WO2022040530A2 - Compositions de virus pour traiter la mucopolysaccharidose ii - Google Patents

Compositions de virus pour traiter la mucopolysaccharidose ii Download PDF

Info

Publication number
WO2022040530A2
WO2022040530A2 PCT/US2021/046907 US2021046907W WO2022040530A2 WO 2022040530 A2 WO2022040530 A2 WO 2022040530A2 US 2021046907 W US2021046907 W US 2021046907W WO 2022040530 A2 WO2022040530 A2 WO 2022040530A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
raav
promoter
further provided
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/046907
Other languages
English (en)
Other versions
WO2022040530A3 (fr
Inventor
Nicholas C. FLYTZANIS
Nicholas S. GOEDEN
Allegra KAUFMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Capsida Inc
Original Assignee
Capsida Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Capsida Inc filed Critical Capsida Inc
Publication of WO2022040530A2 publication Critical patent/WO2022040530A2/fr
Publication of WO2022040530A3 publication Critical patent/WO2022040530A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the invention generally relates to virus compositions for treating mucopolysaccharidosis II.
  • Mucopolysaccharidosis type II (Hunter Syndrome) is a lysosomal storage disease caused by a deficiency in the lysosomal enzyme iduronate-2- sulfatase (I2S). That deficiency allows glycosaminoglycans to build up in tissues causing a variety of symptoms of varying severity. Depending on the severity, resultant health issues can range from behavioral disturbances, joint stiffness, progressive declines in cardiac and pulmonary function from thickening of the heart and airways, physical abnormalities, to death at an early age. Mucopolysaccharidosis type II is caused by a mutation to the iduronate-2- sulfatase (IDS) gene on the X chromosome.
  • IDS iduronate-2- sulfatase
  • adeno-associated viruses are widely used as vectors for gene delivery in therapeutic applications because of their ability to transduce both dividing and nondividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These characteristics make them appealing for applications in therapeutic applications, such as gene therapy.
  • AAV adeno-associated viral
  • compositions and methods of the invention use recombinant adeno-associated viruses (rAAV) to deliver a viral vector comprising a human iduronate-2-sulfatase (IDS) gene encoding a functional iduronate-2- sulfatase enzyme (I2S).
  • IDS human iduronate-2-sulfatase
  • I2S functional iduronate-2- sulfatase enzyme
  • modified rAAVs are used to improve gene delivery and expression and target the central nervous system (CNS) for gene delivery.
  • CNS central nervous system
  • modified rAAVs of the invention may exhibit increased specificity and transduction in the CNS, allowing for systemic delivery thereof with reduced risk of off-target effects.
  • Such modified rAAVs may exhibit specificity engineered into the capsid structure through iterative rounds of selection in non-human primates (NHPs), yielding variants with tropisms having an increased specificity and transduction efficiency in the CNS, and in some cases, a decreased specificity and transduction efficiency in an off-target environment.
  • the rAAVs described herein achieve widespread transduction to the CNS (e.g., CNS cell types or tissues) in a subject upon systemic delivery (e.g., intravenous, intrathecal, intraarterial, intracranial, intraventricular, intracerebroventricular, or subcutaneous).
  • rAAVs may include one or more promoters, regulatory elements, polyadenylations signals, and/or microRNA signals to improve gene expression in the target cells.
  • aspects of the invention may include a recombinant adeno-associated virus (rAAV) comprising a capsid containing an AAV vector comprising a promoter, a human iduronate-2- sulfatase (IDS) sequence comprising SEQ ID NO: 655, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a polyadenylation signal.
  • the promoter may be a CAG synthetic promoter, a CBh synthetic promoter, or a human synapsin I promoter.
  • the promoter may be a CAG synthetic promoter comprising SEQ ID NO: 656. In some embodiments, the promoter may be a CBh synthetic promoter comprising SEQ ID NO: 657. In certain embodiments, the promoter can be a human synapsin I promoter comprising SEQ ID NO: 658.
  • the WPRE may comprise SEQ ID NO: 659.
  • the polyadenylation signal may be selected from the group consisting of a human growth hormone polyadenylation signal (hGH Poly A) and a simian virus 40 polyadenylation signal (SV40 Poly A). In some embodiments, the polyadenylation signal can be hGH PolyA comprising SEQ ID NO: 660 or SV40 PolyA comprising SEQ ID NO: 661.
  • the rAAV may comprise an AAV capsid protein comprising an amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1.
  • the rAAV may comprise AAV9.
  • the AAV capsid protein may comprise a peptide insertion comprising an amino-acid sequence as provided in Tables 1 and 4-30, FIG. 5 and/or Formulas I- XXXIIII.
  • the insertion may be at the residues corresponding to amino acids 588-589 of the AAV9 native sequence of SEQ ID NO: 1.
  • modified capsid proteins for delivery of expression vectors as described herein may comprise engineered specificity in their capsid structure developed through iterative rounds of selection in non-human primates (NHPs) to yield variants with tropisms having an increased specificity and transduction efficiency in the CNS.
  • NEPs non-human primates
  • the rAAV may include a capsid comprising an insertion at amino acid positions 588-589 of SEQ ID NO: 1.
  • the capsid may comprise an insertion at position 588-589 of SEQ ID NO: 1 comprising ISREFYK (SEQ ID NO: 38), EDNLSYV (SEQ ID NO: 77), NLANIPN (SEQ ID NO: 84), LNTTKPI (SEQ ID NO: 241), ENHTKND (SEQ ID NO: 246), GNTTRDY (SEQ ID NO: 255), TNSVKNL (SEQ ID NO: 257), QNSTKLI (SEQ ID NO:264), SNVIKNV (SEQ ID NO: 279) or NVRDLNL (SEQ ID NO: 283).
  • the capsid may comprise both a substitution at amino acid positions 452-458 as well as an insertion at position 588-589 such as ISREFYK (SEQ ID NO: 38), EDNLSYV (SEQ ID NO: 77), NLANIPN (SEQ ID NO: 84), LNTTKPI (SEQ ID NO: 241), -ENHTKND (SEQ ID NO: 246), GNTTRDY (SEQ ID NO: 255), TNSVKNL (SEQ ID NO: 257), QNSTKLI (SEQ ID NO:264), SNVIKNV (SEQ ID NO: 279) or NVRDLNL (SEQ ID NO: 283).
  • ISREFYK SEQ ID NO: 38
  • EDNLSYV SEQ ID NO: 77
  • NLANIPN SEQ ID NO: 84
  • LNTTKPI SEQ ID NO: 241
  • -ENHTKND SEQ ID NO: 246
  • GNTTRDY SEQ ID NO: 255
  • TNSVKNL S
  • the AAV vector may comprise a microRNA signal.
  • the microRNA signal may be miR-183 comprising SEQ ID NO: _.
  • Aspects of the invention may include methods for treating mucopolysaccharidosis type II in a subject. Such methods may include administering to said subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) comprising a capsid containing an AAV vector that may comprise a promoter; a human iduronate-2- sulfatase (IDS) sequence comprising SEQ ID NO: 655; a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and a poly adenylation signal
  • rAAV recombinant adeno-associated virus
  • the therapeutically effective amount of the rAAV may be administered systemically (e.g., intracranial, intraventricular, intracerebroventricular, intravenous, intraarterial, intranasal, intrathecal, intracistemae magna administration, or subcutaneously).
  • the rAAV is administered intrathecally or intracisternally.
  • FIG. 1 shows an exemplary AAV vector for expression of I2S according to certain embodiments.
  • FIG. 2 shows staining against the HA tag fused to hFXN transcripts virally expressed in the macaque brain. Robust and broad expression was achieved by a pool of eight viruses throughout the brain. Stained sections from each coronal block of the brain were imaged in their entirety at a 4x magnification (FIG. 2A). Sub-regions identified within various major brain areas, the four main cortical lobes, hippocampus, caudate, putamen, thalamus and midbrain, were imaged at a lOx magnification across a z-thickness of 25 pm (Fig. 2B).
