WO2024238448A2 - Virus adéno-associé recombinant de capside chimérique avec pénétration de bbb et ciblage de microglie (aav-bm) - Google Patents

Virus adéno-associé recombinant de capside chimérique avec pénétration de bbb et ciblage de microglie (aav-bm) Download PDF

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WO2024238448A2
WO2024238448A2 PCT/US2024/029057 US2024029057W WO2024238448A2 WO 2024238448 A2 WO2024238448 A2 WO 2024238448A2 US 2024029057 W US2024029057 W US 2024029057W WO 2024238448 A2 WO2024238448 A2 WO 2024238448A2
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aav
nucleic acid
disease
microglia
virus
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WO2024238448A3 (fr
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Wenhui Hu
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Temple Univ School of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors

Definitions

  • HPE HIV-associated progressive encephalopathy
  • HAND HIV-Associated Neurocognitive Disorder
  • CNS cells mainly microglia (MG), harboring integrated HIV sequences appear to be sufficient to bring about the cognitive decline, opportunistic infections, autoimmunity, and muscle wasting observed in the clinic.
  • Ridding patients of this reservoir of chronically infected cells is the most direct route toward eradication of HAND and HIV.
  • BBB Blood-Brain Barrier
  • the long-lived myeloid cells such as perivascular macrophages and resident microglia in CNS persistently harbor HIV provirus and could contribute to the source of residual viremia during long-term ART or rebounding virus upon ART cessation.
  • the long-lived myeloid cells such as perivascular macrophages and resident microglia in CNS persistently harbor HIV provirus and could contribute to the source 1 158458212.1 of residual viremia during long-term ART or rebounding virus upon ART cessation. It is undoubtfully and urgently need-ed to develop novel strategies to specifically target CNS myeloid cells for HIV eradication and a cure.
  • Adeno-Associated Virus AAV
  • Embodiments relate to recombinant Adeno-Associated Virus (AAV) chimeric capsids to treat HIV- and non-HIV-associated diseases of nervous system.
  • an adeno-associated virus comprises a nucleic acid sequence encoding: (i) a capsid protein comprising a BBB-penetrating peptide, and (ii) a microglia-tropic peptide.
  • the AAV transfer vector comprises a microglial tropic promoter, a mRNA expression-boosting 5’-UTR (from SARS-CoV-2), a transgene, a woodchuck hepatitis virus post-transcriptional response element (WPRE) short version WPRE3, 4x miRNA-9 target sites (miR9T), and poly(A) sequence.
  • the BBB-penetrating peptide is derived from an AAV serotype (AAV-B) that efficiently crosses the BBB relative to an AAV serotype that lacks ability to cross the BBB.
  • the AAV serotype that efficiently crosses the BBB comprises AAV8, AAV9, AAV10 or derivatives thereof.
  • the AAV9 derivatives comprise those with reported BBB-penetrating efficiency, such as AAV-PhP.B, AAV-PhP-eB, AAV-F, AAV9-Retro, AAV-Cap-MAC, AAV-C2, AAV9P31, AAV-B10, AAV-B22, AAV-DJ8, AAV-CPP21, AAV-CPP16, AAV-cc47, AAV- Pal1, AAV-MDV1A, AAVBI28, AV-BI62, AAV-BI65, and others, or combinations thereof.
  • BBB-penetrating efficiency such as AAV-PhP.B, AAV-PhP-eB, AAV-F, AAV9-Retro, AAV-Cap-MAC, AAV-C2, AAV9P31, AAV-B10, AAV-B22, AAV-DJ8, AAV-CPP21, AAV-CPP16, AAV-cc47, AAV- Pal1, AAV-
  • the BBB penetrating peptide is inserted into a capsid VP1 protein of microglia-tropism AAV (AAV-M), for example, AAV1, AAV2, AAV5, AAV6, AAV-DJ and their derivatives (including mutations).
  • AAV VP1 capsid protein is an AAV2 VP1 capsid protein.
  • the BBB penetrating peptide is inserted at 2 158458212.1 amino acid position N587 of AAV2 VP1 protein.
  • the transgene encodes a therapeutic agent.
  • the transgene encodes a gene-editing complex.
  • the gene editing complex comprises at least two isolated nucleic acid sequences comprising: (i) an isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and (ii) at least one guide RNA (gRNA), the gRNA being complementary to a target sequence in a virus or host genome.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • gRNA guide RNA
  • the virus comprises a retrovirus.
  • the virus is a human immunodeficiency virus (HIV).
  • the first and second target sequences comprise one or more nucleic acid sequences in a HIV comprising: long terminal repeat (LTR) nucleic acid sequences, nucleic acid sequences encoding structural proteins, non-structural proteins, or combinations thereof.
  • LTR long terminal repeat
  • the sequences encoding structural proteins comprise nucleic acid sequences encoding: Gag, Gag-Pol precursor, Pro (protease), Reverse Transcriptase (RT), integrase (In), Env or combinations thereof
  • the sequences encoding non-structural proteins comprise nucleic acid sequences encoding: regulatory proteins, accessory proteins or combinations thereof.
  • the regulatory proteins comprise: Tat, Rev or combinations thereof, and wherein accessory proteins comprise Nef, Vpr, Vpu, Vif or combinations thereof.
  • the endonuclease comprises Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, ARMAN 1, ARMAN 4, saCas9, cjCas9, Cas12a, Cas12f, mutants, variants, high-fidelity variants, orthologs, analogs, fragments, or combinations thereof.
  • the amino acid sequence encoding the BBB penetrating peptide comprises SAQLNTTKPI (SEQ ID NO: 1) for AAV2-Rep-Cap-MAC (R2-MAC).
  • the BBB penetrating peptide comprises poly-L-arginine, a transferrin or lactoferrin ligand, transactivator of transcription (tat), penetratin, insulin peptide or lipoprotein.
  • the therapeutic agent is a treatment for neurological disorders, e.g. AAV-saCas9, AAV-cjCas9, AAV-Cas12f, and others.
  • the neurological disorders comprise: AIDS dementia complex, Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alper's disease, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal 3 158458212.1 lobar degeneration, Huntington's disease, Kennedy's disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinoclastic sclerosis, spinocerebellar ataxia, subacute combined de
  • the therapeutic agent comprises antibodies, aptamers, peptides, proteins, natural ligands of one or more CNS target(s), modified versions of natural ligands of one or more CNS target(s), aptamers, inhibitory nucleic acids, small inhibitory RNAs (siRNA), short hairpin RNAs (shRNA)), miRNA, dsDNA, lncRNA, antisense oligonucleotides ribozymes, and small molecules, or active fragments of thereof.
  • the microglia specific peptide comprises M1, M2 or the combination thereof.
  • a method of treating a subject suffering from a virus mediated neurological disorder or a neurological disorder comprising: administering to the subject a therapeutically effective amount of an AAV comprising a nucleic acid sequence encoding: (i) a capsid protein comprising a BBB penetrating peptide, (ii) a microglial specific peptide, (iii) a microglial tropic promoter and (iv) a transgene.
  • the AAV further comprises a woodchuck hepatitis virus post-transcriptional response element (WPRE).
  • the BBB penetrating peptide is derived from an AAV serotype that efficiently crosses the BBB relative to an AAV serotype that lacks ability to cross the blood brain barrier.
  • the AAV serotype that efficiently crosses the BBB comprises AAV2, AAV8, AAV9, AAV10 or derivatives thereof.
  • the AAV9 derivatives comprise AAV-PhP.B, AAV-PhP-eB, AAV-F, AAV9-Retro, AAV-Cap-MAC, AAV-C2, AAV9P31, AAV-B10, AAV-B22, AAV-DJ8, or combinations thereof.
  • the BBB penetrating peptide is inserted into an AAV2, capsid VP1 protein. In certain embodiments, the BBB penetrating peptide is inserted at amino acid position Q587.
  • the transgene encodes a therapeutic agent. In certain embodiments, the transgene encodes a gene- editing complex. In certain embodiments, the gene editing complex comprises one or more isolated nucleic acid sequences encoding a CRISPR-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a target sequence in a virus genome.
  • gRNA guide RNA
  • the gene editing complex comprises at least two isolated nucleic acid sequences 4 158458212.1 comprising: (i) a first isolated nucleic acid sequence encoding a first Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a first target sequence in a virus genome, and (ii) a second isolated nucleic acid sequence encoding a second Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a second target sequence in a virus genome.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • gRNA guide RNA
  • the first and second target sequences are different.
  • the virus comprises a retrovirus.
  • the virus is a human immunodeficiency virus (HIV).
  • the first and second target sequences comprise one or more nucleic acid sequences in a human immunodeficiency virus (HIV) comprising: long terminal repeat (LTR) nucleic acid sequences, nucleic acid sequences encoding structural proteins, non- structural proteins, or combinations thereof.
  • LTR long terminal repeat
  • sequences encoding structural proteins comprise nucleic acid sequences encoding: Gag, Gag-Pol precursor, Pro (protease), Reverse Transcriptase (RT), integrase (In), Env or combinations thereof, and wherein the sequences encoding non-structural proteins comprise nucleic acid sequences encoding: regulatory proteins, accessory proteins or combinations thereof.
  • regulatory proteins comprise: Tat, Rev or combinations thereof, and wherein accessory proteins comprise Nef, Vpr, Vpu, Vif or combinations thereof.
  • endonuclease comprises Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, saCas9, cjCas9, Cas12a, Cas12f, ARMAN 1, ARMAN 4, mutants, variants, high-fidelity variants, orthologs, analogs, fragments, or combinations thereof.
  • the nucleic acid sequence encoding the blood brain barrier (BBB) penetrating peptide comprises at least an 80% sequence identity to SAQLNTTKPI (SEQ ID NO: 1).
  • the amino acid sequence encoding the blood brain barrier (BBB) penetrating peptide comprises SAQLNTTKPI (SEQ ID NO: 1) for AAV2-Rep-Cap- MAC (R2-MAC).
  • the blood brain barrier (BBB) penetrating peptide comprises poly-L-arginine, a transferrin or lactoferrin ligand, transactivator of transcription (tat), penetratin, insulin peptide or lipoprotein.
  • the therapeutic agent is a treatment for neurological disorders.
  • the neurological disorders comprise: AIDS dementia complex, Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alper's disease, ataxia telangiectasia, Batten disease, 5 158458212.1 bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy's disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinoclastic sclerosis, spinocerebellar ataxia, subacute combined de
  • BSE
  • the therapeutic agent comprises antibodies, aptamers, peptides, proteins, natural ligands of one or more CNS target(s), modified versions of natural ligands of one or more CNS target(s), aptamers, inhibitory nucleic acids, small inhibitory RNAs (siRNA), short hairpin RNAs (shRNA), ribozymes, and small molecules, or active fragments of thereof.
  • the microglia specific peptide comprises M1, M2 or the combination thereof.
  • the treatment further comprises administering to the subject one or more secondary treatments.
  • the secondary treatments comprise anti-virus agents, chemotherapeutic agents, immunotherapy, growth inhibitory agents, cytotoxic agents, radiation therapy, surgery, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents and combinations thereof.
  • the anti-virus agent comprises antibodies, aptamers, adjuvants, anti-sense oligonucleotides, chemokines, cytokines, immune stimulating agents, immune modulating agents, B-cell modulators, T-cell modulators, NK cell modulators, antigen presenting cell modulators, enzymes, siRNA’s, ribavirin, protease inhibitors, helicase inhibitors, polymerase inhibitors, helicase inhibitors, neuraminidase inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, purine nucleosides, chemokine receptor antagonists, interleukins, or combinations thereof.
  • an AAV transfer vector for microglia-specific transgene expression comprises a nucleic acid including: (i) a microglial tropic minimal/essential promoter, (ii) a mRNA expression-boosting UTR, (iii) a transgene, a mRNA stabilizing minimal Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element 3 (WPRE3) and (iv) a microglia- illuminating miR9T and (V) a poly(A) sequence.
  • a microglial tropic minimal/essential promoter e.g., a mRNA expression-boosting UTR
  • a transgene a mRNA stabilizing minimal Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element 3 (WPRE3)
  • WPRE3 Woodchuck Hepatitis Virus
  • a recombinant adeno-associated virus comprises a nucleic acid sequence encoding: (i) a capsid protein comprising a BBB penetrating peptide, (ii) at least two 6 158458212.1 microglial specific peptides, (iii) a microglial tropic promoter and (iv) a transgene.
  • the method further comprises a nucleic acid encoding: (i) a microglial tropic minimal/essential promoter, (ii) a mRNA expression-boosting UTR, (iii) a mRNA stabilizing minimal Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element 3 (WPRE3) and (iv) a poly(A) sequence.
  • a bar-coded (bc) adeno- associated virus (AAV) library comprises bar- coded AAV transfer vectors wherein each bar coded AAV transfer vector comprises two CD68 or two CD11b promoters one or a plurality of microglial specific peptides.
  • each microglial specific peptide specifically targets the bc AAV to microglia. In certain embodiments, each microglial specific peptide specifically binds to different microglial targets.
  • the bc AAV comprises adeno-associated virus serotype B (AAV-B).
  • AAV-B adeno-associated virus serotype B
  • a method of screening for adeno-associated virus target specificity comprising, screening the bar coded AAV library against microglial specific markers to determine specificty.
  • the microglial specific markers comprise Tmem119, P2ry12, HexB, Fcrls, Sall1, C1q, Gpr34, Olfml3, Mertk, Pros1, Tyro3, Tgfbr1 or combinations thereof.
  • the amino acid sequence encoding a blood brain barrier (BBB) penetrating peptide comprises an amino acid sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SAQLNTTKPI (SEQ ID NO: 1).
  • the BBB peptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1. In some embodiments, the BBB comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 1. In some embodiments, the BBB comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 1. In some embodiments, the BBB comprises the amino acid sequence of SEQ ID NO: 1.
  • the nucleotide sequence of an AAV-R2-MAC comprises a sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 2.
  • the AAV-R2-MAC comprises a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 2.
  • the AAV- R2-MAC comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID 7 158458212.1 NO: 2.
  • the AAV-R2-MAC comprises a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the AAV-R2-MAC comprises a nucleotide sequence of SEQ ID NO: 2.
  • the amino acid sequence encoding an R2-MAC, REP protein comprises an amino acid sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 3. In some embodiments, the R2-MAC, REP protein comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 3.
  • the R2-MAC, REP protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 3. In some embodiments, the R2-MAC, REP protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3. In some embodiments, the R2- MAC, REP protein comprises the amino acid sequence of SEQ ID NO: 3. [0016] In another aspect, the amino acid sequence encoding an R2-MAC, VP1(SAQ-MAC) protein comprises an amino acid sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 4.
