed gRNA scaffold can comprise the sequence set forth in SEQ ID NO: 129, SEQ ID NO: 130, or a fragment thereof.
  • a disclosed isolated nucleic acid or disclosed viral vector comprises one or more microglia specific regulatory elements.
  • the one or more microglia specific regulatory elements can comprise a microglia specific wild-type promoter, a microglia specific enhancer, a synthetic promoter, a synthetic enhancer, a transcriptional or translational regulatory sequence or any combination thereof.
  • the one or more microglia specific regulatory elements comprise at least one microglia specific promoter (e.g., a microglia specific wild-type promoter or a microglia specific synthetic promoter).
  • the microglia specific regulatory element may be specific for a certain type of microglial cell (e.g., a dopaminergic or a cholinergic microglial cell).
  • the microglia specific regulatory element may include a dopaminergic specific regulatory element (z.e., a dopaminergic promoter) and/or a cholinergic microglia specific regulatory element (z.e., a cholinergic promoter).
  • the microglia specific regulatory element can comprise a microglia specific promoter.
  • a disclosed microglia specific promoter can comprise a F4/80 promoter, CD68 promoter, FCGR2A promoter, HexB promoter or IBA1 promoter, a derivative thereof, a fragment thereof, or any combination thereof.
  • the promoter is a CD68 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one or more of the sequences set forth in SEQ ID NO: 1-3, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 1-3.
  • the promoter is a F4/80 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 4, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 4.
  • the promoter is a IBA1 promoter, or a derivative thereof.
  • the promoter may comprise a nucleic acid sequence having at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 5-7, or a fragment, or a derivative thereof.
  • the promoter comprises a sequence as set forth in any one of SEQ ID NO: 5-7.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Des méthodes d'administration de thérapie génique de précision, de traitement et/ou de prévention de la progression de la maladie d'Alzheimer et de réduction de l'expression de APOE et APOE e4 sont divulguées dans la présente invention. Sont divulgués également des molécules d'acide nucléique isolées, des vecteurs viraux, des vecteurs AAV, des formulations pharmaceutiques, des cellules hôtes, des ARN guides et des plasmides destinés à être utilisés dans les méthodes divulguées.
PCT/US2024/020591 2023-03-20 2024-03-19 Thérapie génique spécifique de la microglie ciblant l'apoe contre maladies neurologiques Ceased WO2024196955A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363453186P 2023-03-20 2023-03-20
US63/453,186 2023-03-20
US202363524024P 2023-06-29 2023-06-29
US63/524,024 2023-06-29

Publications (2)

Publication Number Publication Date
WO2024196955A2 true WO2024196955A2 (fr) 2024-09-26
WO2024196955A3 WO2024196955A3 (fr) 2024-12-19

Family

ID=92842640

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/020591 Ceased WO2024196955A2 (fr) 2023-03-20 2024-03-19 Thérapie génique spécifique de la microglie ciblant l'apoe contre maladies neurologiques

Country Status (1)

Country Link
WO (1) WO2024196955A2 (fr)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006122141A2 (fr) * 2005-05-10 2006-11-16 Biogen Idec Ma Inc. Methodes et produits permettant de determiner l'expression du gene f4/80 dans des cellules microgliales
EP3497221A4 (fr) * 2016-08-10 2020-02-05 Duke University Compositions, systèmes et procédés de programmation d'une fonction de cellules immunitaires par régulation ciblée de gènes
JP2022540446A (ja) * 2019-07-10 2022-09-15 ロックアネイビオ， インコーポレイテッド Ｒｎａ標的ノックダウン及び置換の組成物及び使用方法
AU2021364596A1 (en) * 2020-10-22 2023-06-08 Duke University Compositions and methods relating to alzheimer's disease
EP4288534A1 (fr) * 2021-02-05 2023-12-13 Ospedale San Raffaele S.r.l. Transposase modifiée et ses utilisations

Also Published As

Publication number Publication date
WO2024196955A3 (fr) 2024-12-19

Similar Documents

Publication Publication Date Title
US20250161383A1 (en) Viral gene therapy as treatment for cholesterol storage disease or disorder
US20250082778A1 (en) Rna guided eradication of human jc virus and other polyomaviruses
JP7634507B2 (ja) 操作されたｄｎａ結合タンパク質
AU2020282352B2 (en) Compositions and methods for selective gene regulation
US20240409948A1 (en) Codon-optimized human npc1 genes for the treatment of niemann-pick type c1 deficiency and related conditions
AU2017234929A1 (en) Therapeutic for treatment of diseases including the central nervous system
AU2019392679A1 (en) Methods of detecting, preventing, reversing, and treating neurological diseases
US20250177568A1 (en) Compositions and methods for modulating alpha-synuclein expression
US20230392133A1 (en) Compositions and Methods Relating to Alzheimer's Disease
Shen et al. Behavioral abnormalities, cognitive impairments, synaptic deficits, and gene replacement therapy in a CRISPR engineered rat model of 5p15. 2 deletion associated with cri du chat syndrome
Lin et al. AAVLINK: A potent DNA-recombination method for large cargo delivery in gene therapy
WO2023069923A1 (fr) Compositions et procédés se rapportant à la modulation épigénétique
US20240424135A1 (en) Compositions For and Methods of Improving Viral Vectors
EP3897695B1 (fr) Vecteurs recombinant pour le traitement à long terme de la mucopolysaccharidose
WO2024196752A2 (fr) Compositions pour thérapie génique ciblant snca spécifique à une cellule et leurs méthodes de production et d'utilisation
US20240366684A1 (en) Compositions comprising a zim3 effector and methods of use thereof
US20230211014A1 (en) AAV Gene Therapy for Spastic Paraplegia
WO2025016362A1 (fr) Thérapie à base de promoteur de l'homéostasie intracérébrale du phosphate
WO2025015328A2 (fr) Augmentation de l'activité ou de l'expression de nfe2l1 en tant que thérapie pour des troubles oculaires
WO2024147114A1 (fr) Compositions et procédés pour le traitement de la maladie de parkinson

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24775586

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 24775586

Country of ref document: EP

Kind code of ref document: A2