  • FIG. 3 shows a 3-dimensional scatter plot of the distribution of engineered rAAV sequences in liver, spinal cord or brain tissue after administration of a viral library to marmosets and next-generation sequencing of the variants pulled out from tissue.
  • FIG. 4 shows the result of further refinement of the data in the scatter plot in Fig. 3 focusing on the expression of the sequences that express in the spinal cord.
  • FIG. 5 shows AAV capsid protein insertion amino acid sequences and DNA sequences encoding the amino acid sequences which were found in the non-human primate CNS after two rounds of selection of an engineered AAV library.
  • FIG. 6 shows the expression achieved by the eight AAV variants from the pool in Fig. 1 throughout the macaque brain (Fig. 5A), spinal cord (Fig. 5B) and liver (Fig. 5C).
  • the relative viral genomes and transcript expression levels of each of the barcoded viruses were normalized to those of AAV9 and averaged across two animals.
  • FIG. 7A shows the concentration of I2S enzyme measured in the brain, spinal cord, dorsal root ganglia and liver after intravenous injection of 7.5E+13 vg/kg of a variant [E] or saline control in 8-month old cynomolgus macaques.
  • FIG. 7B shows the I2S enzyme activity measured in the brain, spinal cord, dorsal root ganglia and liver after intravenous injection of 7.5E+13 vg/kg of the variant.
  • FIG. 8 shows AAV variant packaging a human IDS cDNA under control of a ubiquitous CAG promoter normalized I2S enzyme activity in the brain and most peripheral organs in a Hunter syndrome mouse model lacking endogenous I2S.
  • I2S enzyme activity is measured from homogenized tissue using a 2-step assay after intra-cisterna magna injection of 5E+10 vg of the AAV variant.
  • FIG. 9 shows an AAV variant packaging a human IDS cDNA under control of a ubiquitous CAG promoter can normalize glucosaminoglycan (GAG) substrate levels in the brain and most peripheral organs in a Hunter syndrome mouse model. GAGs were measured from homogenized tissue using the Blyscan GAG assay after intra-cisterna magna injection of 5E+10 vg of the variant.
  • GAG glucosaminoglycan
  • compositions and methods of the invention provide rAAVs for the delivery of vectors encoding human I2S useful in the treatment of mucopolysaccharidosis type II (Hunter syndrome).
  • Genes encoding I2S along with optional combinations of promoters, regulatory elements, polyadenylation signals, and miRNA may be included in modified rAAVs having higher efficiency and specificity for transduction in specific cell-types (e.g., cells of the central nervous system such as brain endothelial cells, neurons, and astrocytes).
  • cell-types e.g., cells of the central nervous system such as brain endothelial cells, neurons, and astrocytes.
  • functional I2S can be preferentially expressed in the cells affected by mucopolysaccharidosis type II to alleviate symptoms thereof with diminished off-target effects.
  • vectors of the invention may include a human IDS gene expressing human I2S.
  • vectors comprise an IDS gene comprising the following nucleotide sequence (SEQ ID NO: 655): atgccgccaccccggaccggccgaggccttctctggctgggtctggtttctgagctccgtctgcgtcgccctcggatccgaaacgcaggcc aactcgaccacagatgctctgaacgttcttctcatcatcgtggatgacctgcgccccccctgggctgttatggggataagctggtgaggtcc ccaaatattgaccaactggcatcccacagcctcctcttccagaatgccttttgcgcagcaagcagtgtgtgcgccccc
  • the gene may express I2S comprising the following peptide sequence (SEQ ID NO: 663):
  • the IDS gene may be split between two AAV vectors, the first with 3’ splice donor and the second with a 5’ splice acceptor.
  • concatemers form, which are spliced together to express a full-length IDS gene.
  • the vector may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue/cell specific promoter.
  • the promoter may be CMV promoter, a CMV-P-Actin-intron-P-Globin hybrid promoter (CAG), CBA promoter, FRDA or FXN promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 promoter, GFAP promoter, Hl promoter, U6 promoter, NFL promoter, NFH promoter, SCN8A promoter, or PGK promoter.
  • promoters can be tissue- specific expression elements include, but are not limited to, human elongation factor la-subunit (EFla), immediate-early cytomegalovirus (CMV), chicken P-actin (CBA) and its derivative CAG, the P glucuronidase (GUSB), and ubiquitin C (UBC).
  • EFla human elongation factor la-subunit
  • CMV immediate-early cytomegalovirus
  • CBA chicken P-actin
  • GUSB the P glucuronidase
  • UBC ubiquitin C
  • the vector may include a tissue-specific expression elements for neurons such as, but not limited to, neuronspecific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B- chain (PDGF-P), the synapsin (Syn), the methyl-CpG binding protein 2 (MeCP2), Ca2+/calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2 promoters.
  • NSE neuronspecific enolase
  • PDGF platelet-derived growth factor
  • PDGF-P platelet-derived growth factor B- chain
  • Syn the synapsin
  • MeCP2 methyl-CpG binding protein 2
  • CaMKII Ca2+/calmodulin-dependent protein kinase II
  • mGluR2 metabotropic glutamate receptor 2
  • NFL NFH, n
  • the vector may comprise a tissue-specific expression element for astrocytes such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters.
  • the vector may comprise tissue-specific expression elements for oligodendrocytes such as, but not limited to, the myelin basic protein (MBP) promoter.
  • GFAP glial fibrillary acidic protein
  • MBP myelin basic protein
  • the promoter is less than 1 kb.
  • the promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,
  • the promoter may have a length between 200-300, 200- 400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400- 500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800.
  • the promoter may provide expression of the therapeutic gene expression product for a period of time in targeted tissues such as, but not limited to, the central nervous system and peripheral organs (e.g., lung).
  • Expression of the therapeutic gene expression product may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years,
  • Expression of the payload may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years or 5-10 years or 10-15 years, or 15-20 years, or 20-25 years, or 25-30 years, or 30-35 years, or 35-40 years, or 40-45 years, or 45-50 years, or 50-55 years, or 55-60 years, or 60-65 years.
  • Promoters are DNA regions that initiate gene transcription by controlling the binding of RNA polymerase to the vector DNA to begin the process toward expression of the encoded protein. Promoters control the binding of RNA polymerase to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately translated into a functional protein. Thus the promoter region controls when and where in the organism your gene of interest is expressed. Exemplary promoters include CMV, CBh, human synapsin I, EFla, SV40, PGK1, Ubc, human beta actin, and CAG.
  • the vector comprises a promoter selected from a CAG synthetic promoter, a CBh synthetic promoter, and a human synapsin I promoter.
  • CAG synthetic promoter may comprise the following nucleotide sequence (SEQ ID NO: 656): TTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCG CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC ATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTACGCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTA GTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCT CCCCCCTCCCCACCCCCAATTTTGTATTTATTTTTGTGCAGCG ATGGGGGCGGGGTCAGTTGTTCCCCATCT CCCCC
  • the CBh synthetic promoter may comprise the following nucleotide sequence (SEQ ID NO: 657): cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagg gactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattg acgtcaatgacggtaaatggcccgctggcattgtgcccagtacatgaccttatgggactttcctacttggcagtacatctattagtcatc gctattaccatggtcgaggtgagccccacgttcgtcgtcgtt
  • the human synapsin I promoter may comprise the following nucleotide sequence (SEQ ID NO: 658):
  • Expression vectors may comprise optional microRNA-encoding sequence.
  • MicroRNA or miRNA refers to small non-coding RNA molecules (about 22 nucleotides in length) that function in post-transcriptional regulation of gene expression. miRNAs function via base-pairing with complementary sequences within mRNA molecules to effectively disrupt expression of those mRNA molecules.
  • expression vectors may encode miRNA- 183.