  • an R2-MAC, VP1(SAQ-MAC) comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 4. In some embodiments, an R2-MAC, VP1(SAQ-MAC) comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 4. In some embodiments, an R2-MAC, VP1(SAQ- MAC) comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:4. In some embodiments, an R2-MAC, VP1(SAQ-MAC) comprises the amino acid sequence of SEQ ID NO: 4.
  • an AAV-R2e-MAC nucleotide sequence (enhanced version of R2- MAC with 4 YF mutations and 1 TV mutation; (AAV2-REP-link-VP1(4YF/TV and SAQ-MAC)) comprises a nucleotide sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 5.
  • an AAV-R2e-MAC comprises a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 5.
  • an AAV-R2e-MAC comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 5. In some embodiments, an AAV-R2e-MAC comprises a nucleotide sequence having at least about 8 158458212.1 95% sequence identity to SEQ ID NO: 5. In some embodiments, an AAV-R2e-MAC comprises a nucleotide sequence of SEQ ID NO: 5.
  • the amino acid sequence encoding an R2e-MAC, REP protein comprises an amino acid sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 6.
  • an R2e-MAC, REP protein comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 6.
  • the an R2e-MAC, REP protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 6.
  • an R2e-MAC, REP protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:6. In some embodiments, an R2e-MAC, REP protein comprises the amino acid sequence of SEQ ID NO: 6. [0019] In another aspect, the amino acid sequence encoding an R2e-MAC, VP1(4YF/TV and SAQ-MAC) protein comprises an amino acid sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 7.
  • an R2e-MAC, VP1 comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 7. In some embodiments, an R2e-MAC, VP1 comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 7. In some embodiments, an R2e-MAC, VP1 comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 7. In some embodiments, an R2e-MAC, VP1 comprises the amino acid sequence of SEQ ID NO: 7.
  • the nucleotide sequence encoding TP2018 comprises a nucleotide sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 8.
  • the TP2018 comprises a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 8.
  • the TP2018 comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 8.
  • TP2018 comprises a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 8. In some embodimentsTP2018 comprises a nucleotide sequence of SEQ ID NO: 8. 9 158458212.1 [0021]
  • the nucleotide sequence encoding an AAV barcode reporter vector comprises a nucleotide sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 9.
  • an AAV barcode reporter vector comprises a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 9. In some embodiments, an AAV barcode reporter vector comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 9. In some embodiments, an AAV barcode reporter vector comprises a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 9. In some embodiments, an AAV barcode reporter vector comprises a nucleotide sequence of SEQ ID NO: 9.
  • a nucleotide sequence encoding an AAV barcode reporter vector comprises a nucleotide sequence having at least about 70% (such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to SEQ ID NO: 10.
  • an AAV barcode reporter vector comprises a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 10.
  • an AAV barcode reporter vector comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 10. In some embodiments, an AAV barcode reporter vector comprises a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO: 10. In some embodiments, an AAV barcode reporter vector comprises a nucleotide sequence of SEQ ID NO: 10. [0023] In another aspect, a pharmaceutical composition comprises any one or more SEQ ID NOs: 1-9, 10 and combinations thereof. [0024] Definitions [0025] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
  • the term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.
  • the AAV genome is built of single stranded DNA and comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames: rep and cap, encoding replication and capsid proteins, respectively.
  • ITRs inverted terminal repeats
  • rep and cap two open reading frames
  • a foreign polynucleotide can replace the native rep and cap genes.
  • AAVs can be made with a variety of different serotype capsids which have varying transduction profiles or as used herein, “tropism” for different tissue types.
  • serotype refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ or DJ/8.
  • serotype AAV2 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV2 and a genome containing 5' and 3' ITR sequences from the same AAV2 serotype.
  • Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs of a second serotype.
  • Pseudotyped rAAV would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype.
  • Pseudotyped rAAV are produced using standard techniques described in the art.
  • the AAVs comprise mutations in the capsid proteins. These mutations comprise one or more amino acid changes at one or multiple locations of the capsid proteins.
  • administering is meant a method of giving a dosage of a composition described herein (e.g., an AAV and/or a pharmaceutical composition thereof) to a subject.
  • compositions utilized in the methods described herein can be administered by any suitable route, including, for example, by inhalation, nebulization, aerosolization, intranasally, intratracheally, 11 158458212.1 intrabronchially, orally, parenterally (e.g., intravenously, subcutaneously, or intramuscularly), orally, nasally, rectally, topically, or buccally.
  • parenterally e.g., intravenously, subcutaneously, or intramuscularly
  • nasally, rectally, topically, or buccally e.g., amino acid
  • amino acid refers to naturally occurring and synthetic ⁇ , ⁇ , ⁇ , and ⁇ amino acids, and includes but is not limited to, amino acids found in proteins, i.e.
  • glycine alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine.
  • the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, ⁇ -alanyl, ⁇ - valinyl, ⁇ -leucinyl, ⁇ -isoleucinyl, ⁇ -prolinyl, ⁇ -phenylalaninyl, ⁇ -tryptophanyl, ⁇ -methioninyl, ⁇ - glycinyl, ⁇ -serinyl, ⁇ -threoninyl, ⁇ -cystein
  • amino acid When the term amino acid is used, it is considered to be a specific and independent disclosure of each of the esters of ⁇ , ⁇ , ⁇ , and ⁇ glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L-configurations.
  • blood-brain barrier refers to the physiological barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Daltons).
  • the BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the CNS and are herein collectively referred to as the blood-brain barrier or BBB.
  • the BBB also encompasses the blood-CSF barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.
  • blood-brain barrier receptor abbreviated “R/BBB” herein
  • R/BBB blood-brain barrier receptor
  • R/BBB examples include transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low density lipoprotein receptors including without limitation low density lipoprotein receptor-related protein 1 (LRP1) and low density lipoprotein receptor-related protein 12 158458212.1 8 (LRP8), and heparin-binding epidermal growth factor-like growth factor (HB-EGF).
  • TfR transferrin receptor
  • IGF-R insulin-like growth factor receptor
  • LRP1 low density lipoprotein receptor-related protein 1
  • LRP8 low density lipoprotein receptor-related protein 12 158458212.1 8
  • HB-EGF heparin-binding epidermal growth factor-like growth factor
  • the “transferrin receptor” (“TfR”) is a transmembrane glycoprotein (with a molecular weight of about 180,000) composed of two disulphide-bonded sub-units (each of apparent molecular weight of about 90,000) involved in iron uptake in vertebrates.
  • the amino acid sequence encoding the blood brain barrier (BBB) penetrating peptide comprises SAQLNTTKPI (SEQ ID NO: 1).
  • BBB blood brain barrier
  • CNS central nervous system
  • CNS central nervous system
  • the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements--or, as appropriate, equivalents thereof--and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.
  • a “comparison window” refers to a segment of any one of the number of contiguous positions (e.g., least about 10 to about 100, about 20 to about 75, about 30 to about 50, 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250) in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • a comparison window is the entire length of one or both of two aligned sequences.
  • two sequences being compared comprise different lengths, and the comparison window is the entire length of the longer or the shorter of the two sequences.
  • the comparison window includes the entire length of the shorter of the two sequences. In embodiments relating to two sequences of different lengths, the comparison window includes the entire length of the longer of the two sequences.
  • “Diagnostic” or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”).
  • false negatives Diseased individuals not detected by the assay are “false negatives.” Subjects who 13 158458212.1 are not diseased and who test negative in the assay, are termed “true negatives.”
  • the “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • predisposition as used herein means that a subject does not currently present with the dysfunction, but is liable to be affected by the dysfunction in time. Methods of diagnosis according to the disclosure are useful to confirm the existence of a dysfunction, or predisposition thereto.
  • microglial activation can refer to processes associated with innate activation or adaptive activation of the microglia. Such activation may include morphological changes of the microglial cells, including shortening of cellular processes and enlargement of their soma, as well as the release of proinflammatory cytokines and chemokines, reactive oxygen and/or nitrogen intermediates, proteinases and complement proteins, and upregulation of cell surface activation antigens.
  • a “neurological disorder” as used herein refers to a disease or disorder which affects the CNS and/or which has an etiology in the CNS.
  • exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.
  • the CNS will be understood to include the eye, which is normally sequestered from the rest of the body by the blood-retina barrier.
  • neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy- Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, 14 158458212.1 Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-
  • the terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g., of an entire polypeptide sequence or an individual domain thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • a specified region e.g., of an entire polypeptide sequence or an individual domain thereof
  • two sequences are 100% identical. 15 158458212.1 In embodiments, two sequences are 100% identical over the entire length of one of the sequences (e.g., the shorter of the two sequences where the sequences have different lengths).
  • identity may refer to the complement of a test sequence. In embodiments, the identity exists over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length.
  • the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250 or more amino acids or nucleotides in length.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol.
  • BLAST and BLAST 2.0 may be used, with the parameters described herein, to determine percent sequence identity for nucleic acids and proteins.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI), as is known in the art.
  • An exemplary BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of 16 158458212.1 matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the NCBI BLASTN or BLASTP program is used to align sequences.
  • the BLASTN or BLASTP program uses the defaults used by the NCBI.
  • the BLASTN program (for nucleotide sequences) uses as defaults: a word size (W) of 28; an expectation threshold (E) of 10; max matches in a query range set to 0; match/mismatch scores of 1,-2; linear gap costs; the filter for low complexity regions used; and mask for lookup table only used.
  • the BLASTP program (for amino acid sequences) uses as defaults: a word size (W) of 3; an expectation threshold (E) of 10; max matches in a query range set to 0; the BLOSUM62 matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)); gap costs of existence: 11 and extension: 1; and conditional compositional score matrix adjustment.
  • W word size
  • E expectation threshold
  • max max matches in a query range set to 0
  • the BLOSUM62 matrix see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)
  • gap costs of existence 11 and extension: 1
  • conditional compositional score matrix adjustment typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • recombinant adeno-associated virus AAV
  • rAAV vector a recombinantly-produced AAV or AAV particle that comprises a polynucleotide sequence not of AAV origin (e.g., a polynucleotide comprising a transgene, which may be operably linked to one or more enhancer and/or promoters) to be delivered into a cell, either in vivo, ex vivo, or in vitro.
  • Non-naturally occurring (e.g., chimeric) capsids may be used in the rAAVs.
  • the terms “specifically binds to”, “specific for”, “targets” and related grammatical variants refer to that binding which occurs between such paired species as enzyme/substrate, receptor/agonist, antibody/antigen, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions.
  • the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions.
  • “specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction.
  • the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.
  • an antibody typically binds to a single epitope and to no other epitope within the family of proteins.
  • specific binding between an antigen and an antibody will have a binding affinity of at least 10 -6 M.
  • the antigen and antibody will bind with affinities of at least 10 -7 M, 10 -8 M to 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M.
  • affinities of at least 10 -7 M, 10 -8 M to 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M.
  • the terms “specific binding” or “specifically binding” when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., the epitope) on the protein.
  • a “therapeutic gene,” “prophylactic gene,” “target polynucleotide,” “transgene,” “gene of interest,” “exogenous gene” and the like generally refer to a gene or genes to be transferred using a vector.
  • rAAV vector which vector is flanked by inverted terminal repeat (ITR) regions and thus can be replicated and encapsidated into rAAV particles.
  • ITR inverted terminal repeat
  • Target polynucleotides can be used to generate rAAV vectors for a number of different applications.
  • polynucleotides include, but are not limited to: (i) polynucleotides encoding proteins useful in other forms of gene therapy to relieve deficiencies caused by missing, defective or sub-optimal levels of a structural protein or enzyme; (ii) polynucleotides that are transcribed into anti-sense molecules; (iii) polynucleotides that are transcribed into decoys that bind transcription or translation factors; (iv) polynucleotides that encode cellular modulators such as cytokines; (v) polynucleotides that can make recipient cells susceptible to specific drugs, such as the herpes virus thymidine kinase gene; (vi) polynucleotides for cancer therapy, such as E1A tumor suppressor genes or p53 tumor suppressor genes for the treatment of various cancers; and (vii) polynucleotides for gene editing (e.g., CRISPR).
  • CRISPR CRISPR
  • the transgene in a recipient host cell it is in one embodiment operably linked to a promoter, either its own or a heterologous promoter.
  • a promoter either its own or a heterologous promoter.
  • suitable promoters are known in the art, the choice of which depends on the desired level of expression of the target polynucleotide, whether one desires 18 158458212.1 constitutive expression, inducible expression, cell-specific or tissue-specific expression, etc.
  • the rAAV vector may also contain a selectable marker.
  • the term “variant,” when used in the context of a polynucleotide sequence may encompass a polynucleotide sequence related to a wild type gene.
  • This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the disclosure are variants of wild type gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered.
  • Any given natural or recombinant gene may have none, one, or many allelic forms.
  • Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FIGS.1A, 1B demonstrate the AAV serotype screening for infectivity in human ad monkey primary microglia identified 8 AAV serotypes with >80% transduction efficiency at MOI of 10 5 vg/cell (>90% efficiency at MOI 10 6 vg/cell, data not shown).
  • Cells in 96-well plate (5x10 3 /well) were infected with indicated AAV carrying CMV-EGFP (10 8 vg per well for 3 wells.
  • FIGS.2A- 2C is a diagram of AAV-BM generation and summary of functional test in vitro.
  • FIG.2A Reported peptides from BBB-crossing serotypes (AAV-B) were inserted into the heparan sulfate (HS) binding sites (R585-R588) of AAV2 CAP VP1 and underlined within the 19 158458212.1 colored inserted amino acid sequences.
  • FIG.2B Microglia-specific peptide (highlighted red) was inserted into the 455-loop of AAV-PhP.eB.
  • FIG.2C Transduction efficiency of AAV-BM in various cell types in vitro.
  • FIG.3 demonstrates that R2-MAC shows highest packaging titer but all tested AAV- BM lost various degrees of transduction efficiency in HEK293T cells.
  • Cells in 96-well were transfected with helper vector, transfer vector TP1152 (scAAV-CB-EGFP) and indicated serotype vectors.
  • TP1152 transfer vector
  • 10 ⁇ l of crude supernatant was used to infect HEK293T cells (1x10 4 /well of 96-well).
  • Two clones of each AAV-BM were sequenced and tested for the functional packaging. The number (red) indicates transduction unit (TU/ml).