  • Expression vectors may, for example, comprise the following sequence encoding miRNA- 183 (SEQ ID NO: 662): CCGCAGAGTGTGACTCCTGTTCTGTGTATGGCACTGGTAGAATTCACTGTGAACAGT CTCAGTCAGTGAATTACCGAAGGGCCATAAACAGAGCAGAGACAGATCCACGA
  • PREs posttranscriptional regulatory elements
  • HPRE hepatitis B virus
  • WPRE woodchuck hepatitis virus
  • Hsp70 human heat shock protein 70 mRNA
  • SP163 the vascular endothelial growth factor
  • SP163 the tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer (TM)
  • TM major late promoter enhancer
  • Intron A the first intron of human cytomegalovirus immediate early gene
  • Posttranscriptional regulatory elements can help enhance gene expression when included in expression vectors such as those described herein.
  • Particular PREs may exhibit cellspecific and/or gene-specific regulatory enhancement and those factors are considered when selecting a PRE.
  • vectors of the invention may comprise a WPRE which may comprise (SEQ ID NO: 659): gatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctat gtggatacgctgctttaatgcctttgtatcatgctattgctttcccgtatggcttttcattttctcctcttgtataaatcctggttgctgtctctttatgag gagttgtggcccgttgtcaggcaacgtggcgtggtgtgtgcactgtgttttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttc
  • vectors of the invention may include a polyadenylation signals or terminator to define the end of the transcriptional unit.
  • the selected terminator or poly(A) signal can impact gene expression.
  • Exemplary poly(A) signals that may be included in expression vectors described herein may be derived from SV40, hGH, BGH, and rbGlob.
  • vectors may include a poly (A) signal selected from Human Growth Hormone Gene Polyadenylation Signal (hGH poly A) and Simian Virus 40 Polyadenylation Signal (SV40 poly A).
  • the hGH Poly A may comprise the following nucleotide sequence (SEQ ID NO: 660): acgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaat aaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaag acaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctcccgctctgggttca agcgattctcctgctcagcctccgagcgattctg
  • the SV40 polyA may comprise the following nucleotide sequence (SEQ ID NO: 661): ttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaatgctttatttgtgaaatttgtgatgct attgcttttttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggagatgtgggagg tttttaaagcaagtaaacctctacaaatgtggtaaatctaaaaatctaaaatctggtaaaatctaaaaatctggtaaaatctaaaaatctggtaaaatctaaaaatctggt
  • FIG. 1 An exemplary expression vector 101 of the invention is shown in FIG. 1.
  • Such expression vectors 101 may be delivered in an rAAV construct as described below.
  • a vector may comprise a promoter 103 followed by cDNA 105 encoding the protein to be expressed (e.g., IDS encoding I2S).
  • the cDNA 105 may be followed by an optional miRNA signal 107 such as miRNA-183.
  • a posttranscriptional regulatory element 109 such as WPRE can then be included followed by a poly(A) 111 or other terminator such as SV40 or hGH poly(A).
  • the order of the components in FIG. 1 is preferred although, in certain embodiments, the miRNA signal 107 may be omitted such that the PRE 109 follows the cDNA 105.
  • the individual components may follow directly in order or may be linked together by additional sequences.
  • vectors of the invention may be included in rAAVs or modified rAAVs as described in PCT/2019/052969, WG/2020/028751, or U.S. Pat. Pub. Nos. 2020/0165576, 2019/0292230, or 2017/0166926, the content of each of which is incorporated herein by reference.
  • Vectors may be delivered using various modified adeno-associated (AAV) virus capsid compositions useful for integrating a transgene into a target cell or environment (e.g., a cell-type or tissue) in a subject when they are administered systemically (e.g., intracranial, intraventricular, intracerebroventricular, intravenous, intraarterial, intranasal, intrathecal, intracisternae magna administration, or subcutaneously ) to the subject.
  • the modified AAV capsid proteins of the present disclosure may comprise at least one insertion or substitution of an amino acid in a corresponding parental AAV capsid protein that confers a desired tropism such as an increased transgene transduction.
  • the disclosure provides rAAVs with high expression levels in the CNS.
  • the disclosure provides rAAVs with a peptide insertion comprising or consisting of an amino-acid sequence set forth in any one of Tables 1 and 4-30, Figure 5 and/or Formulas I- XXXIII, as defined below in greater detail.
  • Some aspects disclosed herein provide AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula I
  • X ⁇ X ⁇ X ⁇ X ⁇ X ⁇ X ⁇ X 7 (I) (SEQ ID NO: 2) wherein: X 1 is an amino acid selected from I, L, M and V;
  • X 2 is an amino acid selected from A, S and T;
  • X 3 is an amino acid selected from K and R;
  • X 4 is an amino acid selected from D, E, N and Q;
  • X 5 is an amino acid selected from F, W and Y ;
  • X 6 is an amino acid selected from F, W and Y ;
  • X 7 is an amino acid selected from K and R.
  • AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula II
  • X 8 -X 9 -X 10 -X n -X 12 -P-X 13 (II) (SEQ ID NO: 3) wherein: X 8 is an amino acid selected from I, L, M and V;
  • X 9 is an amino acid selected from D, E, N, and Q;
  • X 10 is an amino acid selected from A, S and T;
  • X 11 is an amino acid selected from A, S and T;
  • X 12 is an amino acid selected from K and R;
  • X 13 is an amino acid selected from I, L, M and V.
  • AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula III
  • X 14 -X 15 -H-X 16 -X 17 -X 18 -X 19 (III) (SEQ ID NO: 4) wherein: X 14 is an amino acid selected from D, E, N and Q;
  • X 15 is an amino acid selected from D, E, N and Q;
  • X 16 is an amino acid selected from A, S and T;
  • X 17 is an amino acid selected from K and R;
  • X 18 is an amino acid selected from D, E, N and Q;
  • X 19 is an amino acid selected from D, E, N and Q.
  • AAV capsids with greater expression in brain comprising an AAV capsid protein comprising an insertion sequence of Formula IV
  • X 21 is an amino acid selected from Q, N, S, T, F, L, A and E;
  • X 22 is an amino acid selected from T, S, G, R, N, and D;
  • X 23 is an amino acid selected from D, E, S, T, G, I, M, H and N;
  • X 24 is an amino acid selected from I, L, F, R, T, S, N and Q;
  • X 25 is an amino acid selected from A, L, Q, G, K, S, P and Y;
  • X 26 is an amino acid selected from D, K, H, M, Y, T, L, and I ;
  • X 22 is not S when X 24 is R or S; further provided X 21 is not S when X 23 is S or when X 25 is S; further provided X 25 is not S when X 24 is T or F or when X 26 is L; further provided X 23 is not T when X 24 is Q or when X 25 is P; further provided X 22 is not G when X 20 is S or when X 26 is M; further provided X 25 is not L when X 23 is S or when X 26 is T or K; further provided X 22 is not T when X 24 is S or when X 25 is P; further provided X 24 is not S when X 22 is D or R; further provided X 25 is not G when X 22 is G or T; further provided X 20 is not G when X 25 is P; further provided X 25 is not A or X 23 is T when X 26 is T; further provided X 20 is not Y when X 22 is A; further provided X 20 is not R when X 23 is D; further provided
  • the insertion sequence comprises a sequence of Formula IV wherein X 22 is R.
  • the insertion sequence as described in Table 4 is selected from AFGGIAD (SEQ ID NO: 37), ISREFYK (SEQ ID NO: 38), GTDMRQT (SEQ ID NO: 39), HLTSNQL (SEQ ID NO: 40), PSSNNPH (SEQ ID NO: 41), NARSTGM (SEQ ID NO: 42), SNRTLSI (SEQ ID NO: 43), SQSIQKD (SEQ ID NO: 44), REDHNLY (SEQ ID NO: 45) and YQNDSGK (SEQ ID NO: 46).
  • AAV capsids with greater enrichment in the BRAIN over that found in the LIVER comprising an AAV capsid protein comprising an insertion sequence of Formula V X 27 -X 28 -X 29 -X 30 -X 31 -X 32 -X 33 (V) (SEQ ID NO: 6) wherein: X 27 is an amino acid selected from I, G, L, T, V, D, S and N;
  • X 28 is an amino acid selected from D, A, L, I, H, Y, F and N;
  • X 29 is an amino acid selected from S, T, M, E, V, L, I and N;
  • X 30 is an amino acid selected from P, G, L, I, V, E and D;
  • X 31 is an amino acid selected from T, E, S, G, I, M, Q and N;
  • X 32 is an amino acid selected from P, S, M, H, I, V, E and D;
  • X 33 is an amino acid selected from G, L, K, H, T and D; provided X 27 is not S when X 32 is S; further provided X 27 is not T when X 29 is I or S; further provided X 27 is not V when X 29 is S; further provided X 27 is not L when X 31 is N; further provided X 28 is not N when X 32 is P; further provided X 29 is not V when X 30 is P; further provided X 29 is not N when X 30 is V; further provided X 30 is not G when X 31 is Q; further provided X 29 is not S when X 32 is P; further provided X 31 is not T when X 32 is S or V; and further provided X 32 is not S when X 33 is K or L.
  • the insertion sequence comprises a sequence of Formula V wherein X 27 is I or L.
  • the insertion sequence as described in Table 7, is selected from IDVDTPT (SEQ ID NO: 47), GASGEDL (SEQ ID NO: 48), LDNLSVT (SEQ ID NO: 49), TLMEGMK (SEQ ID NO: 50), VNEIIEK (SEQ ID NO: 51), LHLGMID (SEQ ID NO: 52), DHEVTDH (SEQ ID NO: 53), SYIPGHK (SEQ ID NO: 54), NIEDNMG (SEQ ID NO: 55) and IFTLQSG (SEQ ID NO: 56).
  • AAV capsids having greater enrichment in the BRAIN over that found in the SPINAL CORD comprising an AAV capsid protein comprising an insertion sequence of Formula VI
  • X 34 -X 35 -X 36 -X 37 -X 38 -X 39 -X 40 (VI) (SEQ ID NO: 7) wherein: X 34 is an amino acid selected from T, K, N, A, V and L;
  • X 35 is an amino acid selected from T, S, A, L, P and N;
  • X 36 is an amino acid selected from T, S, I, A, N and P;
  • X 37 is an amino acid selected from S, T, D, E, N, V, I and L;
  • X 38 is an amino acid selected from S, T, K, R, P, V, L, A and G
  • X 39 is an amino acid selected from N, T, S, K, D, E and G
  • X 40 is an amino acid selected from S, T, K, N, Q, D, L and E;
  • X 40 is not S when X 34 is A or N or when X 35 is N; further provided X 39 is not S when X 34 is T or L; further provided X 40 is not N or when X 35 is A or when X 36 is S; further provided X 36 is not S when X 39 is T or when X 40 is L; further provided X 35 is not S when X 39 is G or when X 40 is D or K; further provided X 38 is not S when X 34 is V or when X 40 is K; further provided X 35 is not P when X 36 is P or when X 37 is L; further provided X 39 is not T when X 34 is not L or when X 36 is A; further provided X 37 is not S when X 36 is A or N; further provided X 37 is not V when X 34 is T or K; further provided X 35 is not T when X 34 is K or when X 39 is K; further provided X 34 is not V when X 35 is A or when X 40 is Q;
  • the insertion sequence comprises a sequence of Formula VI wherein X 41 is L, X 43 is T, and X 47 is V.
  • the insertion sequence as described in Table 8 is selected from TTISSTS (SEQ ID NO: 57), KSSDKDS (SEQ ID NO: 58), NSNVPKN (SEQ ID NO: 59), AAAEVNK (SEQ ID NO: 60), VLTTLSK (SEQ ID NO: 61), VTTNREL (SEQ ID NO: 62), NPTVANT (SEQ ID NO: 63), TLNILNQ (SEQ ID NO: 64), NNPLTGD (SEQ ID NO: 65) and LSTSGNE (SEQ ID NO: 66).
  • X 4 I -X 42 -X 43 -X ⁇ -X 45 -X 46 -X 47 (VII) (SEQ ID NO: 8) wherein: X 41 is an amino acid selected from Q, G, A, S, C, E, P and L;
  • X 42 is an amino acid selected from D, P, H, S, G, V, L and N;
  • X 43 is an amino acid selected from N, E, Q, S, T, V, G and D;
  • X 44 is an amino acid selected from G, T, S, M, Y and E;
  • X 45 is an amino acid selected from P, F, T, K, E, M, A and G;
  • X 46 is an amino acid selected from V, E, D, M, K, S and Y ;
  • X 47 is an amino acid selected from R, K, N, A, T, V and W; Provided X 41 is not G when X 46 is S; further provided X 41 is not S when X 46 is Y or S; further provided X 41 is not A when X 45 is A; further provided X 41 is not P when X 43 is N; further provided X 42 is not P when X 46 is S; further provided X 42 is not S when X 46 is D; further provided X 42 is not H when X 47 is K; further provided X 43 is not S when X 44 is G; further provided X 43 is not G when X 45 is P; further provided X 44 is not T when X 47 is T ; further provided X 44 is not S when X 46 is V; and further provided X 45 is not G when X 47 is V.
  • the insertion sequence comprises a sequence of Formula VII wherein X 41 is A. In some embodiments, the insertion sequence comprises a sequence of Formula VII wherein X 43 is D. In some embodiments, the insertion sequence comprises a sequence of Formula VII wherein X 41 is L, X 43 is T, and X 47 is V. In some embodiments, the insertion sequence comprises a sequence of Formula VII wherein X 46 is E or D, and X 47 is K or R.
  • the insertion sequence as described in Table 9, is selected from QVDGPVR (SEQ ID NO: 67), GDNGFYK (SEQ ID NO: 68), APVTGEN (SEQ ID NO: 69), SNDMTEK (SEQ ID NO: 70), CNEEMKA (SEQ ID NO: 71), ENQSAST (SEQ ID NO: 72), PHSEGDN (SEQ ID NO: 73), LSTETMV (SEQ ID NO: 74), AGDYKEW (SEQ ID NO: 75) and ALGEEST (SEQ ID NO: 76).
  • AAV capsids having greater enrichment in the SPINAL CORD comprising an AAV capsid protein comprising an insertion sequence of Formula VIII
  • X 49 is an amino acid selected from D, S, K, N, I and L;
  • X 50 is an amino acid selected from N, S, T, G, V, A and R;
  • X 51 is an amino acid selected from L, T, G, N, D, R and A;
  • X 52 is an amino acid selected from S, A, P, E, I, T and M;
  • X 53 is an amino acid selected from Y, F, T, N, G, E, P and Q;
  • X 54 is an amino acid selected from V, I, D, A, Y, N, E and T;
  • X 52 is not S when X 49 is L or S; further provided X 48 is not S when X 49 is K, further provided X 48 is not S, when X 52 is T or when X 53 is P; further provided X 48 is not P when X 53 is N, further provided X 48 is not G when X 53 is T, further provided X 49 is not S when X 52 is M or X 51 is N; further provided X 49 is not N when X 53 is T; further provided X 50 is not G when X 51 is L, further provided X 49 is not N when X 54 is V, and further provided X 53 is not N when X 54 is A.
  • the insertion sequence comprises a sequence of Formula VIII wherein X 48 is E or S. In some embodiments, the insertion sequence comprises a sequence of Formula VIII wherein X 49 is D.
  • the insertion sequence as described in Table 5 is selected from EDNLSYV (SEQ ID NO: 77), SDSTAFI (SEQ ID NO: 78), SSNGPTD (SEQ ID NO: 79), EKTNEND (SEQ ID NO: 80), SNTDSGT (SEQ ID NO: 81), GIGTSEA (SEQ ID NO: 82), AIVAAGY (SEQ ID NO: 83), NLANIPN (SEQ ID NO: 84), PLRTTQE (SEQ ID NO: 85) and SDRRMNT (SEQ ID NO: 86).