  • FIG.4 demonstrates that R2-MAC shows almost similar efficiency in transducing mouse neural stem cells (NSC) to the original AAV2-RC serotype but the other three AAV-BM lost the transduction compared with the original AAV serotype (AAV2 and PhP-eB).
  • HEK293T cells in 96-well were transfected with helper vector, transfer vector TP1921 (AAV-Cre-CD63-MG1MG2- MCP) and indicated serotype vector.
  • FIG.5 demonstrates that R2-MAC transduces mouse primary microglia but the other three AAV-BM does not.
  • HEK293T cells in 96-well were transfected with helper vector, transfer vector TP1921 (AAV-Cre-CD63-MG1MG2-MCP) and indicated serotype vector.
  • FIG.6 demonstrates that all the tested AAV-BM can transduce mouse fibroblast from AI14 mice.
  • HEK293T cells in 96-well were transfected with helper vector, transfer vector TP1921 (AAV-Cre-CD63-MG1MG2-MCP) and indicated serotype vector.
  • FIGS.7A-7F demonstrate that R2-MAC shows stronger transduction efficiency than AAV2 (normalized by MOI) in human primary microglia to the original AAV2-RC serotype but the other three AAV-BM lost the transduction.
  • HEK293T cells in 10-cm dish were transfected 20 158458212.1 with helper vector, transfer vector TP1152 (scAAV-CB-GFP, FIGS.7A-7E) or TP1987 (scAAV- CD68-135-UTR-EGFP, FIG.7F) and indicated serotype vector.
  • transfer vector TP1152 scAAV-CB-GFP, FIGS.7A-7E
  • TP1987 scAAV- CD68-135-UTR-EGFP, FIG.7F
  • serotype vector indicated serotype vector.
  • PEG Polyethylene glycol
  • Chloroform purification and buffer exchange via ultrafiltration (30KD) were performed.1 ⁇ l was used to infect human primary microglia (1x10 4 /well of 96-well).
  • FIGS.8A-8F demonstrate that R2-MAC shows stronger transduction efficiency than AAV2 (normalized by moi) in human primary microglia to the original AAV2-RC serotype but the other three AAV-BM lost the transduction.
  • HEK293T cells in 10-cm dish were transfected with helper vector, transfer vector TP1152 (scAAV-CB-GFP, FIGS.8A-8E) or TP1987 (scAAV- CD68-135-UTR-EGFP, FIG.8F) and indicated serotype vector.
  • TP1152 scAAV-CB-GFP
  • TP1987 scAAV- CD68-135-UTR-EGFP
  • FIGS.9A-9D demonstrate that R2-MAC shows high transduction efficiency in mouse NSC to AAV-DJ8.
  • HEK293T cells in 10-cm dish were transfected with helper vector, transfer vector TP1987 (scAAV-hCD68-135-UTR-EGFP) and indicated serotype vectors AAV2-MAC and AAV-DJ8.
  • FIG.10 demonstrates that R2-MAC crosses BBB and transduces microglia. P23 mice were injected via jugular vein with purified AAV-R2-MAC carrying hCD68-135-UTR-eGFP (2e10 vg per animal).
  • FIGS.12A-12D demonstrate that R2-MAC shows moderate transduction efficiency in mouse neural stem cells (NSC) compared to AAV-DJ8.
  • HEK293T cells in 1x10-cm dish were transfected with helper vector, transfer vector TP1960 (AAV-Cre-GFP-CD63-MG1MG2-MCP) and indicated serotype vectors AAV2-MAC and AAV-DJ8.
  • transfer vector TP1960 AAV-Cre-GFP-CD63-MG1MG2-MCP
  • serotype vectors AAV2-MAC and AAV-DJ8.
  • PEG/Chloroform purification and buffer exchange via ultrafiltration (30KD) were performed.1 and 10 ⁇ l of purified virus (in 1x DPBS) was used to infect mouse NSC (1x10 4 /well of 96-well).
  • FIGS.13A-13F demonstrate that intracranial microinjection of AAV-R2-MAC induce effective delivery of Cre to microglia (arrows) and neurons but DJ8 transduced only neurons.
  • R2-MAC FIGS.13A-13D
  • AAV-DJ8 FIGS.13E, 13F
  • AAV-CMV-Cre TP1960
  • FIGS.14A-14D demonstrate that an I.V. injection of AAV-R2-MAC can cross BBB and transduce microglia (white arrow), neurons and microglia-like (red arrow).
  • R2-MAC FIGS. 14A-14C
  • AAV-DJ8 FIG.14D
  • AAV-CMV-Cre TP1960
  • FIGS.15A-15D demonstrate that R2-MAC shows strong transduction efficiency in mouse neural stem cells (NSC).
  • NSC mouse neural stem cells
  • HEK293T cells in 3x10-cm dishes were transfected with helper vector, TP1989 (R2-MAC) and indicated transfer vectors.
  • PEG/Chloroform purification and buffer exchange via ultrafiltration (30KD) were performed.1 and 9 ⁇ l of purified virus (in 1x DPBS) was used to infect mouse NSC (1x10 4 /well of 96-well).
  • confocal images were taken at 10x objective.
  • FIG.16 demonstrates that the higher titer of AAV-R2-MAC-CMV-Cre induce extensive transduction throughout the brain, mainly in microglia-like cells (white arrows), neurons, astrocytes based on morphology.
  • HEK293T cells in 3x10-cm dishes were transfected with helper vector, TP1989 (R2-MAC) and TP1921 transfer vector (AAV-CMV-Cre-CD63- MG1MG2-MCP).
  • helper vector TP1989 (R2-MAC)
  • AAV-CMV-Cre-CD63- MG1MG2-MCP TP1921 transfer vector
  • FIG.17 demonstrates that AAV-R2-MAC-CMV-Cre also induces transduction of macrophage in various organs.
  • Various organs of the same animals as described in FIG.16 were collected for cryosection and tdTomato positive cells without immunostaining were imaged under confocal microscope.
  • FIG.18 demonstrates that AAV-R2-MAC-CMV-Cre induce transduction throughout the brain, in microglia-like cells (white arrows), neurons, astrocytes based on morphology.
  • FIG.19 demonstrates that extensive tdT expression throughout the brain was not colocalized with IBA1 expression.
  • FIG.20 demonstrates that higher titer of AAV-R2-MAC-hHexB-134-UTR-eGFP induces effective transduction in microglia-like cells.
  • HEK293T cells in 3x10-cm dishes were transfected with helper vector, TP1989 (R2-MAC) and TP1986 transfer vector (scAAV-hHexB- 134-UTR-eGFP).
  • helper vector TP1989
  • TP1986 transfer vector scAAV-hHexB- 134-UTR-eGFP.
  • PEG/Chloroform purification and buffer exchange via ultrafiltration (30K) were performed.
  • Around 50 ⁇ l of purified virus (9.6e13 vg/kg) was injected intravenously of adult wildtype mice (Tg26Q115, 25.9g, male, 3 months old).
  • animals were perfused with 4% PAF and brain sections were immunostained with Chicken anti-GFP and Rabbit anti-GFP antibodies.
  • Scale bar 100 ⁇ m.
  • FIG.21 demonstrates that inclusion of miRNA 9 target (miR9T) in AAV-R2-MAC- hHexB-134-UTR-eGFP virus induces specific transgene expression in microglia but not neural stem cells (NSC).
  • HEK293T cells in 6-well plate were transfected with helper vector, TP1989 23 158458212.1 (R2-MAC) and indicated transfer vector.
  • R2-MAC neural stem cells
  • FIG.22 demonstrates that inclusion of miRNA 9 target (miR9T) in AAV-PhP.eB- hHexB-134-UTR-eGFP virus induces specific transgene expression in microglia but not neural stem cells (NSC).
  • HEK293T cells in 6-well plate were transfected with helper vector, TP1190 (AAV-PhP.eB) and indicated transfer vector.
  • helper vector AAV-PhP.eB
  • TP1190 AAV-PhP.eB
  • FIG.23 demonstrates that AAV2 mutations at 4YF (Tyrosine to Phenylalanine) plus TV (Threonine to Valine), designated R2e for enhanced AAV2, robustly increase transgene expression in mouse NSC and human microglia but miR9T addition illuminates microglia expression.
  • HEK293T cells in a 6-well plate were transfected with helper vector, indicated capsids and transfer vectors.
  • FIGS.24A-24C demonstrate that R2-MAC mutations at 4YF plus TV (R2e-MAC) robustly increase transgene expression with miR9T in human primary microglia.
  • FIG.24A Combined mutation (4YF/TV) significantly improved the transduction efficiency.
  • FIG.24B R2e-MAC mutants showed similar transduction efficiency to R2e mutant.
  • FIGS.25A-25E show that the 4x repeated miRNA-9 target site (miR9T) was added downstream of WPRE3 to illuminate microglia expression.
  • FIG.25A Mouse primary microglia validated by immunocytochemistry with anti-IBA1 antibody.
  • FIG.25B R2-MAC-4YF/TV efficient delivery of scAAV-HexB-UTR-EGFP-miR9T-BGH (TP2018) to mouse microglia with 24 158458212.1 or without miR9T.
  • FIG.25C Diagram of the improved Cre (iCre)-mediated LoxP recombination to induce tdTomato (tdT) expression in Ai14 transgenic mice.
  • R2-MAC-4YF/TV increases transgene expression in mouse NSC and human microglia without miR9T (TP2057: scAAV-hCD68-iCre-3xHA-WPRE3-BGH) but miR9T addition illuminates microglia expression (TP2058: scAAV-hCD68-iCre-3xHA-WPRE3-miR9T-BGH).
  • FIG.26 is a schematic representation showing the screening of AAV-B-bc-Library in mouse microglia cultured from LSL-tdT reporter mice (Ai14) identified 7 AAV-B serotypes (highlighted red) that can transduce microglia at the efficiency of >50 positive cells per well of 96-well plate.
  • Four barcoded AAV transfer vectors with microglia-specific promoter (duplicate) and miR9T were packaged into selected AAV-B at a large scale.
  • Viruses were purified by ultracentrifugation and titer was quantified via qPCR assay.
  • Microglia (1e4 cells/well) were infected with 1 ⁇ l of purified AAV-B virus.
  • FIG.27 shows representative images for AAV-B-bc-Library transduction in mouse microglia cultured from LSL-tdT reporter mice (Ai14).
  • AAV transfer vectors with microglia-specific promoter (duplicate) and miR9T were packaged into selected AAV-B at a large scale.
  • Viruses were purified by ultracentrifugation and titer was quantified via qPCR assay.
  • Microglia (1e4 cells/well) were infected with 1 ul of purified AAV-B virus.
  • FIGS.28A-28D demonstrate that addition of miR9T to barcoded (bc) AAV-B library induces specific transgene expression in microglia in vivo.
  • FIG.28A Brain injection of mixed 8 AAV serotype (0.3-2.5e+10 GC, 2 ⁇ l/site) carrying 4 bc iCre-miR9T vectors in LSL-tdT mice (M, 3 months old) induces microglia tdT expression in the injection sites but not contralateral sites.
  • FIGS.28B-28D Representative images in other brain regions showing transgenes mainly in microglia but also some neuron-like cells.
  • brain sections were immunostained with anti-GFAP and anti-RFP (FIG.28A) or anti-Cre (FIGS.28B-28D) antibodies, and confocal images were taken.3V, third ventricles.
  • Scale bar 100 ⁇ m. 25 158458212.1
  • FIGS.29A-29D show that the fluorescent immunohistochemistry verifies the specificity of tdT positive cells.
  • FIGS.30A-30B demonstrate that brain injection of individual barcoded (bc) AAV-B induces specific iCre transduction in microglia in vivo.
  • FIG.31 shows a further illustration of microglia transduction by AAV-DJ8 as described in FIGS.30A-30B.
  • FIG.32 demonstrates that systemic (i.v., 50 ⁇ l) injection of individual barcoded (bc) AAV-B induces specific iCre transduction in microglia in vivo.
  • AAV- GTX (TCP101, F, 9 months old, 25.7g, 4.71e12 GC), AAV-1RX (TCP100, F, 9-month old, 34.8g, 3.55e12GC), AAV-PhP-C2 (TCP102, F, 9 month old, 29g, 3.98e12GC), AAVv66 (TCP105, F, 9 months old, 32.5g, 3.45e12GC), AAV-B1 (TCP109, F, 9 months old, 25.4g, 2.145e12GC), AAV-Rh39 (TCP115, F, 8 month old, 20.5g, 3.65e12GC), AAV-cc47 (TCP122, M, 4 months old, 32.2g, 4.44e12GC) and AAV1_P5 (TCP127, F, 4 months old, 23.2g, 3.85e12GC) in LSL-tdT mice.
  • AAV-1RX TCP100, F, 9-month old, 34.8g, 3.55e12GC
  • FIGS.33A-33D demonstrate that systemic (i.v., 150 ⁇ l) injection of individual barcoded (bc) AAV-B induces specific iCre transduction in microglia in vivo.
  • Jugular vein 26 158458212.1 injection of AAVRh10 (TCP107, F, 7 months old, 23.2g, 6.435e12 GC), AAV-Pal2 (TCP114, F, 6 months old, 23g, 1.251e13 GC), AAV6-TM (TCP104, F, 7-month old, 26g, 1.065e13GC) and AAV-HSC16 (TCP108, F, 7-month old, 24.2g, 1.485e13GC) in LSL-tdT mice.
  • TCP107, F, 7 months old, 23.2g, 6.435e12 GC AAV-Pal2
  • AAV6-TM (TCP104, F, 7-month old, 26g, 1.065e13GC)
  • AAV-HSC16 (TCP108, F, 7-month old, 24.2g, 1.485e13GC) in LSL-tdT mice.
  • brain sections were immunostained with anti-RFP (tdT) and anti-GF
  • FIG.34 demonstrates that AAV-CPP21-iCre brain injection induces expression of tdT mainly in neurons but rarely in astrocytes and microglia.
  • Mouse brain stereotactic injection of AAV-CPP21-hCD68-iCre-3xHA-WPRE3 viruses (6.4e+9 GC, 2 ⁇ l/site).
  • brain sections were immunostained with anti-GFAP (astrocytes) and anti-IBA1 (microglia) antibodies, and confocal images were taken.
  • FIG.35 demonstrates that large scale of packaging and purification of R2e-MAC with different transfer vector retain high transduction in microglia.
  • FIGS.36A-36B demonstrate that systemic (i.v., 150 ⁇ l) injection of AAVone-R2e- MAC-scAAV-CMV-EGFP (3.66e13GC/ml) in C57BL6 mice (22g, male) induced extensive transduction mainly in microglia-like cells and rarely in astrocytes and neurons.
  • FIG.37 is a series of representative confocal Z-stack images demonstrating extensive transduction in microglia-like cells as well as some neurons and astrocytes after i.v. injection as described in FIGS.36A, 36B.
  • the inset of hippocampus shows colocalization of microglia-like cells with IBA1+ cells in most cases (white arrows). Red arrow shows astrocyte colocalization.
  • FIG.38 is a series of representative confocal Z-stack images showing extensive transduction in microglia-like cells as well as some neurons and astrocytes in pre-cortex and olfactory tubercle after i.v. injection as described in FIGS.36A, 36B.
  • White arrows show the colocalization of eGFP-labeled microglia-like cells with IBA1+ cells in most cases.
  • Red arrow 27 158458212.1 shows astrocyte colocalization. Maximized images from 12-14 Z-stacks at interval thickness of 1 ⁇ m.
  • FIG.39 is a series of representative confocal Z-stack images showing extensive transduction in microglia-like cells as well as some neurons and astrocytes in amygdala and lateral striatum after i.v. injection as described in FIGS.36A-36B.
  • White arrows show the colocalization of eGFP-labeled microglia-like cells with IBA1+ cells in most cases.
  • Red arrow shows astrocyte colocalization. Maximized images from 12-14 Z-stacks at interval thickness of 1 ⁇ m.
  • FIG.40 shows the AAV-R2-MAC sequence (SEQ ID NO: 2) and the plasmid encoding AAV-R2-MAC.
  • FIG.40 shows the AAV-R2-MAC sequence (SEQ ID NO: 2) and the plasmid encoding AAV-R2-MAC.
  • FIG. 41 shows the R2-MAC, REP amino acid sequence (SEQ ID NO: 3) and the R2- MAC, VP1(SAQ-MAC) protein (SEQ ID NO: 4).
  • FIG.42 shows the AAV-R2e-MAC nucleotide sequence (enhanced version of R2-MAC with 4 YF mutations and 1 TV mutation; SEQ ID NO: 5) and the plasmid encoding AAV-R2e- MAC.
  • FIG.43 shows the R2e-MAC, REP amino acid sequence (SEQ ID NO: 6) and the R2e- MAC, VP1 (4YF/TV and SAQ-MAC) protein (SEQ ID NO: 7).
  • FIG. 42 shows the AAV-R2e-MAC nucleotide sequence (enhanced version of R2-MAC with 4 YF mutations and 1 TV mutation; SEQ ID NO: 5) and the plasmid encoding AAV-R2e- MAC.
  • FIG.43 shows the R2e-
  • FIG. 44 shows the nucleic acid sequence of TP2018 (ITR-pdsAAV-hHexB-134-UTR- EGFP-4xmiR9T-BGH-ITR; SEQ ID NO: 8) and the TP2018 plasmid.
  • FIG. 45 the AAV barcode reporter vector (ITR-pDsAAV-hHexB134-UTR-NLS-iCre- HA-WPRE3-bc25-miR9T-bGH-ITR; SEQ ID NO: 9) nucleotide sequence and the plasmid encoding the AAV barcode reporter.
  • FIG. 45 the AAV barcode reporter vector (ITR-pDsAAV-hHexB134-UTR-NLS-iCre- HA-WPRE3-bc25-miR9T-bGH-ITR; SEQ ID NO: 9) nucleotide sequence and the plasmid encoding the AAV barcode reporter.
  • AAV barcode reporter vector pDsAAV-hCD68-135-UTR-NLS-iCre-HA- WPRE3-bc25-miR9T-bGH-ITR; SEQ ID NO: 10.
  • AAV barcode reporter vector pDsAAV-hCD68-135-UTR-NLS-iCre-HA- WPRE3-bc25-miR9T-bGH-ITR; SEQ ID NO: 10.
  • this disclosure provides novel AAV vectors for the treatment of HIV- Associated Neurocognitive Disorder (HAND) and other neurological disorders.
  • the AAVs are engineered to specifically target the microglia.
  • Microglia are a population of Central Nervous System (CNS) cells that serve an immune function. According to Lopez, et al., (Microglia: The Real Foe in HIV-Associated Neurocognitive Disorders? Biomedicines. 2021 Jul 30;9(8):925. doi: 10.3390/biomedicines9080925.
  • microglia are the principal targets of HIV infection in the CNS. Development of therapies which can (i) cross the Blood-Brain Barrier (BBB), and (ii) specifically target infected microglia will go some way toward treatment of HAND, and will directly address the pathophysiology of the disease, unlike the current standard of care.
  • BBB Blood-Brain Barrier
  • HAND according to a February 2022 study may have a prevalence of up to 42.6% among HIV patients receiving HAART.
  • a 2020 meta-analysis of 5,588 records revealed an estimated 16,145,400 patients suffer with HAND worldwide.
  • This disclosure also provides targeting the root cause of cognitive defects: residual components of the HIV genome in the microglia.
  • the instant disclosure also provides for Adeno-Associated Virus serotype 2 (AAV2) that has been mutated to enforce high level expression of the target genes while at the same time restricting expression to only the microglia.
  • AAV2 Adeno-Associated Virus serotype 2
  • intravenous administration of purified virus will lead to migration to within the CNS space, where individual virus particles will attempt to infect all cell types. Infection will be limited to microglia based upon modifications made to the virus capsid protein made by the investigators. Infection of target cells harboring integrated HIV sequences will then engage the Cas9/CRISPR system which will selectively excise the HIV sequences.
  • Adeno-associated viruses are small, non-pathogenic, ssDNA packaging viruses, capable of infecting a wide range of vertebrate hosts, including humans. AAVs belong to the Parvovirinae subfamily of the Parvoviridae, and Dependoparvovirus genus. As the name implies, they require co-infection with adeno- or herpesviruses as helpers for replication (Cotmore, S. F. et al.
  • ICTV virus taxonomy profile parvoviridae. J. Gen. Virol.100, 367–368 (2019); Buller, R. M. & Rose, J. A. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J. Virol.25, 331–338 (1978); McPherson, R. A., et al., Human cytomegalovirus completely helps adeno-associated virus replication. Virology 147, 217–222 (1985); Weindler, F. W. & Heilbronn, R., A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication. J.
  • AAVs package a 4.7 ⁇ kb genome encoding non-structural (rep), structural (cap), assembly activating (aap), and membrane associated accessory (maap) proteins (Wistuba, A., et al., Intermediates of adeno- associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. J. Virol.69, 5311–5319 (1995); supra, F., Schmidt, K. & Kleinschmidt, J. A.
  • a viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc. Natl Acad. Sci. USA 107, 10220–10225 (2010); Ogden, P. J. et al., AAV capsid fitness landscape reveals a viral gene and enables machine-guided design. Science 366, 1139–1143 (2019)).
  • AAVs have become widely used for gene therapy applications, with several advantages over other viral vectors, including a lower toxicity and the availability of over 150 naturally occurring genotypes and serotypes (Zinn, E. & Vandenberghe, L. H. Adeno-associated virus: fit to serve. Curr. Opin. Virol.8, 90–97 (2014); 30 158458212.1 Gao, G. P.
  • rAAVs Recombinant AAVs
  • GOI gene of interest
  • rAAV-based biologics have been approved: Luxturna by the FDA and EMA (FDA STN#125610; EMEA/H/C/004451), Zolgensma by the FDA (FDA STN #125694), and Glybera by the EMA; and several other products are presently being reviewed (clinicaltrials.gov/) Naso, M. F. et al. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 31, 317–334 (2017)).
  • a gene of interest replaces the natural AAV genome for delivery to tissues or cells to treat a monogenic disease.
  • rAAVs are widely used research tools for transgene expression in tissue culture and preclinical animal models (Kimura, T., et al. Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci. Rep. 9 (2019), doi.org/10.1038/s41598-019- 49624-w)).
  • the VPs are generated through alternative splicing of the mRNA and use of an alternate translational start codon (Srivastava, A et al. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J.
  • the VP3 (59–61 ⁇ kDa, 524–544aa) sequence is shared among all VPs and is referred to as the VP3 common region.
  • VP2 (64–67 ⁇ kDa, 580–601aa) is approximately 57aa longer than VP3 and the VP2 N-terminal region is referred to as the VP1/VP2 common region.
  • VP1 (79–82 ⁇ kDa, 713–738aa) is approximately 137 aa longer than VP2 and this region is called the VP1 unique (VP1u) region.
  • the VP3 common region assembles the icosahedral capsid.
  • the VP1u contains an essential phospholipase A2 (PLA2) enzyme, and VP1u and VP1/VP2 common region contain nuclear localization sequences (NLSs) (Sonntag, F et al. Adeno- associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior 31 158458212.1 to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. J. Virol. 80, 11040–11054 (2006)). These N-terminal extensions of VP1 and VP2 are reported to play crucial roles in endosomal trafficking and escape, nuclear localization, and genome release (Agbandje-McKenna, M.
  • the disclosure provides an adeno-associated virus (AAV) comprising a nucleic acid sequence encoding: (i) a capsid protein comprising a blood brain barrier penetrating peptide, (ii) a microglial specific peptide, (iii) a microglial tropic promoter and (iv) a transgene.
  • AAV adeno-associated virus
  • the AAV further comprises a woodchuck hepatitis virus post- transcriptional response element (WPRE).
  • WPRE woodchuck hepatitis virus post- transcriptional response element
  • Adeno-associated virus is a member of the Parvoviridae family comprises a linear, single-stranded DNA genome of less than about 5,000 nucleotides.
  • AAV requires co-infection with a helper virus (i.e., an adenovirus or a herpes virus), or expression of helper genes, for efficient replication.
  • helper virus i.e., an adenovirus or a herpes virus
  • helper genes for efficient replication.
  • AAV vectors used for administration of therapeutic nucleic acids typically have approximately 96% of the parental genome deleted, such that only the terminal repeats (ITRs), which contain recognition signals for DNA replication and packaging, remain. This eliminates immunologic or toxic side effects due to expression of viral genes.
  • AAV ITRs flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)).
  • the terminal 145 nucleotides are self-complementary and are organized so that an 32 158458212.1 energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication by serving as primers for the cellular DNA polymerase complex.
  • the Rep genes encode the Rep proteins Rep78. Rep68, Rep62, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the p19 promoter.
  • the Rep78 and Rep68 proteins are multifunctional DNA binding proteins that perform helicase and nickase functions during productive replication to allow for the resolution of AAV termini (see. e.g., Im et al., Cell.
  • the AAV vector may be generated using any AAV serotype known in the art. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or nonhuman primate tissues (reviewed in.
  • AAV serotypes have genomic sequences of significant homology at the nucleic acid sequence and amino acid sequence levels, such that different serotypes have an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent and replicate and assemble by practically identical mechanisms.
  • AAV serotypes 1-5 and 7-9 are defined as “true” serotypes, in that they do not efficiently cross-read with neutralizing sera specific for all other existing and characterized serotypes.
  • AAV serotypes 6, 10 also referred to as Rh10), and 11 are considered “variant” serotypes as they do not adhere to the definition of a “true” serotype.
  • AAV serotype 2 (AAV2) has been used extensively for gene therapy due to its lack of pathogenicity, wide range of infectivity, and ability to establish long- term transgene expression (see, e.g., Carter, Hum. Gone Ther., 15:541 (2005). Genome sequences of various AAV serotypes and comparisons thereof are disclosed in, for example, GenBank Accession numbers U89790, J01901, AF043303, and AF085716: Chiorini et al., J.
  • AAV rep and ITR sequences are particularly conserved across most AAV serotypes.
  • Rep78 proteins of AAV2, AAV3A, AAV3B, AAV4, and AAV6 are reportedly about 89-93% identical (see Bantel-Schaal et al., J. Virol., 73(2):939 (1999)).
  • the AAV vector can comprise a mixture of serotypes and thereby be a ⁇ chimeric ⁇ or ⁇ pseudotyped ⁇ AAV vector.
  • a chimeric AAV vector typically comprises AAV capsid proteins derived from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes.
  • a pseudotyped AAV vector comprises one or more ITRs of one AAV serotype packaged into a capsid of another AAV serotype. Chimeric and pseudotyped AAV vectors are further described in, for example, U.S. Pat. No.6,723,551: Flotte, Mol.
  • the AAV comprises one or more mutations in the capsid protein at one or more locations in the capsid protein.
  • the AAV vector is generated using an AAV that infects humans (e.g., AAV2).
  • the AAV vector is generated using an AAV that infects non-human primates, such as, for example, the great apes (e.g., chimpanzees). Old World monkeys (e.g., macaques) and New World monkeys (e.g., marmosets).
  • the AAV vector is generated using an AAV that infects a non-human primate pseudotyped with an AAV that infects humans. Examples of such pseudotyped AAV vectors are disclosed in, e.g., Cearley et al., Molecular Therapy.12:528 (2006).
  • the AAV vector may comprise expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the nucleic acid sequence in a host cell.
  • expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology. Methods in Enzymology. Vol. 185. Academic Press. San Diego. Calif. (1990).
  • the AAV further comprises a woodchuck hepatitis virus post-transcriptional response element (WPRE).
  • WPRE woodchuck hepatitis virus post-transcriptional response element
  • the woodchuck hepatitis virus post-transcriptional response element (WPRE) is thought to promote nuclear export of viral genomic mRNA (Donello JE et al. Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J Virol. 1998 Jun;72(6):5085-92). As a result, the WPRE is often included in viral gene transfer vectors where it can improve viral titer and subsequent transgene expression (Zufferey R et al. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol.1999 Apr;73(4):2886-922).
  • promoters including constitutive, inducible, and repressive promoters, from a variety of different sources are well known in the art.
  • Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources.
  • Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3' or 5' direction).
  • Non limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter.
  • Inducible promoters include, for example, the Tet system (U.S. Pat. Nos.5,464,758 and 5,814.618), the Ecdysone inducible system (No et al., Proc. Natl. Acad.
  • AAV vectors are produced using well characterized plasmids.
  • human embryonic kidney 293T cells are transfected with one of the transgene specific plasmids and another plasmid containing the adenovirus helper and AAV rep and cap genes. After 72 hours, the cells are harvested, and the vector is released from the cells by five freeze/thaw cycles. Subsequent centrifugation and berizonase treatment remove cellular debris and unencapsidated DNA. Iodixanol gradients and ion exchange columns may be used to further purify each AAV vector. Next the purified vector is concentrated by a size exclusion centrifuge spin column to the required concentration.