  • EDNLSYV SEQ ID NO: 77
  • SDSTAFI SEQ ID NO: 78
  • SSNGPTD SEQ ID NO: 79
  • EKTNEND SEQ ID NO: 80
  • SNTDSGT SEQ ID NO: 81
  • GIGTSEA SEQ ID NO: 82
  • AIVAAGY SEQ ID NO: 83
  • NLANIPN SEQ ID NO: 84
  • PLRTTQE
  • AAV capsids having greater enrichment in the SPINAL CORD over that found in the LIVER comprising an AAV capsid protein comprising an insertion sequence of Formula IX
  • X 55 is an amino acid selected from N, E, M, G, S, P, D, C and V;
  • X 56 is an amino acid selected from Q, L, A, I, G, R, T, S, and V;
  • X 57 is an amino acid selected from K, N, V, L, G, A and E;
  • X 58 is an amino acid selected from P, T, G, M, S and E;
  • X 59 is an amino acid selected from D, S, A, P, R, I, M, Q and L;
  • X 60 is an amino acid selected from A, M, E, P, T, V, L and Q;
  • X 61 is an amino acid selected from K, P, T, M, H, N and Y ;
  • X 55 is not V when X 56 is G or L or when X 57 is N; further provided X 55 is not P when X 57 is K or when X 58 is P; further provided X 58 is not S when X 55 is S or E or when X 60 is A; further provided X 57 is not L when X 59 is R or when X 61 is P; further provided X 57 is not G when X 59 is L or when X 61 is P; further provided X 61 is not T when X 57 is A or G; further provided X 59 is not P when X 56 is R or when X 61 is M; further provided X 59 is not S when X 57 is A or when X 61 is K; further provided X 55 is not D when X 56 is V; further provided X 55 is not N when X 57 is V; further provided X 58 is not T when X 56 is T; further provided X 57 is not E when X 61 is H
  • the insertion sequence comprises a sequence of Formula IX wherein X 59 is S.
  • the insertion sequence as described in Table 12 is selected from NSEPDAN (SEQ ID NO: 87), ELGTAEM (SEQ ID NO: 88), STLEMPH (SEQ ID NO: 89), VQVGSMT (SEQ ID NO: 90), PTNMPPT (SEQ ID NO: 91), DAVSRVP (SEQ ID NO: 92), CGKTILT (SEQ ID NO: 93), MVNELTP (SEQ ID NO: 94), NIAEQPK(SEQ ID NO: 95) and GREPSQY (SEQ ID NO: 96).
  • AAV capsids having a greater enrichment in the SPINAL CORD over that found in BRAIN comprising an AAV capsid protein comprising an insertion sequence of Formula X
  • X 63 is an amino acid selected from Q, N, T, P, L, I, G and Y ;
  • X 64 is an amino acid selected from T, S, M, G, A and F;
  • X 65 is an amino acid selected from N, T, H, G and P;
  • X 66 is an amino acid selected from S, D, Q, N and R;
  • X 67 is an amino acid selected from T, G, A, Y, E, D, K and N;
  • X 68 is an amino acid selected from H, A, F, Y, P, N, I and V ; provided X 64 is not S when X 62 is T; further provided X 65 is not N or T when X 66 is R; further provided X 63 is not P when X 62 is T or M; further provided X 62 is not G when X 65 is N; further provided X 65 is not G when X 67 is T; further provided X 63 is not Y when X 67 is A; further provided X 64 is not S when X 68 is N; and further provided X 64 is not T when X 66 is N.
  • the insertion sequence comprises a sequence of Formula X wherein X 65 is N. In some embodiments, the insertion sequence comprises a sequence of Formula X wherein X 66 is S. In some embodiments, the insertion sequence comprises a sequence of Formula X wherein X 63 is Q or N.
  • the insertion sequence as described in Table 11, is selected from DQTNSTH (SEQ ID NO: 97), MQMNSGA (SEQ ID NO: 98), NTMNSYP (SEQ ID NO: 99), ILSNQAF (SEQ ID NO: 100), GYSTSEV (SEQ ID NO: 101), ANSHDKI (SEQ ID NO: 102), GPGTSDN (SEQ ID NO: 103), TGFNNKI (SEQ ID NO: 104), DIAGRNP (SEQ ID NO: 105) and KQSPSNY (SEQ ID NO: 106).
  • AAV capsids having greater enrichment in the SPINAL CORD over that found in the LIVER and BRAIN comprising an AAV capsid protein comprising an insertion sequence of Formula XI
  • X 69 -X 70 -X 71 -X 72 -X 73 -X 74 -X 75 (SEQ ID NO: 12) wherein: X 69 is an amino acid selected from S, G, M, Q, H, V, I, A and E;
  • X 70 is an amino acid selected from T, E, N, H, A, L, D, and R;
  • X 71 is an amino acid selected from H, M, D, E, and A;
  • X 72 is an amino acid selected from D, K, F, G, L, N, and T;
  • X 73 is an amino acid selected from R, D, V, S, T, G, N, and H;
  • X 74 is an amino acid selected from D, M, S, R, T, and G;
  • X 75 is an amino acid selected from F, S, T, L, A, G, H, W, and P;
  • X 71 is not A or M when X 74 is S; further provided X 72 is not G or T when X 74 is T; further provided X 70 is not R when X73 is V or when X 69 is Q; further provided X 73 is not R when X 69 is I or when X 71 is M; further provided X 71 is not E when X 69 is S or when X 72 is L; further provided X 73 is not S when X 70 is L or when X 69 is G; further provided X 70 is not H when X 73 is G; further provided X 71 is not A when X 74 is D; further provided X 71 is not H when X 72 is L; further provided X 72 is not T when X 74 is R; and further provided X73 is not T when X 74 is G.
  • the insertion sequence comprises a sequence of Formula XI wherein X 71 is D or E. In some embodiments, the insertion sequence comprises a sequence of Formula XI wherein X 72 is K.
  • the insertion sequence as described in Table 10 is selected from STHDRDF (SEQ ID NO: 107), GEMKDMS (SEQ ID NO: 108), MNDFVSL (SEQ ID NO: 109), QHDGSML (SEQ ID NO: 110), HADLRDG (SEQ ID NO: 111), GLEFTRH (SEQ ID NO: 112), VDANGTW (SEQ ID NO: 113), IEEKNGT (SEQ ID NO: 114), ARDTDDA (SEQ ID NO: 115) and ETDKHGP (SEQ ID NO: 116).
  • AAV capsids having improved enrichment in both the BRAIN and in the SPINAL CORD comprising an AAV capsid protein comprising an insertion sequence of Formula XII
  • X 76 is an amino acid selected from S, G, P, V, A and E;
  • X 77 is an amino acid selected from N, G, A, L, V, D, and K;
  • X 78 is an amino acid selected from I, N, Q, T, S, E, and G;
  • X 79 is an amino acid selected from G, P, F, K, S, Q, N, and T;
  • X 80 is an amino acid selected from K, R, T, S, Y, G, V and N;
  • X 81 is an amino acid selected from H, E, S, T, V and N;
  • X 82 is an amino acid selected from I, N, L, H, K, D, Y, and T; provided X 77 is not L when X 78 is I or when X 80 is G or when X 82 is T; further provided X 76 is not S when X 78 is G or when X 79 is S; further provided X 76 is not P when X 77 is V or when X 80 is S; further provided X 77 is not A when X 79 is P or when X 82 is I; further provided X 78 is not S when X 79 is G or when X 81 is S; further provided X 81 is not S when X 79 is G or when X 80 is S; further provided X 81 is not N when X 77 is N or when X 80 is T; further provided X 81 is not T when X 82 is L; further provided X 79 is not N when X 81 is V; further provided X 77 is R
  • the insertion sequence comprises a sequence of Formula XII wherein X 76 is S. In some embodiments, the insertion sequence comprises a sequence of Formula XII wherein X 77 is A, L or V. In some embodiments, the insertion sequence comprises a sequence of Formula XII wherein X 81 is N.