  • the present disclosure provides AAV capsid proteins comprising at least one mutation (i.e., a modification, which can be a substitution or an insertion or a deletion) in the amino acid sequence and virus capsids and virus vectors comprising the modified AAV capsid protein.
  • the modifications at the one or more amino acid positions described can confer one or more desirable properties to virus vectors comprising the modified AAV capsid protein including without limitation: (i) enhancing the penetration of the blood-brain barrier; (ii) enhancing tropism to specifically target a desired cell, e.g. microglia; (iii) enhancing intracellular trafficking to a target cell nucleus; (iv) enhancing the interactions with target cells; (v) enhancing penetration through the mucus gel layer.
  • the tropism is specific for microglial cells. Any microglial marker can be targeted. See, for example Table 1.
  • the modified AAV capsid protein of the disclosure comprises one or more mutations (i.e., modifications) in the amino acid sequence of the native, capsid protein, such as, for example, a blood brain barrier penetrating. Those skilled in the art will appreciate that for some AAV capsid proteins the corresponding modification will be an insertion and/or a substitution, depending on whether the corresponding amino acid positions are partially or completely present in the virus or, alternatively, are completely absent.
  • a “mutation” or “modification” in an amino acid sequence includes substitutions, insertions and/or deletions, each of which can involve one, two, three, four, five, six, seven, eight, nine, ten or more amino acids.
  • the modification is a substitution.
  • the modification is an insertion e.g., a blood brain barrier penetrating peptide.
  • the modification is a deletion.
  • the modification is a modified amino acid. 36 158458212.1 [00118] It is to be understood that the substitutions and insertions described in the AAV capsid proteins of this disclosure can include substitutions and/or insertions with conservative amino acid residues.
  • Such conservative substitutions are well known in the art and include, e.g., nonpolar amino acids Gly, Ala, Val, Leu, Ile, Met, Phe, Trp and Pro can be substituted for one another; polar amino acids Ser, Thr, Cys, Tyr, Asn and Gln can be substituted for one another; negatively charged amino acids Asp and Glu can be substituted for one another; and positively charged amino acids Lys, Arg and His can be substituted for one another, in any combination.
  • the present disclosure also provides an AAV capsid comprising one or more mutations as well as a virus vector comprising one or more mutations.
  • recombinant AAV genomes comprise, an adeno-associated virus (AAV) comprises a nucleic acid sequence encoding: (i) a capsid protein comprising a blood brain barrier penetrating peptide (the capsid can be derived from pseudotyped AAV or AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ or DJ/8), (ii) a microglial specific peptide, (iii) a microglial tropic promoter, (iv) a transgene, (v) a woodchuck hepatitis virus post-transcriptional response element (WPRE) and one or more AAV ITRs flanking a nucleic acid molecule.
  • AAV adeno-associated virus
  • the capsid protein and microglial specific peptide are related to the AAV serotype.
  • the AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ or DJ/8. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).
  • an AAV transfer vector further comprises a microglial tropic promoter, (iv) a transgene, (v) a woodchuck hepatitis virus post-transcriptional response element (WPRE) and one or more AAV ITRs flanking a nucleic acid molecule.
  • a microglial tropic promoter iv
  • a transgene iv
  • a woodchuck hepatitis virus post-transcriptional response element WPRE
  • AAV ITRs flanking a nucleic acid molecule.
  • the AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV 37 158458212.1 genome ITRs, including, but not limited to, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ or DJ/8.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.
  • Methods of generating a packaging cell comprise creating a cell line that stably expresses all the necessary components for AAV particle production.
  • a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6.
  • the packaging cell line is then infected with a helper virus such as adenovirus.
  • a helper virus such as adenovirus.
  • packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line).
  • packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).
  • an AAV-R2-MAC(AAV2-REP-link-VP1(SAQ-MAC)) nucleotide sequence comprises SEQ ID NO: 2: [00125] ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAG CATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGA GTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGT GGCCGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCC CGGAGGCCCTTTTCTTTGTGCAATTTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGA TTCGCGAAAAACTGATTCAGAAAACTGATTCAGAAAACTGATTCAGAAAACT GGTTCGGTCACAAAGACC
  • Microglial pathology precedes and even drives the development of multiple neurodegenerative conditions. These resident brain immune cells are divergent from the peripherally infiltrated macrophages. Microglial heterogeneity in the brain is especially visible in their morphology and cell density in particular brain structures but also in the expression of cellular markers. This often determines their role in physiology or pathology of brain functioning. Furthermore, due to activation, microglia show a broad spectrum of phenotypes ranging from the pro-inflammatory, potentially cytotoxic M1 to the anti- inflammatory, scavenging, and regenerative M2. [00143] Microglia markers include surface, intracellular (cytosolic proteins, gene transcripts), and released molecules.
  • the general microglia markers can be detected irrespective of current cell phenotype.
  • the most widely used markers are ionized calcium-binding adapter molecule 1 (IBA- 1), cluster of differentiation receptors (CD68, CD11b, CD14, CD45, CD80, and CD115), fractalkine receptor (CX3CR1), ferritin, F4/80, high-affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) and vimentin.
  • IBA-1 calcium-binding adapter molecule 1
  • CD68, CD11b, CD14, CD45, CD80, and CD115 fractalkine receptor
  • ferritin ferritin
  • F4/80 high-affinity immunoglobulin epsilon receptor subunit gamma
  • FCER1G high-affinity immunoglobulin epsilon receptor subunit gamma
  • FCER1G high-affinity immunoglobulin epsilon receptor subunit gamma
  • TMEM119 transmembrane protein 119
  • P2Y12R purinergic receptor
  • ionized calcium allograft inflammation factor nding adapter 1 (AIF-1 reorganization of microglial IBA-1 bi ), microglia response factor cytoskeleton, supporting the molecule 1 (MRF-1), daintain phagocytosis process responsible for the degradation of GM2 HexB ⁇ -hexosaminidase N-acetyl-beta- gangliosides and other molecules s ubunit ⁇ glucosaminidase subunit beta containing terminal N-acetyl hexosamines VIMENTIN vimentin fibroblast intermediate f ilament key controller for microglia activation responsible for iron storage and its Ferritin ferritin homeostasis, which is downregulated in inflammation m aintenance of microglia homeostasis; Sall1 Sal-like protein 1 its inactivation resulted in the conversion of microglia from resting 48 158458212.1 Marker Full name Synonyms Functions tissue macrophages
  • CD44 marker reported to be expressed only by infiltrating cells and not on resident microglia (Bennett et al., 2016). Another study examining the gene transcription 50 158458212.1 of adult microglia compared to peripheral cells has suggested that microglia lack CD169 (Butovsky O., et al. (2012). Modulation of inflammatory monocytes with a unique microRNA- gene signature ameliorates ALS mice. J. Neuroimmunol.253:63 10.1172/JCI62636).
  • Siglec-H was also indicated as a marker for microglia in mice, absent from CNS-associated macrophages and CNS-infiltrating monocytes except for a minor subset of cells (Konishi H., et al. (2017). Siglec-H is a microglia-specific marker that discriminates microglia from CNS-associated macrophages and CNS-infiltrating monocytes. Glia 65, 1927–1943.10.1002/glia.23204 (2017). [00146] Quantitative Markers: In some cases, the quantitative differentiation of markers is conducted.
  • CD45 protein amount can be detected between microglia expressing CD11b + /CD45 low and macrophages expressing CD11b + /CD45 high (Grabert K., et al. (2016). Microglial brain region-dependent diversity and selective regional sensitivities to ageing. Nat. Neurosci.19, 504–516.10.1038/nn.4222).
  • a comparison of different phenotypes of human CNS-resident microglia and peripheral immune cells showed characteristic patterns of markers.
  • CD11b + /CD206 high /CD163 + Three main markers were chosen to distinguish perivascular macrophages (CD11b + /CD206 high /CD163 + ) from resident microglia (CD11b + /CD206 low/ ⁇ / CD163 ⁇ ).
  • CD206 and CD163 are expressed in the activated M2 anti-inflammatory microglial phenotype (Böttcher C., et al. (2019).
  • TGF ⁇ Transforming growth factor ⁇
  • FCRLS Fc receptor-like S, scavenger receptor
  • HexB ⁇ -hexosaminidase subunit ⁇
  • P2Y12R P2Y12R
  • P2Y12R is a metabotropic purinoceptor detecting nucleotides like ATP, being released during injuries. It is considered along with TMEM119 as one of the most specific microglial markers.
  • Microglia may be activated by various factors present in their surroundings and spotted during the “surveilling” process. Those factors may be exogenous, such as pathogen-associated molecular patterns (PAMPs), bacterial LPS, or pathogen genetic material or viruses. Activating signals can also be endogenous, presented by stressed surrounding cells such as danger/damage-associated molecular patterns (DAMPs, for example, nucleotides) and protein aggregates like amyloid ⁇ (A ⁇ ) senile plaques or can be released by other microglial cells and astrocytes. Recognition of harmful vs.
  • PAMPs pathogen-associated molecular patterns
  • DAMPs danger/damage-associated molecular patterns
  • a ⁇ amyloid ⁇
  • microglia e.g., vitronectin receptor (VNR), MER receptor tyrosine kinase (MerTK), CX3CR1, or complement receptor 3 (CR3)].
  • VNR vitronectin receptor
  • MerTK MER receptor tyrosine kinase
  • CX3CR1 CX3CR1, or complement receptor 3 (CR3).
  • Microglia also respond to so-called “eat-me” signals from the target cells.
  • phosphatidylserine or calreticulin present in, e.g., disrupted neuronal membrane, secreted fractalkine or opsonins (growth arrest-specific 6, Gas6), milk fat globule epidermal growth factor 8 (MFG-E8), or complement factors.
  • the target cells can also send “do not eat me” signals protecting them from being phagocytosed by microglia. Those signals are recognized by specific microglial receptors.
  • neuronal CD47 and sialylation are cell-surface proteins recognized by microglial signal regulatory protein ⁇ (SIRP ⁇ ; Barclay A. N., Van den Berg T. K. (2014).
  • the transgene encodes a therapeutic agent. In certain embodiments, the transgene encodes a gene-editing complex.
  • the gene editing complex comprises at least two isolated nucleic acid sequences comprising: (i) a first isolated nucleic acid sequence encoding a first Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a first target sequence in a virus genome, and (ii) a second isolated nucleic acid sequence encoding a second Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a second target sequence in a virus genome.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • gRNA guide RNA
  • the CRISPR-Cas system includes a gene editing complex comprising a CRISPR- associated nuclease, e.g., Cas9, and a guide RNA complementary to a target sequence situated on a DNA strand, such as a target sequence in proviral DNA integrated into a mammalian genome, a receptor used by a virus to infect a cell, e.g., HIV.
  • the gene editing complex can cleave the DNA within the target sequence. This cleavage can in turn cause the introduction of various mutations into the proviral DNA, resulting in inactivation of HIV provirus.
  • the mechanism by which such mutations inactivate the provirus can vary. For example, the mutation can affect proviral replication, and viral gene expression.
  • the mutations may be located in regulatory sequences or structural gene sequences and result in defective production of HIV.
  • the mutation can comprise a deletion.
  • the size of the deletion can vary from a single nucleotide base pair to about 10,000 base pairs.
  • the deletion can include all or substantially all of the integrated retroviral DNA sequence.
  • the deletion can include the entire integrated retroviral DNA sequence.
  • the mutation can comprise an insertion, that is, the addition of one or more nucleotide base pairs to the pro-viral sequence.
  • the size of the inserted sequence also may vary, for example from about one base pair to about 300 nucleotide base pairs.
  • the mutation can comprise a point mutation, that is, the replacement of a single nucleotide with another nucleotide.
  • the CRISPR/Cas system can be a type I, a type II, or a type III system.
  • Non-limiting examples of suitable CRISPR/Cas proteins include Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, saCas9, cjCas9, Cas12a, Cas12f, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, ARMAN 1, ARMAN 4, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, saCas9, cjCas9, Cas12a, Cas12f, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse
  • the Cas9 can be orthologous. Six smaller Cas9 orthologues have been used and reports have shown that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter.
  • Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter.
  • embodiments of the disclosure also encompass CRISPR systems including “enhanced specificity” S. pyogenes Cas9 variants (eSpCas9), which dramatically reduce off target cleavage. These variants are engineered with alanine substitutions to neutralize positively charged sites in a groove that interacts with the non-target strand of DNA.
  • This aim of this modification is to reduce interaction of Cas9 with the non-target strand, thereby encouraging re-hybridization between target and non-target strands.
  • the effect of this modification is a requirement for more stringent Watson-Crick pairing between the gRNA and the target DNA strand, which limits off-target cleavage (Slaymaker, I.M. et al. (2015) DOI:10.1126/science.aad5227).
  • three variants found to have the best cleavage efficiency and fewest off-target effects: SpCas9 (K855A), SpCas9 (K810A/K1003A/R1060A) (a.k.a.
  • eSpCas9 1.0 and SpCas9(K848A/K1003A/R1060A) (a.k.a. eSPCas9 1.1) are employed in the compositions.
  • the disclosure is by no means limited to these variants, and also encompasses all Cas9 variants (Slaymaker, I.M. et al. Science. 2016 Jan 1;351(6268):84-8. doi: 10.1126/science.aad5227. Epub 2015 Dec 1).
  • the present disclosure also includes another type of enhanced specificity Cas9 variant, “high fidelity” spCas9 variants (HF-Cas9).
  • Examples of 54 158458212.1 high-fidelity variants include SpCas9-HF1 (N497A/R661A/Q695A/Q926A), SpCas9-HF2 (N497A/R661A/Q695A/Q926A/D1135E), SpCas9-HF3 (N497A/R661A /Q695A/ Q926A/ L169A), SpCas9-HF4 (N497A/R661A/Q695A/Q926A/Y450A).
  • the term “Cas” is meant to include all Cas molecules comprising variants, mutants, orthologues, high-fidelity variants and the like.
  • the endonuclease is derived from a type II CRISPR/Cas system.
  • the endonuclease is derived from a Cas9 protein and includes Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, saCas9, cjCas9, Cas12a, Cas12f, ARMAN 1, ARMAN 4, mutants, variants, high-fidelity variants, orthologs, analogs, fragments, or combinations thereof.
  • the Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, 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., Acetohalobium arab
  • CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with guide RNAs.
  • CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains.