  • the insertion sequence as described in Table 16 is selected from SDIGKTH (SEQ ID NO: 117), PNEGGHN (SEQ ID NO: 118), AGNPGVI (SEQ ID NO: 119), VVGSTVL (SEQ ID NO: 120), GAITNNY (SEQ ID NO: 121), SLNNVTN (SEQ ID NO: 122), EKTSVNT (SEQ ID NO: 123), SLSQYEK (SEQ ID NO: 124), GAQFRSD (SEQ ID NO: 125) and VASKSNH (SEQ ID NO: 126).
  • AAV capsids having improved enrichment in the SPINAL CORD AND BRAIN over that found in the LIVER comprising an AAV capsid protein comprising an insertion sequence of Formula XIII X 83 -X 84 -X 85 -X 86 -X 87 -X 88 -X 89 (XIII) (SEQ ID NO: 14) wherein: X 83 is an amino acid selected from F, I, A, N, E, D, N, Q, K and T;
  • X 84 is an amino acid selected from G, T, I, W, S, D, A, and H;
  • X 85 is an amino acid selected from E, D, T, I, N, M, and S;
  • X 86 is an amino acid selected from I, N, P, E, D, H, V, and A;
  • X 87 is an amino acid selected from T, R, V, H, G, A, and K;
  • X 88 is an amino acid selected from P, I, A, Q, E, K, G, and H;
  • X 89 is an amino acid selected from G, V, H, E, S, K, A, P, and N;
  • X 84 is not S when X 85 is S or N or when X 83 is F; further provided X 85 is not T when X 87 is R or X 84 is S; further provided X 84 is not H when X 88 or X 86 is P; further provided X 83 is not A when X 87 is R; further provided X 86 is not A when X 89 is P; further provided X 84 is not T when X 89 is S; further provided X 85 is not S when X 87 is G; further provided X 85 is not T when X 84 is A or when X 87 is G; further provided X 83 is not T when X 84 is G or W; further provided X 83 is not E when X 84 is A; further provided X 86 is not V when X 88 is Q; and further provided X 88 is not P when X 89 is V.
  • the insertion sequence comprises a sequence of Formula XIII wherein X 85 is D. In some embodiments, the insertion sequence comprises a sequence of Formula XIII wherein X 86 is N.
  • the insertion sequence as described in Table 29, is selected from FGEITPG (SEQ ID NO: 127), ITDNRIV (SEQ ID NO: 128), AITPVAH (SEQ ID NO: 129), NGIERQE (SEQ ID NO: 130), EWNNHES (SEQ ID NO: 131), DSMDGKK (SEQ ID NO: 132), NDNNAGA (SEQ ID NO: 133), KDDHKEP (SEQ ID NO: 134), QADVGAN (SEQ ID NO: 135) and THSAVHH (SEQ ID NO: 136).
  • AAV capsids having an improved enrichment in the SPINAL CORD comprising an AAV capsid protein comprising an insertion of Formula XIV
  • X 90 -X 91 -X 92 -X 93 -X 94 -X 95 -X 96 (XIV) (SEQ ID NO: 15) wherein: X 90 is an amino acid selected from E, N, D, T, S, I, N, and K; X 91 is an amino acid selected from G, S, Q, I, L, P, and V;
  • X 92 is an amino acid selected from K, D, T, S, A and Q;
  • X 93 is an amino acid selected from L, P, A, T, S and N;
  • X 94 is an amino acid selected from H, P, I, A, S, T, Q, E and R;
  • X 95 is an amino acid selected from V, A, T, S, G, N, Q and E;
  • X 96 is an amino acid selected from I, T, N, R, H, and Y ;
  • X 90 is not S when X 92 is T or when X 93 is S or when X 91 is G; further provided X 92 is not S or A or X 93 is not A when X 96 is T ; further provided X 92 is not A when X 93 is A or when X 95 is Q; further provided X 90 is not N when X 95 is G; further provided X 90 is not T when X 94 is A; further provided X 90 is not D when X 96 is N; further provided X 92 is not S when X 94 is S; further provided X 95 is not S when X 96 is I; further provided X 91 is not V when X 92 is Q; further provided X 92 is not T when X 96 is H; further provided X 91 is not S when X 90 is I or T; and further provided X 94 is not S when X 90 is D or when X 93 is T.
  • the insertion sequence comprises a sequence of Formula XIV wherein X 91 is G, I, L or V. In some embodiments, the insertion sequence comprises a sequence of Formula XIV wherein X 93 is N.
  • the insertion sequence as described in Table 14, is selected from EGKNEVI (SEQ ID NO: 137), NSDNHNI (SEQ ID NO: 138), DQKLPAT (SEQ ID NO: 139), TITPITN (SEQ ID NO: 140), ILTASER (SEQ ID NO: 141), IGTTQTN (SEQ ID NO: 142), SPATASH (SEQ ID NO: 143), SVDNRGN (SEQ ID NO: 144), NVSSRSN (SEQ ID NO: 145) and KSQATQY (SEQ ID NO: 146).
  • AAV capsids having improved enrichment in the SPINAL CORD over the LIVER comprising an AAV capsid protein comprising an insertion sequence of Formula XV
  • X 98 is an amino acid selected from N, T, I, V, F, P, R and G;
  • X 99 is an amino acid selected from G, E, M, A, I, F, S and V;
  • X 100 is an amino acid selected from V, I, A, L, K, G, S, E, and D;
  • X 101 is an amino acid selected from K, V, I, A, G, Y, E and T;
  • X 102 is an amino acid selected from E, S, D, N, K, P, A and G;
  • X 103 is an amino acid selected from K, R, A, V, I, G and L;
  • X 102 is not S when X 97 is T or when X 101 is T; further provided X 97 is not N when X 100 is G or S or when X 98 is N; further provided X 97 is not G when X 98 is R or when X 99 is G; further provided X 99 is not S when X 101 is T or A or when X 98 is G; further provided X 98 is not R when X 103 is A; further provided X 100 is not G when X 102 is P; further provided X 100 is not S when X 102 is A; further provided X 99 is not A when X 100 is L; further provided X 97 is not M when X 101 is A or when X 98 is I or when X 102 is S; further provided X 101 is not T when X 103 is V; further provided X 97 is not I when X 98 is G or when X 100 is A; further provided X 98 is not T when X 97 is A or T; further provided
  • the insertion sequence comprises a sequence of Formula XV wherein X 100 is G, A, I or L.
  • the insertion sequence as described in Table 18, is selected from DNGVKEK (SEQ ID NO: 147), GTELVSR (SEQ ID NO: 148), AIMKIDA (SEQ ID NO: 149), AFAGANV (SEQ ID NO: 150), MNFAGPI (SEQ ID NO: 151), GVSSIDK (SEQ ID NO: 152), IVSEYAG (SEQ ID NO: 153), NPIAESR (SEQ ID NO: 154), NREDTKL (SEQ ID NO: 155) and TGVIEGL (SEQ ID NO: 156).
  • DNGVKEK SEQ ID NO: 147
  • GTELVSR SEQ ID NO: 148
  • AIMKIDA SEQ ID NO: 149
  • AFAGANV SEQ ID NO: 150
  • MNFAGPI SEQ ID NO: 151
  • GVSSIDK SEQ ID NO: 152
  • IVSEYAG SEQ ID NO: 153
  • NPIAESR SEQ ID NO: 154
  • NREDTKL
  • AAV capsids having improved enrichment in the SPINAL CORD over that found in BRAIN comprising an AAV capsid protein comprising an insertion sequence of Formula XVI
  • X 104 -X 105 -X 106 -X 107 -X 108 -X 109 -X 110 (XVI) (SEQ ID NO: 17) wherein: X 104 is an amino acid selected from N, Q, K, M, T, L, I, V and G; X 105 is an amino acid selected from G, E, S, T, A, Q and H;
  • X 106 is an amino acid selected from S, T, N, K, R, I and L;
  • X 107 is an amino acid selected from T, S, A, N, E, R and G;
  • X 108 is an amino acid selected from D, R, K, T, S, P, A and V;
  • X 109 is an amino acid selected from H, G, N, P, V, T, S and F;
  • X 110 is an amino acid selected from D, S, T, I, A, L, F and Y;
  • X 104 is not V when X 105 is A or when X 106 is S; further provided X 104 is not N when X 108 is D or P; further provided X 104 is not L when X 105 is A or S; further provided X 105 is not E when X 104 is M or V; further provided X 105 is not S when X 104 is Q or when X 110 is A; further provided X 105 is not T when X 107 is A or when X 110 is A; further provided X 105 is not H when X 108 is S or when X 109 is P; further provided X 106 is not S when X 104 is G or when X 105 is S; further provided X 106 is not T when X 104 is L or when X 109 is V; further provided X 106 is not R when X 104 is L or when X 105 is A; further provided X 108 is not A when X 109 is F or when X 110 is T; further provided
  • the insertion sequence comprises a sequence of Formula XVI wherein X 104 is G and X 105 is S. In some embodiments, the insertion sequence comprises a sequence of Formula XVI wherein X 105 is S. In some embodiments, the insertion sequence comprises a sequence of Formula XVI wherein X 109 is S.