  • Active DNA-targeting CRISPR-Cas systems use 2 to 4 nucleotide protospacer-adjacent motifs (PAMs) located next to target sequences for self versus non-self discrimination.
  • PARMs nucleotide protospacer-adjacent motifs located next to target sequences for self versus non-self discrimination.
  • ARMAN-1 has a strong ‘NGG’ PAM preference.
  • Cas9 also employs two separate transcripts, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), for RNA-guided DNA cleavage.
  • Embodiments of the disclosure also include a new type of class 2 CRISPR-Cas system found in the genomes of two bacteria recovered from groundwater and sediment samples. This system includes Cas1, Cas2, Cas4 and an approximately ⁇ 980 amino acid protein that is referred to as CasX.
  • the CRISPR arrays associated with each CasX has highly similar repeats (86% identity) of 37 nucleotides (nt), spacers of 33–34 nt, and a putative tracrRNA between the Cas operon and the CRISPR array.
  • Distant homology detection and protein modeling identified a RuvC domain near the CasX C-terminal end, with organization reminiscent of that found in type V CRISPR-Cas systems.
  • CasX protein 630 N-terminal amino acids
  • the rest of the CasX protein showed no detectable similarity to any known protein, suggesting this is a novel class 2 effector.
  • the combination of tracrRNA and separate Cas1, Cas2 and Cas4 proteins is unique among type V systems, and phylogenetic analyses indicate that the Cas1 from the CRISPR-CasX system is distant from those of any other known type V.
  • CasX is considerably smaller than any known type V proteins: 980 aa compared to a typical size of about 1,200 amino acids for Cpf1, C2c1 and C2c3 (Burstein, D. et al., 2017 supra).
  • CasY Another new class 2 Cas protein is encoded in the genomes of certain candidate phyla radiation (CPR) bacteria.
  • CPR phyla radiation
  • CasY This approximately 1,200 amino acid Cas protein, termed CasY, appears to be part of a minimal CRISPR-Cas system that includes Cas1 and a CRISPR array.
  • Most of the 56 158458212.1 CRISPR arrays have unusually short spacers of 17–19 nt, but one system, which lacks Cas1 (CasY.5), has longer spacers (27–29 nt).
  • the CasY molecules comprise CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, mutants, variants, analogs or fragments thereof.
  • the CRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein.
  • the CRISPR/Cas-like protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
  • nuclease domains of the CRISPR/Cas-like protein can be modified, deleted, or inactivated.
  • the CRISPR/Cas-like protein can be truncated to remove domains that are not essential for the function of the fusion protein.
  • the CRISPR/Cas-like protein can also be truncated or modified to optimize the activity of the effector domain of the fusion protein.
  • the CRISPR/Cas-like protein can be derived from a wild type Cas protein or fragment thereof. In other embodiments, the CRISPR/Cas-like protein can be derived from modified Cas proteins.
  • the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein.
  • properties e.g., nuclease activity, affinity, stability, etc.
  • domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.
  • the CRISPR-associated endonuclease can be a sequence from another species, for example, other bacterial species, bacteria genomes and archaea, or other prokaryotic microorganisms.
  • the wild type Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, ARMAN 1, ARMAN 4, sequences can be modified.
  • the nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., “humanized.”
  • a humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in GENBANK accession numbers KM099231.1, GI:669193757; KM099232.1 GI:669193761; or KM099233.1 GI:669193765.
  • the Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, ARMAN 1, ARMAN 4 sequences can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, MA).
  • the Cas9 57 158458212.1 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of GENBANK accession numbers KM099231.1 GI:669193757; KM099232.1 GI:669193761; or KM099233.1 GI:669193765, or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, MA).
  • the wild type Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, ARMAN 1, ARMAN 4, sequences can be a mutated sequence.
  • the Cas9 nuclease can be mutated in the conserved HNH and RuvC domains, which are involved in strand specific cleavage.
  • an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single-stranded breaks, and the subsequent preferential repair through HDR can potentially decrease the frequency of unwanted indel mutations from off-target double-stranded breaks.
  • these variants can have or can include, for example, an amino acid sequence that differs from a wild type by virtue of containing one or more mutations (e.
  • substitution mutations can be a substitution (e.g., a conservative amino acid substitution).
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
  • amino acid sequence can be non-naturally occurring amino acid residues.
  • Naturally 58 158458212.1 occurring amino acid residues include those naturally encoded by the genetic code as well as non- standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration).
  • the present peptides can also include amino acid residues that are modified versions of standard residues (e.g. pyrrolysine can be used in place of lysine and selenocysteine can be used in place of cysteine).
  • Non-naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid and L- cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid.
  • Two nucleic acids or the polypeptides they encode may be described as having a certain degree of identity to one another.
  • a Cas9 protein and a biologically active variant thereof may be described as exhibiting a certain degree of identity. Alignments may be assembled by locating short Cas9 sequences in the Protein Information Research (PIR) site (pir.georgetown.edu), followed by analysis with the “short nearly identical sequences” Basic Local Alignment Search Tool (BLAST) algorithm on the NCBI website (ncbi.nlm.nih.gov/blast).
  • PIR Protein Information Research
  • BLAST Basic Local Alignment Search Tool
  • a percent sequence identity to Cas9 can be determined and the identified variants may be utilized as a CRISPR-associated endonuclease and/or assayed for their efficacy as a pharmaceutical composition.
  • a naturally occurring Cas9 can be the query sequence and a fragment of a Cas9 protein can be the subject sequence.
  • a fragment of a Cas9 protein can be the query sequence and a biologically active variant thereof can be the subject sequence.
  • a query nucleic acid or amino acid sequence can be aligned to one or more subject nucleic acid or amino acid sequences, respectively, using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). See Chenna et al., Nucleic Acids Res.31:3497-3500, 2003.
  • the isolated nucleic acids sequences can be encoded by the same construct with one or more isolated nucleic acids sequences directed toward a first and second 59 158458212.1 retroviral target sequence, and one or more isolated nucleic acids sequences directed toward one or more target sequences.
  • the first and second target sequences comprise one or more nucleic acid sequences in a human immunodeficiency virus (HIV) comprising: long terminal repeat (LTR) nucleic acid sequences, nucleic acid sequences encoding structural proteins, non- structural proteins, or combinations thereof.
  • the sequences encoding structural proteins comprise nucleic acid sequences encoding: Gag, Gag-Pol precursor, Pro (protease), Reverse Transcriptase (RT), integrase (In), Env or combinations thereof
  • sequences encoding non-structural proteins comprise nucleic acid sequences encoding: regulatory proteins, accessory proteins or combinations thereof.
  • the regulatory proteins comprise: Tat, Rev or combinations thereof, and wherein accessory proteins comprise Nef, Vpr, Vpu, Vif or combinations thereof.
  • the disclosure features compositions for use in inactivating a proviral DNA integrated into a host cell, including an isolated nucleic acid sequence encoding a CRISPR- associated endonuclease and one or more isolated nucleic acid sequences encoding one or more gRNAs complementary to a target sequence in HIV or another retrovirus.
  • a second isolated nucleic acid sequence encoding a CRISPR-associated endonuclease and one or more isolated nucleic acid sequences encoding one or more gRNAs complementary to a target sequence encoding a second target virus sequence.
  • the isolated nucleic acid can include one gRNA, two gRNAs, three gRNAs etc. Furthermore, the isolated nucleic acid can include one or more gRNAs complementary to target sequences in the retrovirus and a second isolated nucleic acid can include one or more gRNAs complementary to target virus sequences such as LTR, Gag, pol etc.
  • gRNAs complementary to target virus sequences such as LTR, Gag, pol etc.
  • target virus sequences such as LTR, Gag, pol etc.
  • At least one gRNA is complementary to a target sequence in the integrated retroviral DNA and at least one gRNA is complementary to a target sequence in a gene encoding for at least one receptor used by a retrovirus for attachment and/or infection of a cell.
  • two or more gRNAs are complementary to two or more different target sequences in the integrated retroviral DNA and two or more guide RNAs (gRNAs), are 60 158458212.1 complementary to two or more target sequences in a gene encoding for at least one receptor used by a retrovirus for attachment and/or infection of a cell in vitro or in vivo.
  • the isolated nucleic acid encodes at least one gRNA complementary to a target sequence in the integrated retroviral DNA and at least a first gRNA that is complementary to a first target sequence in a gene encoding for at least one receptor used by a retrovirus for attachment and/or infection of a cell; and a second gRNA that is complementary to a second target sequence in a gene encoding for at least one receptor used by a retrovirus for attachment and/or infection of a cell.
  • the isolated nucleic acid encodes at least a first gRNA that is complementary to a first target sequence in the integrated retroviral DNA and at least a second gRNA that is complementary to a second target sequence in the integrated retroviral DNA. Accordingly, any number and combinations of gRNAs with different target sequences can be used to target desired target sequences.
  • gRNA targets comprise one or more target sequences in an LTR region of an HIV proviral DNA and one or more targets in a structural gene of the HIV proviral DNA; or, one or more targets in a second gene; or, one or more targets in a first gene and one or more targets in a second gene; or, one or more targets in a first gene and one or more targets in a second gene and one or more targets in a third gene; or, one or more targets in a second gene and one or more targets in a third gene or fourth gene; or, any combinations thereof.
  • gRNA targets comprise one or more target sequences in a gene encoding at least one receptor used by a retrovirus for attachment and/or infection of a cell and one or more targets in another gene associated with a viral infection; or, one or more targets in a second gene; or, one or more targets in a first gene and one or more targets in a second gene; or, one or more targets in a first gene and one or more targets in a second gene and one or more targets in a third gene; or, one or more targets in a second gene and one or more targets in a third gene or fourth gene; or, any combinations thereof.
  • a composition for preventing or treating a retroviral infection in vitro or in vivo comprises at least two isolated nucleic acid sequences wherein the first isolated nucleic acid sequences encodes a first Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the 61 158458212.1 gRNA being complementary to a target sequence in the integrated retroviral DNA; the second isolated nucleic acid sequences encodes a second Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a target sequence in a gene encoding for at least one receptor used by a retrovirus for attachment and/or infection of a cell in vitro or in vivo.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • gRNA guide RNA
  • the target sequence comprises one or more nucleic acid sequences in coding and non-coding nucleic acid sequences of the retrovirus genome.
  • the target sequences comprise one or more nucleic acid sequences in HIV comprising: long terminal repeat (LTR) nucleic acid sequences, nucleic acid sequences encoding structural proteins, non-structural proteins or combinations thereof.
  • the sequences encoding structural proteins comprise nucleic acid sequences encoding: Gag, Gag-Pol precursor, Pro (protease), Reverse Transcriptase (RT), integrase (In), Env or combinations thereof.
  • sequences encoding non-structural proteins comprise nucleic acid sequences encoding: regulatory proteins, accessory proteins or combinations thereof.
  • the regulatory proteins comprise: Tat, Rev or combinations thereof.
  • the accessory proteins comprise Nef, Vpr, Vpu, Vif or combinations thereof.
  • the gRNA target sequences comprise one or more target sequences in an LTR region of an HIV proviral DNA and one or more target sequences in a structural gene of the HIV proviral DNA; or, one or more targets in a second gene; or, one or more targets in a first gene and one or more targets in a second gene; or, one or more targets in a first gene and one or more targets in a second gene and one or more targets in a third gene; or, one or more targets in a second gene and one or more targets in a third gene or fourth gene; or, any combinations thereof.
  • the compositions and methods of the present disclosure may include a sequence encoding a guide RNA that is complementary to a target sequence in HIV.
  • the genetic variability of HIV is reflected in the multiple groups and subtypes that have been described.
  • a collection of HIV sequences is compiled in the Los Alamos HIV databases and compendiums (hiv.lanl.gov).
  • the methods and compositions of the disclosure can be applied to HIV from any of those various groups, subtypes, and circulating recombinant forms.
  • a gRNA includes a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease III-aided processing of pre-crRNA.
  • the crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA.
  • Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rd nucleotide from PAM).
  • NVG trinucleotide
  • PAM protospacer adjacent motif
  • the crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion gRNA via a synthetic stem loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex.
  • AAU synthetic stem loop
  • Such gRNA can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6 or H1-promoted RNA expression vector.
  • each gRNA includes a sequence that is complementary to a target sequence in a retrovirus.
  • the exemplary target retrovirus is HIV, but the compositions of the present disclosure are also useful for targeting other retroviruses, such as HIV-2 and simian immunodeficiency virus (SIV)-1.
  • the guide RNA can be a sequence complimentary to a coding or a non-coding sequence (i.e., a target sequence).
  • the guide RNA can be a sequence that is complementary to a HIV long terminal repeat (LTR) region.
  • LTR HIV long terminal repeat
  • Some of the exemplary gRNAs of the present disclosure are complimentary to target sequences in the long terminal repeat (LTR) regions of HIV.
  • the LTRs are subdivided into U3, R and U5 regions. LTRs contain all of the required signals for gene expression and are involved in the integration of a provirus into the genome of a host cell. For example, the basal or core promoter, a core enhancer and a modulatory region is found within U3 while the transactivation response element is found within R.
  • the U5 region includes several sub-regions, for example, TAR or trans-acting responsive element, which is involved in transcriptional activation; Poly A, which is involved in dimerization and genome packaging; PBS or primer binding site; Psi or the packaging signal; DIS or dimer initiation site.
  • gRNA targets comprise one or more target sequences in an LTR region of an HIV proviral DNA and one or more targets in a structural gene of the HIV proviral DNA; or, one or more targets in a second 63 158458212.1 gene; or, one or more targets in a first gene and one or more targets in a second gene; or, one or more targets in a first gene and one or more targets in a second gene and one or more targets in a third gene; or, one or more targets in a second gene and one or more targets in a third gene or fourth gene; or, any combinations thereof.
  • Some of the exemplary gRNAs of the present disclosure target sequences in the coding and non-coding protein coding genome of HIV.
  • gRNAs complementary to LTR target sequences include LTR 1, LTR 2, LTR 3, LTR A, LTR B, LTR B’, LTR C, LTR D, LTR E, LTR F, LTR G, LTR H, LTR I, LTR J, LTR K, LTR L, LTR M, LTR N, LTR O, LTR P, LTR Q, LTR R, LTR S, AND LTR T.
  • gRNAs complementary to Gag target sequences include Gag A, Gag B, Gag C, and Gag D.
  • gRNAs complementary to pol target sequences include Pol A and Pol B.
  • compositions of the present disclosure include these exemplary gRNAs, but are not limited to them, and can include gRNAs complimentary to any suitable target site in the protein coding genes of HIV, including but not limited to those encoding the envelope protein env, the structural protein tat, and the accessory proteins vif, willef (negative factor) vpu (Virus protein U) and tev.
  • Guide RNA sequences according to the present disclosure can be sense or anti-sense sequences.
  • the guide RNA sequence generally includes a proto-spacer adjacent motif (PAM).
  • the sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S.
  • the target DNA typically immediately precedes a 5’-NGG proto-spacer adjacent motif (PAM).
  • PAM proto-spacer adjacent motif
  • the PAM sequence can be AGG, TGG, CGG or GGG.
  • Other Cas9 orthologs may have different PAM specificities.
  • Cas9 from S. thermophilus requires 5’-NNAGAA for CRISPR 1 and 5’-NGGNG for CRISPR 3) and Neiseria meningitidis requires 5’-NNNNGATT).
  • the specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV provirus.
  • the length of the guide RNA sequence can vary from about 20 to about 60 or more nucleotides, for example about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotides.
  • Useful selection methods identify regions having extremely low homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA, include bioinformatic screening using 12-bp+NGG target- 64 158458212.1 selection criteria to exclude off-target human transcriptome or (even rarely) untranslated-genomic sites; avoiding transcription factor binding sites within the HIV LTR promoter (potentially conserved in the host genome); and WGS, Sanger sequencing and SURVEYOR assay, to identify and exclude potential off-target effects.
  • the guide RNA sequence can be configured as a single sequence or as a combination of one or more different sequences, e.g., a multiplex configuration.
  • Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs.
  • Combinations of gRNAs are especially effective when expressed in multiplex fashion, that is, simultaneously in the same cell. In many cases, the combinations produce excision of the HIV provirus extending between the target sites. The excisions are attributable to deletions of sequences between the cleavages induced by the endonuclease at each of the multiple target sites.
  • These combinations pairs of gRNAs with one member being complementary to a target site in an LTR of the retrovirus, and the other member being complementary to a gRNA complementary to a target site in a structural gene of the retrovirus.
  • Exemplary effective combinations include Gag D combined with one of LTR 1, LTR 2, LTR 3, LTR A, LTR B, LTR C, LTR D, LTR E, LTR F, LTR G; LTR H, LTR I, LTR J, LTR K, LTR L, LTR M; LTR N, LTR O, LTR P, LTR Q, LTR R, LTR S, or LTR T.
  • Exemplary effective combinations also include LTR 3 combined with one of LTR-1, Gag A; Gag B; Gag C, Gag D, Pol A, or Pol B. [00187] These are only meant as examples and are not to be construed as limiting the disclosure in any way.
  • the CRISPR endonuclease can be encoded by the same nucleic acid or vector as the guide RNA sequences. Alternatively, or in addition, the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the gRNA sequences or in a separate vector.
  • the gRNA sequences according to the present disclosure can be complementary to either the sense or anti-sense strands of the target sequences. They can include additional 5’ and/or 3’ sequences that may or may not be complementary to a target sequence. They can have less than 100% complementarity to a target sequence, for example 75% complementarity.
  • the gRNA sequences can be employed as a combination of one or more different sequences, e.g., a multiplex 65 158458212.1 configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs.
  • Modified or Mutated Nucleic Acid Sequences In some embodiments, any of the nucleic acid sequences may be modified or derived from a native nucleic acid sequence, for example, by introduction of mutations, deletions, substitutions, modification of nucleobases, backbones and the like.
  • the nucleic acid sequences include the vectors, gene-editing agents, gRNAs, etc.
  • modified nucleic acid sequences envisioned for this disclosure include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • modified oligonucleotides comprise those with phosphorothioate backbones and those with heteroatom backbones, CH2 --NH--O--CH2, CH,-- N(CH3)--O--CH2 [known as a methylene(methylimino) or MMI backbone], CH2 --O--N (CH3)-- CH 2 , CH 2 --N (CH 3 )--N (CH 3 )--CH 2 and O--N (CH 3 )---CH 2 --CH 2 backbones, wherein the native phosphodiester backbone is represented as O--P--O--CH,).
  • nucleic acid sequences having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506), peptide nucleic acid (PNA) backbone wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. Science 1991, 254, 1497).
  • the nucleic acid sequences may also comprise one or more substituted sugar moieties.
  • the nucleic acid sequences may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • the nucleic acid sequences may also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2’ deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 66 158458212.1 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7- deazaguanine, N 6 (6-aminohe
  • nucleic acid sequences of the disclosure involves chemically linking to the nucleic acid sequences one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al. Ann.
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. Tetrahedron Lett.1995, 36, 3651; Shea et al. Nucl. Acids Res.1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al. Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid (Manoharan et al.
  • RNA molecules e.g. crRNA, tracrRNA, gRNA are engineered to comprise one or more modified nucleobases.
  • known modifications of RNA molecules can be found, for example, in Genes VI, Chapter 9 (“Interpreting the Genetic Code”), Lewis, ed.
  • RNA components include the following: 2'-O-methylcytidine; N 4 -methylcytidine; N 4 -2'-O- dimethylcytidine; N 4 - acetylcytidine; 5-methylcytidine; 5,2'-O-dimethylcytidine; 5- 67 158458212.1 hydroxymethylcytidine; 5- formylcytidine; 2'-O-methy1-5-formaylcytidine; 3-methylcytidine; 2- thiocytidine; lysidine; 2'-O- methyluridine; 2-thiouridine; 2-thio-2'-O-methyluridine; 3,2'-O- dimethyluridine; 3-(3-amino-3- carboxypropyl)uridine; 4-thiouridine; ribosyl
  • the isolated nucleic acid molecules of the present disclosure can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends 68 158458212.1 of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR polymerase chain reaction
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3’ to 5’ direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
  • the method represents a solution to the problem of latent HIV reservoirs, a solution which is essential to the treatment and prevention of AIDS and other retroviral diseases.
  • HIV-1 replication in the CNS is initiated from invading monocytes and CD4 + T cells, and then spreads to microglial cells and, arguably, astrocytes within the brain parenchyma.
  • Definitive evidence that HIV replicates in myeloid lineage cells within the CNS comes from the observation that HAD patients harbor macrophage-tropic HIV-1 variants that grow selectively in the CNS (Alvarez- Carbonell D, et al., Cross-talk between microglia and neurons regulates HIV latency. PLoS Pathog.
  • HIV-1 replication in longer-lived cell types in the brain, including microglia, is that virus is depleted more slowly in the cerebrospinal fluid (CSF) than virus in the peripheral circulation after the initiation of therapy.
  • CSF cerebrospinal fluid
  • Minimal viral replication still persists in the CNS, especially in microglia and perivascular macrophages, in part because not all anti-HIV drugs are able to cross the blood-brain barrier with high efficiency.
  • Microglia constitute the first barrier of the innate immune response in the CNS. They constantly survey the brain parenchyma to detect physiological changes and then migrate to regions of damage, where they become activated.
  • microglia Normally, the activation of microglia in response to inflammatory stimuli is characterized by a transition from a resting state (M0 cells) to an activated proinflammatory phenotype (M1 cells). It is generally believed that over-activated microglia exacerbate neuronal injury through the synthesis and secretion of proinflammatory and cytotoxic factors. Therefore, in NeuroHIV, microglia-mediated neuronal injury appears to result from excitotoxicity, which disrupts the intrinsic molecular mechanisms that control ion homeostasis and energy production in neurons (Alvarez-Carbonell D, et al., 2019). [00197] HIV-1 neuropathology arises because of the combined neurotoxic effects of the viral proteins and exaggerated inflammatory responses by microglial cells.
  • the HIV proteins Tat, gp120, Vpr, and Nef can directly induce neuronal damage. Additionally, microglia can contribute to neurodegeneration through the release of cytokines and toxins that damage neurons and astrocytes. The natural control mechanisms preventing over-activation of microglial cells are likely to be impaired as a consequence of HIV infection. HIV-infected microglia respond vigorously to proinflammatory signals and produce an excess of cytokines. Importantly, neuronal dysfunction does not correlate with the number of HIV-infected cells or viral antigens in CNS, but rather with elevated inflammatory cytokine levels.
  • the present disclosure when stably expressed in potential host cells, e.g., microglia reduce or prevent new infection by HIV.
  • the present disclosure also provides a method of treatment to reduce the risk of HIV infection in a mammalian subject at risk for infection.
  • the method includes the steps of determining that a mammalian subject is at risk of HIV infection, administering an effective amount of the previously described pharmaceutical composition, and reducing the risk of HIV infection in the mammalian subject.
  • the pharmaceutical composition includes a vector that provides stable and/or inducible expression of at least one of the previously enumerated. 70 158458212.1 [00199]
  • Pharmaceutical compositions according to the present disclosure can be prepared in a variety of ways known to one of ordinary skill in the art.
  • the nucleic acids and vectors described above can be formulated in compositions for application to cells in tissue culture or for administration to a patient or subject.
  • compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral.
  • topical including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery
  • pulmonary e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal
  • ocular oral or parenteral.
  • Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration.
  • Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump.
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like.
  • compositions which contain, as the active ingredient, nucleic acids and vectors described herein, in combination with one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate.
  • compositions of the disclosure includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.
  • the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container.
  • the excipient when it serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), 71 158458212.1 which acts as a vehicle, carrier or medium for the active ingredient.
  • the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the type of diluent can vary depending upon the intended route of administration.
  • the resulting compositions can include additional agents, such as preservatives.
  • the carrier can be, or can include a lipid-based or polymer-based colloid.
  • the carrier material can be a colloid formulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle.
  • the carrier material can form a capsule, and that material may be a polymer-based colloid.
  • a polymeric, biodegradable microparticle or microcapsule delivery vehicle sized to optimize phagocytosis by phagocytic cells such as macrophages.
  • a polymeric, biodegradable microparticle or microcapsule delivery vehicle sized to optimize phagocytosis by phagocytic cells such as macrophages.
  • PLGA poly-lacto-co-glycolide
  • microparticles approximately 1-10 ⁇ m in diameter can be used.
  • the polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell.
  • a second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro- particle through biodegradation.
  • These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 ⁇ m and preferably larger than 20 ⁇ m).
  • Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods.
  • the nucleic acids can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies, for example antibodies that target cell types that are common latently infected reservoirs of HIV infection, for example, brain macrophages, microglia, astrocytes, and gut- associated lymphoid cells.
  • a molecular complex composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces.
  • Poly-L-lysine binds to a ligand that can bind to a receptor on target cells.
  • Delivery of "naked DNA" i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression.
  • the nucleic acid sequence encoding an isolated nucleic acid sequence comprising a sequence encoding a 72 158458212.1 CRISPR-associated endonuclease and a guide RNA is operatively linked to a promoter or enhancer-promoter combination. Promoters and enhancers are described above.
  • the compositions of the disclosure can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol- modified (PEGylated) low molecular weight LPEI.
  • LPEI high molecular weight linear polyethylenimine
  • the nucleic acids and vectors may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device.
  • the nucleic acids and vectors of the disclosure can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline).
  • a pharmaceutically acceptable excipient or carrier e.g., physiological saline
  • the excipient or carrier is selected on the basis of the mode and route of administration.
  • Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary).
  • the compositions can be formulated as a nanoparticle encapsulating a nucleic acid encoding Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, saCas9, cjCas9, Cas12a, Cas12f, ARMAN 1, ARMAN 4, mutants, variants, high-fidelity variants, orthologs, analogs, fragments, or combinations thereof, and at least one gRNA sequence complementary to a target HIV and/or to a receptor target sequence, such as CCR5 ; or it can include a vector encoding these components.
  • compositions can be formulated as a nanoparticle encapsulating the CRISPR-associated endonuclease the polypeptides encoded by one or more of the nucleic acid compositions of the present disclosure.
  • a subject can be identified using standard clinical tests, for example, immunoassays to detect the presence of HIV antibodies or the HIV polypeptide p24 in the subject’s serum, or through HIV nucleic acid amplification assays.
  • An amount of such a composition provided to the subject that results in a complete resolution of the symptoms of the infection, a decrease in the severity of the symptoms of the infection, or a slowing of the infection’s progression is considered a therapeutically effective amount.
  • the present methods may also include a monitoring step to help optimize dosing and scheduling as well as 73 158458212.1 predict outcome.
  • a monitoring step to help optimize dosing and scheduling as well as 73 158458212.1 predict outcome.
  • the methods can further include the step of determining the nucleic acid sequence of the particular HIV harbored by the patient and then designing the guide RNA to be complementary to those particular sequences.
  • the novel gRNAs provided by the present disclosure greatly enhance the chances of formulating an effective treatment.
  • the gRNAs targeted to nucleic acid sequences encoding a receptor used by a virus to infect a cell would prevent further infection.
  • a subject at risk for having an HIV infection can be, for example, any sexually active individual engaging in unprotected sex, i.e., engaging in sexual activity without the use of a condom; a sexually active individual having another sexually transmitted infection; an intravenous drug user; or an uncircumcised man.
  • a subject at risk for having an HIV infection can be, for example, an individual whose occupation may bring him or her into contact with HIV-infected populations, e.g., healthcare workers or first responders.
  • a subject at risk for having an HIV infection can be, for example, an inmate in a correctional setting or a sex worker, that is, an individual who uses sexual activity for income employment or nonmonetary items such as food, drugs, or shelter.
  • COMBINATION THERAPIES the disclosure features compositions which include therapeutically effective amounts of at least one antiretroviral agent administered sequentially or alternately or in conjunction with a composition for inactivating a proviral DNA integrated into a host cell.
  • This composition comprises an isolated nucleic acid sequence encoding a CRISPR-associated endonuclease and one or more isolated nucleic acid sequences encoding one or more gRNAs complementary to a target sequence in HIV or another retrovirus.
  • the antiretroviral agent comprises viral entry inhibitors, reverse transcriptase inhibitors, protease inhibitors, and immune-based therapeutic agents. 74 158458212.1 [00210]
  • the antiretroviral agent or its prodrug or pharmaceutically acceptable salt can be administered in combination or alternation with another anti-HIV agent and/or a gene-editing agent embodied herein.
  • effective dosages of two or more agents are administered together, whereas during alternation therapy, an effective dosage of each agent is administered serially. The dosage will depend on absorption, inactivation and excretion rates of the drug, as well as other factors known to those of skill in the art.
  • the gene-editing compositions embodied herein are administered to a patient in combination with one or more other anti-viral agents or therapeutics.