  • the insertion sequence as described in Table 20 is selected from IGNTDHD (SEQ ID NO: 157), LEISTTS (SEQ ID NO: 158), VSLAPSI (SEQ ID NO: 159), GSKSTFF (SEQ ID NO: 160), NASNASA (SEQ ID NO: 161), QQNNSSL (SEQ ID NO: 162), MHTERGT (SEQ ID NO: 163), KSRSVND (SEQ ID NO: 164), GSLGKPT (SEQ ID NO: 165) and TTNRTVY (SEQ ID NO: 166).
  • IGNTDHD SEQ ID NO: 157
  • LEISTTS SEQ ID NO: 158
  • VSLAPSI SEQ ID NO: 159
  • GSKSTFF SEQ ID NO: 160
  • NASNASA SEQ ID NO: 161
  • QQNNSSL SEQ ID NO: 162
  • MHTERGT SEQ ID NO: 163
  • KSRSVND SEQ ID NO: 164
  • GSLGKPT S
  • AAV capsids having improved enrichment in the SPINAL CORD over that found in the LIVER and BRAIN comprising an AAV capsid protein comprising an insertion sequence of Formula XVII
  • X ni -X 112 -X 113 -X 114 -X 115 -X 116 -X 117 (SEQ ID NO: 18) wherein: X 111 is an amino acid selected from N, H, T, S, G, A, I, Y and F; X 112 is an amino acid selected from N, E, G, L, I, P and S;
  • X 113 is an amino acid selected from G, S, T, R and E;
  • X 114 is an amino acid selected from S, E, D, N, V and L;
  • X 115 is an amino acid selected from S, V, I, R, K, H, D, Q and P;
  • X 116 is an amino acid selected from T, S, G, E, D, I and V; and X 117 is an amino acid selected from S, Y, P, A, V, L, Q and M; Provided X 111 is not Y when X 112 is E or I; further provided X 111 is not N when X 112 is P or when X 113 is T; further provided X 111 is not G when X 112 is L or when X 117 is S; further provided X 111 is not H when X 117 is V; further provided X 111 is not T when X 115 is P; further provided X 111 is not A when X 112 is G; further provided X 111 is not S when X 116 is V; further provided X 112 is not N when X 113 is T or when X 114 is N; further provided X 112 is not S when X 113 is G or when X 116 is T; further provided X 113 is not S when X 114
  • the insertion sequence comprises a sequence of Formula XVII wherein X 113 is T. In some embodiments, the insertion sequence comprises a sequence of Formula XVII wherein X 113 is G. In some embodiments, the insertion sequence comprises a sequence of Formula XVII wherein X 113 is S.
  • the insertion sequence as described in Table 22, is selected from HNGVSIL (SEQ ID NO: 167), NESSVTS (SEQ ID NO: 168), TGTEIGY (SEQ ID NO: 169), SLSDREY (SEQ ID NO: 170), GPGEHSP (SEQ ID NO: 171), TSTSDIA (SEQ ID NO: 172), ASRDSDV (SEQ ID NO: 173), YNSLQGQ (SEQ ID NO: 174), FIENKVA (SEQ ID NO: 175) and IGTLPTM (SEQ ID NO: 176).
  • HNGVSIL SEQ ID NO: 167
  • NESSVTS SEQ ID NO: 168
  • TGTEIGY SEQ ID NO: 169
  • SLSDREY SEQ ID NO: 170
  • GPGEHSP SEQ ID NO: 171
  • TSTSDIA SEQ ID NO: 172
  • ASRDSDV SEQ ID NO: 173
  • YNSLQGQ SEQ ID
  • AAV capsids having significant enrichment in both the BRAIN and in the SPINAL CORD comprising an AAV capsid protein comprising an insertion of Formula XVIII
  • X 118 -X 119 -X 120 -X 121 -X 122 -X 123 -X 124 (SEQ ID NO: 19) wherein: X 118 is an amino acid selected from H, E, N, S, T and V;
  • X 119 is an amino acid selected from G, T, D, S and V;
  • X 120 is an amino acid selected from S, P, A, N and D;
  • X 121 is an amino acid selected from N, D, K, S, G, A, I and P;
  • X 122 is an amino acid selected from A, I, V, L, H, N, S and T;
  • X 123 is an amino acid selected from R, D, A, I, H, T, Q, F and P; and X 124 is an amino acid selected from D, R, K, G, S, F, V, P and Y;
  • X 119 is not S when X 120 is S or when X 122 is A or when X 118 is T; further provided X 119 is not V when X 118 is S or when X 120 is S; further provided X 121 is not S when X 122 is S or when X 119 is D or G; further provided X 120 is not D when X 123 is R; further provided X 119 is not T when X 118 is V or N; further provided X 120 is not P when X— is A or when X 123 is R; further provided X— is not I when X 122 is N; further provided X 118 is not T when X 123 is P; further provided X 118 is not V when X 119 is G or D; further X 119 is not D when X 124 is P; and further provided X 118 is not H when X 120 is N.
  • the insertion sequence comprises a sequence of Formula XVIII wherein X 118 is N and X 119 is D. In some embodiments, the insertion sequence comprises a sequence of Formula XVIII wherein X 118 is E and X 119 is T. In some embodiments, the insertion sequence comprises a sequence of Formula XVIII wherein X 119 is S.
  • the insertion sequence as described in Table 17, is selected from HGSDIRD (SEQ ID NO: 177), ETPNHDG (SEQ ID NO: 178), NDSGAAS (SEQ ID NO: 179), ETASVHF (SEQ ID NO: 180), NDNANTK (SEQ ID NO: 181), SSNALQV (SEQ ID NO: 182), SGANHFS (SEQ ID NO: 183), TGSPNIP (SEQ ID NO: 184), VSNISRY (SEQ ID NO: 185) and NVDKTPR (SEQ ID NO: 186).
  • AAV capsids having significant enrichment in the SPINAL CORD and BRAIN over that found in the LIVER comprising an AAV capsid protein comprising an insertion sequence of Formula XIX
  • X 129_ X 13O_ X 131 ( XIX) SE Q J D N Q. 20 ) wherein: X 125 is an amino acid selected from P, G, K, E, T and A;
  • X 126 is an amino acid selected from R, T, G, N, P and V;
  • X 127 is an amino acid selected from D, Q, E, N, V, I, A and P;
  • X 128 is an amino acid selected from L, I, V, N, D, Q, K and S;
  • X 129 is an amino acid selected from N, D, E, G, S, T and I;
  • X 130 is an amino acid selected from D, N, Q, F, T, G, L and V;
  • X 131 is an amino acid selected from P, M, I, G, T, H and K;
  • X 125 is not P when X 127 is I or when X 126 is V; further provided X 126 is not P when X 125 is E or G; further provided X 129 is not S when X 126 is R or when X 130 is T; further provided X 128 is not S when X 131 is T or when X 126 is P; further provided X 126 is not G when X 125 is G or when X 127 is P; further provided X 127 is not A when X 128 is L; further provided X 125 is not K when X 126 is T; and further provided X 125 is not T when X 127 is N.