  • examples include any molecules that are used for the treatment of a virus and include agents which alleviate any symptoms associated with the virus, for example, anti-pyretic agents, anti-inflammatory agents, chemotherapeutic agents, and the like.
  • An antiviral agent includes, without limitation: antibodies, aptamers, adjuvants, anti-sense oligonucleotides, chemokines, cytokines, immune stimulating agents, immune modulating agents, B-cell modulators, T-cell modulators, NK cell modulators, antigen presenting cell modulators, enzymes, siRNA’s, ribavirin, protease inhibitors, helicase inhibitors, polymerase inhibitors, helicase inhibitors, neuraminidase inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, purine nucleosides, chemokine receptor antagonists, interleukins, or combinations thereof.
  • the gene-editing compositions embodied herein are administered with one or more compositions comprising a therapeutically effective amount of a non-nucleoside reverse transcriptase inhibitor (NNRTI) and/or a nucleoside reverse transcriptase inhibitor (NRTI), analogs, variants or combinations thereof.
  • NNRTI non-nucleoside reverse transcriptase inhibitor
  • NRTI nucleoside reverse transcriptase inhibitor
  • an NNRTI comprises: etravirine, efavirenz, nevirapine, rilpivirine, delavirdine, or nevirapine.
  • an NRTI comprises: lamivudine, zidovudine, emtricitabine, abacavir, zalcitabine, dideoxycytidine, azidothymidine, tenofovir disoproxil fumarate, didanosine (ddI EC), dideoxyinosine, stavudine, abacavir sulfate or combinations thereof.
  • a composition comprises a therapeutically effective amount of at least one NNRTI or a combination of NNRTI’s, analogs, 75 158458212.1 variants or combinations thereof.
  • the NNRTI is rilpivirine.
  • an NRTI comprises: lamivudine, zidovudine, emtricitabine, abacavir, zalcitabine, dideoxycytidine, azidothymidine, tenofovir disoproxil fumarate, didanosine (ddI EC), dideoxyinosine, stavudine, abacavir sulfate or combinations thereof.
  • the composition comprises a therapeutically effective amount of at least one or a combination of NRTI’s, analogs, variants or combinations thereof.
  • KITS also includes a kit including an isolated nucleic acid sequence encoding a CRISPR-associated endonuclease, for example, a Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, saCas9, cjCas9, Cas12a, Cas12f, ARMAN 1, ARMAN 4 endonucleases, and at least one isolated nucleic acid sequence encoding a gRNA complementary to a target sequence in an HIV provirus and at least one isolated nucleic acid sequence encoding a gRNA complementary to a target sequence in a gene or nucleic acid sequence encoding a receptor that is used by a virus to infect a
  • the isolated nucleic acid sequences can be encoded in a vector, such as an expression vector.
  • a vector such as an expression vector.
  • Possible uses of the kit include the treatment or prophylaxis of HIV infection.
  • the kit includes instructions for use, syringes, delivery devices, buffers sterile containers and diluents, or other reagents for required for treatment or prophylaxis.
  • the kit can also include a suitable stabilizer, a carrier molecule, a flavoring, or the like, as appropriate for the intended use.
  • AAV9 and its derivatives such as AAV-PhP.B, AAV-PhP-eB, AAV-F, AAV9-Retro, AAV9-HR, AAV-Cap- MAC or C2, AAV9P31, AAV-B10 and series, AAV-cc47, AAV-Pal, AAV-MDV1A, AAV.CPP.16 or 21, AAV-BI28 and series, as well as AAV8, AAV10 and their derivatives, 76 158458212.1 exhibiting potent capability of crossing BBB in both neonatal and adult animals.
  • AAV-B serotypes have high efficiency to transduce neurons, astrocytes, oligodendrocytes and/or blood vessels.
  • AAV serotypes have been identified for their potential tropism to myeloid cells (AAV-M). For example, early studies showed that AAV2/2 and AAV2/5 can transduce mouse microglia both in vitro and in vivo (local striatum injection) but the transduction efficiency was ⁇ 20%. By screening several AAV serotypes (AAV1, 2, 5, 6, 8, 9, etc.), AAV2/6 was found to be the most effective while AAV9 and the others with less efficiency in transducing microglia in vitro.
  • the triply mutated AAV2/6 exhibits 95% transduction efficiency in cultured primary microglia but achieves 10-80% transduction efficiency in vivo (local injection) in a promoter-dependent manner.
  • Intraspinal injection of 5 AAV serotypes (1, 5, 9, rh10 and hu11) in rats showed 23% transduction efficiency of AAV-rh10 in microglia/macrophages.
  • AAV9 can transduce microglia/macrophages despite of low efficiency.
  • AAV-BM AAV serotype
  • R2-MAC AAV serotype
  • the primary one AAV-BM1 may transduce not only microglia but also non-microglia neural cells such as neurons, astrocytes, oligodendrocytes and endothelial cells. This non-specific targeting may be beneficial in some situations but detrimental in other cases such as the delivery of gene editors.
  • this disclosure also relates to the inclusion of microglia-specific promoter such as hCD68, HexB, CD11b, hIBA1, TMEM119, CX3CR1, as well as miR9T for microglia illumination in the transfer vector.
  • microglia-specific promoter such as hCD68, HexB, CD11b, hIBA1, TMEM119, CX3CR1, as well as miR9T for microglia illumination in the transfer vector.
  • AAV9 and AAV-PhP.eB failed to transduce human and monkey primary microglia.
  • 8 AAV-M serotypes were discovered having >90% transduction efficiency at multiplicities of infection (MOI) of 1 ⁇ 10 6 viral genome (vg)/cell (FIGS. 1A, 1B).
  • MOI multiplicities of infection
  • AAV2 was selected as the initial screening.
  • the AAV2 mutants such as AAV2.7m8 and AAV2- QuadYF showed very high transduction efficiency (99%).
  • AAV-CAP-MAC, AAV-9P31 and 77 158458212.1 AAV-PhP.eB were selected because they were highly efficient at crossing the blood-brain barrier (BBB) at relatively low dosage (1 ⁇ 10 13 vg/kg) in multiple species such as monkeys, mice.
  • BBB blood-brain barrier
  • a BBB-penetrating 9-mer epitopes of these AAV-Bs were inserted into AAV2 capsid VP1 protein at position N587 to generate R2-MAC, R2-9P31 and R2-eB (FIGS.2A-2C).
  • a microglia-specific peptide MG1 was inserted into AAV-PhP.eB at position N452- Q458 (eB-MG1) to explore if MG1 can direct the BBB-crossing AAV-PhP.eB to transduce brain microglia (FIGS.2A-2C).
  • a small scale of AAV packaging (in 96-well plate) was performed using self-complementary AAV transfer vector scAAV-CB-eGFP in a three-plasmid transfection system and found that 4 AAV-BMs had various degrees of reduction in the packaging efficiency compared to the original serotype AAV2 (for R2-MAC, R2-9P31 and R2.eB) or AAV-PhP.eB (for eB-MG1).
  • R2-MAC showed around 1.5-fold reduction while R2-9P31 and R2-eB exhibited about 10-fold reduction as compared with AAV2, while eB- MG1 lost 10-fold transduction compared with AAV-PhP.eB (FIGS.2A-2C, 3).
  • R2- MAC showed very strong transduction (+++++) in mouse neural stem cells (NSC), apparently stronger than AAV2 (+++), while R2-9P31, R2-eB and eB-MG1 had almost no infection (FIG.4).
  • R2-MAC showed the highest transduction in mouse microglia, but all the tested AAV- BMs showed moderate transduction in mouse fibroblast with the original AAV-PhP.eB highest (FIGS. 5, 6). Then, a small scale of AAV packaging (1x10 cm dish) was performed and the transduction efficiency was tested in human primary microglia (FIGS. 7A-7E, 9A-9D). Using universal promoter for the transfer reporter vector (scAAV- CB-eGFP), it was found that R2-MAC showed stronger transduction efficiency in human primary microglia than the original AAV2 serotype but the other three AAV-BMs lost the transduction (FIGS. 7A-7E, 8A-8E).
  • Neonatal mice were selected due to potential higher efficiency to cross BBB.
  • the animals were perfused and all the organs/tissues were collected.
  • Some degree of eGFP expression was 78 158458212.1 observed in brain and other organs in both R2-MAC and AAV-DJ8 groups (FIGS.10, 11), but the transduction efficiency was very low perhaps due to the insufficient dosage.
  • the Cre-mediated recombination in the target cells would dramatically increase the resolution of detecting AAV transduction because the reporter tdTomato expression is driven by universal Rosa26 promoter after Cre recombination even at a very low level of gene delivery.
  • the CMV-Cre-GFP transfer vector was utilized in a parallel study using a small scale of packaging (1x10 cm dish) as described above.
  • a titer of 2.11 ⁇ 10 11 GC/ml for R2-MAC and 18.3 ⁇ 10 11 GC/ml was obtained for AAV-DJ8 in final volume of 150 ⁇ l (FIGS. 12A-12D) and found moderate efficiency of Cre-mediated LoxP recombination in NSCs from AI14 transgenic mice (FIGS.
  • HEK293T cells in 3x10-cm dishes were transfected using another AAV-CMV-Cre transfer vector.
  • the R2- MAC-CMV-Cre virus was purified via PEG/Chloroform precipitation and ultrafiltration.
  • 9 ⁇ l of purified virus (1.5e7 MOI) showed very high transduction efficiency in AI14 mouse NSC.
  • Local injection (3.7e9 vg) in brain induced more transduction of microglia- like cells and neurons (FIGS. 15B-15D).
  • AAV-B BBB- crossing serotypes
  • miR9T inclusion can improve the specificity of R2-MAC to transduce microglia
  • TP1986 scAAV-hHexB-134-UTR-eGFP
  • FIG 21 inclusion of miR9T induced the loss of EGFP expression in mouse NSCs as predicted because non-microglia cells like NSCs endogenously express miR9 that can bind to MiR9T resulting in mRNA degradation.
  • microglia retain the expression of transgene EGFP due to the absence of miR9 (FIG.21). Similar result was observed with AAV-PhP.eB packaging (FIG. 22).
  • This novel vector incorporates several previous technologies: 1) The self-complementary double-stranded AAV transfer vector (scAAV with packaging capacity around 2.4 kb) was selected to increase the transduction and transgene expression; 2) The minimal/essential promoter of microglia-specific gene HexB (135 bp) or hCD68 (135 bp) was used due to its small size, high specificity and pathological upregulation; 3) The SARS-CoV-25’-UTR and the shortest WPRE3 were included to boost the mRNA expression of transgene; 4) The improved Cre (iCre) was chosen to increase the sensitivity of detecting AAV transduction efficiency because single copy of iCre delivered by AAV could remove STOP terminator for strong tdT expression; and 5) Most importantly, the 4x repeated miRNA-9 target site (miR9T) was added downstream of WPRE3 to illuminate microglia expression (due to miR9 absence) but conceal transgene expression in non-microglia cells that endogenously express
  • HexB promoter and miR9T were validated using pscAAV-HexB-GFP-miR9T vector packaged with AAV2, AAV-R2-MAC and AAV-PhP.eB in human microglia and mouse neural stem cells (FIGS.21, 22, 23 and 24A-24C).
  • the packaging efficiency of pscAAV-HexB- UTR-iCre-WPRE3-miR9T-BGH was assessed by standard 3-plasmid packaging in HEK293T cells with the titer at 1.84e13 GC/ml, comparable to the standard pscAAV-Cb-GFP.
  • FIG.25A In cultured mouse primary microglia (FIG.25A), pscAAV-HexB-GFP-miR9T with AAV-R2-MAC-4YFTV (R2e-MAC) showed high transduction (FIG.25B).
  • AAV-iCre-miR9T In cultured neural stem cells from Ai14 mice, AAV-iCre-miR9T showed very few positive cells even at 1e6 MOI, while AAV-iCre without 81 158458212.1 miR9T exhibited high transduction efficiency in a dose-dependent manner (FIGS. 25C-25D.
  • AAV-iCre-miR9T In cultured mouse microglia, AAV-iCre-miR9T induced dose-dependent transduction as determined by tdT positivity (FIG. 25E).
  • AAV-B in transducing microglia in vivo, we initiated brain stereotactic injections of a mixed AAV-B formulation encompassing eight serotypes (1.34e10 GC each, including AAV1/2, AAV-DJ8, AAV6TM, AAV9, AAVrh.10, AAV-HSC16, AAV-Pal2, and AAV-MDV1A) in adult LSL-tdT mice (Male, 3-month-old, 2 ⁇ l/site, 5 sites).
  • tdT + expression was evident solely in microglia-like cells on the injected side, contrasting with the contralateral side, thus providing evidence of successful transduction by at least one of these eight AAV serotypes, and the miR9T addition to these barcoded transfer vectors facilitated highly specific transgene (iCre) expression in microglia in vivo (FIG. 28A).
  • the transgene iCre was validated by immunostaining with anti-Cre antibody (FIGS.28B-28D). Further verification of tdT expression was achieved through immunostaining with anti-RFP or anti- mCherry antibodies (FIGS. 29A-29D).
  • Re2-MAC also transduced neurons and astrocytes but the transduction efficiency is much lower than the original AAV-CAP-MAC (compared to the original publication).
  • eGFP-labeled microglia-like cells are colocalized with IBA1-labeled cells, indicating that other types of microglia-like cells are present, warranting further characterization with different markers for microglia.

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Abstract

Des compositions qui ciblent des maladies liées à la microglie telles que des maladies infectieuses (par exemple le VIH), des maladies neurodégénératives (par exemple, la maladie d'Alzheimer, la maladie de Parkinson, la maladie de Huntington), des troubles neurodéveloppementaux (par exemple l'autisme, la déficience intellectuelle) et une lésion du SNC (par exemple, TBI, SCI), comprennent des complexes d'édition de gènes. De nouveaux sérotypes d'AAV-BM qui peuvent traverser la barrière hémato-encéphalique et transduire la microglie, et des séquences d'acides nucléiques d'emballage, y compris des complexes d'édition de gènes, des ARNsh, des ARNsg, des miARN et des ARNlnc sont utilisés dans le traitement de maladies neurologiques.
PCT/US2024/029057 2023-05-12 2024-05-13 Virus adéno-associé recombinant de capside chimérique avec pénétration de bbb et ciblage de microglie (aav-bm) Ceased WO2024238448A2 (fr)

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CA3092961A1 (fr) * 2018-03-09 2019-09-12 Avrobio, Inc. Compositions et methodes pour le traitement de la maladie de parkinson
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