  • the insertion sequence comprises a sequence of Formula XIX wherein X 125 is P. In some embodiments, the insertion sequence comprises a sequence of Formula XIX wherein X 128 is Q.
  • the insertion sequence as described in Table 30, is selected from PRDLNDP (SEQ ID NO: 187), GTQNDVM (SEQ ID NO: 188), KGVDGDI (SEQ ID NO: 189), ENPSSNG (SEQ ID NO: 190), KGDVTFT (SEQ ID NO: 191), PPNQDQH (SEQ ID NO: 192), TPANELK (SEQ ID NO: 193), GNEQITG (SEQ ID NO: 194), EVIKETG (SEQ ID NO: 195) and ATVINGT (SEQ ID NO: 196).
  • AAV capsids having improved enrichment in the SPINAL CORD comprising an AAV capsid protein comprising an insertion of Formula XX
  • X 133 is an amino acid selected from H, E, S, T, N, G and A;
  • X 134 is an amino acid selected from N, R, D, S, F, L and Y;
  • X 135 is an amino acid selected from L, A, D, E, P, Q, K and S;
  • X 136 is an amino acid selected from L, D, Q, N, R, Y and T;
  • X 137 is an amino acid selected from N, Q, T, S, L and V;
  • X 138 is an amino acid selected from N, S, T, L and A;
  • X 135 is not S when X 137 is S; further provided X 133 is not T when X 136 is T or when X 132 is S; further provided X 132 is not N when X 133 is E or when X 134 is R; further provided X 133 is not H when X 137 is T or when X 132 is S; further provided X 133 is not A when X 134 is N or when X 135 is A; further provided X 134 is not S when X 135 is A or when X 136 is R; further provided X 134 is not L when X 137 is L or when X 132 is A; further provided X 133 is not S when X 132 is P or when X 136 is T; further provided X 132 is not Y when X 136 is N; and further provided X 132 is not T when X 134 is F.
  • the insertion sequence comprises a sequence of Formula XX wherein X 136 is N.
  • the insertion sequence as described in Table 15, is selected from THNDLLN (SEQ ID NO: 197), PERAQVS (SEQ ID NO: 198), YESLTQN (SEQ ID NO: 199), SERPDTL (SEQ ID NO: 200), TNDANTL (SEQ ID NO: 201), SSNEYST (SEQ ID NO: 202), NTFSRNN (SEQ ID NO: 203), YNLQLNS (SEQ ID NO: 204), AGYPNSA (SEQ ID NO: 205) and NADKNNL (SEQ ID NO: 206).
  • AAV capsids having significant enrichment in the SPINAL CORD over the LIVER comprising an AAV capsid protein comprising an insertion sequence of Formula XXI
  • X 139 -X 140 -X 141 -X 142 -X 143 -X 144 -X 145 (SEQ ID NO: 22) wherein: X 139 is an amino acid selected from H, N, S, R, L, V and A;
  • X 140 is an amino acid selected from H, E, D, N, K, S, L and A;
  • X 141 is an amino acid selected from N, A, L, V, E, D and P;
  • X 142 is an amino acid selected from D, S, G, K, L and M;
  • X 143 is an amino acid selected from N, E, Q, R, S and M;
  • X 144 is an amino acid selected from P, T, S, H, Y, I and V;
  • X 145 is an amino acid selected from E, D, P, G, V, L and A;
  • X 143 is not R when X 142 is S or D or when X 144 is T or when X 145 is E; further provided X 142 is not S when X 143 is S or when X 139 is A; further provided X 141 is not A when X 144 is S or when X 139 is S or R; further provided X 143 is not N when X 140 is A and when X 139 is R; further X 142 is not G when X 140 is N or when X 143 is E; further provided X 140 is not H when X 139 is H or L; further provided X 141 is not L when X 144 is H; further provided X 141 is not P when X 145 is A; further provided X 140 is S when X 142 is K; further provided X 139 is not V when X 141 is V.
  • the insertion sequence comprises a sequence of Formula XXI wherein X 139 is V. In some embodiments, the insertion sequence comprises a sequence of Formula XXI wherein X 140 is E. In some embodiments, the insertion sequence comprises a sequence of Formula XXI wherein X 141 or X 142 is D.
  • the insertion sequence as described in Table 19, is selected from NHNDSVE (SEQ ID NO: 207), LEASNTA (SEQ ID NO: 208), VDNDNPL (SEQ ID NO: 209), VELGSSP (SEQ ID NO: 210), VNEKESV (SEQ ID NO: 211), SAVDMSA (SEQ ID NO: 212), RLDLQHD (SEQ ID NO: 213), HEDKSVA (SEQ ID NO: 214), RSPGQIG (SEQ ID NO: 215) and AKEMRYA (SEQ ID NO: 216).
  • AAV capsids having significant enrichment in the SPINAL CORD over that found in BRAIN comprising an AAV capsid protein comprising an insertion sequence of Formula XXII
  • X 146 is an amino acid selected from M, N, Q, T, S, Y and I;
  • X 147 is an amino acid selected from V, G, I, D, Q, T and S;
  • X 148 is an amino acid selected from N, A, L, M, T, S and P;
  • X 149 is an amino acid selected from V, A, S, K, R, Q, N and G;
  • X 150 is an amino acid selected from N, G, V, L, I, S and K;
  • X 151 is an amino acid selected from F, S, T, M, N, P, L, G and V;
  • X 152 is an amino acid selected from K, S, T, P, A, M, N, E and Y;
  • X 146 is not T when X 147 is T or when X 148 is L or when X 150 is L; further provided X 146 is not I when X 148 is N or when X 150 is I; further provided X 146 is not N when X 147 is V or when X 151 is P; further provided X 146 is not S when X 148 is S, L or N or when X 150 is V; further provided X 147 is not S when X 148 is A or when X149 is S or when X 150 is V; further provided X 147 is not T when X 146 is S or when X 152 is A; further provided X 147 is not V when X 150 is I or when X 151 is T; further provided X 148 is not S when X 147 is D or S or when X 151 is G; further provided X 150 is not S when X 148 is T or when X 151 is S; further provided X 150 is not N when X 147 is I
  • the insertion sequence comprises a sequence of Formula XXII wherein X 148 is N.
  • the insertion sequence as described in Table 21, is selected from MVNVNVK (SEQ ID NO: 217), NTLASFS (SEQ ID NO: 218), IGAKGSP (SEQ ID NO: 219), NITSVTA (SEQ ID NO: 220), ITMRSMM (SEQ ID NO: 221), MDNQSNN (SEQ ID NO: 222), YQSGLLE (SEQ ID NO: 223), TGANIGY (SEQ ID NO: 224), QDNSKLS (SEQ ID NO: 225) and SSPAKPT (SEQ ID NO: 226).
  • aspects disclosed herein provide AAV capsids having significant enrichment in the SPINAL CORD over that found in the LIVER and BRAIN comprising an AAV capsid protein comprising an insertion of Formula XXIII
  • X 153 is an amino acid selected from Q, P, W, M, S ,R, D, V and I;
  • X 154 is an amino acid selected from E, D, W, L, P, H, Y, G and S;
  • X 155 is an amino acid selected from N, G, H, F and D;
  • X 156 is an amino acid selected from D, E, P, H, R, T, N and G;
  • X 157 is an amino acid selected from L, H, Q, G, P, Y, T, S and R;
  • X 158 is an amino acid selected from V, T, S, P, H, N and G;
  • X 159 is an amino acid selected from S, T, H, A, L and E;
  • X 155 is not N when X 154 is L or when X 159 is L or when X 158 is N or V; further provided X 155 is not G when X 156 is G or when X 158 is P or when X 159 is A or when X 154 is L; further provided X 155 is not D when X 158 is S; further provided X 155 is not H when X 154 is S or when X 159 is S; further provided X 157 is not S when X 159 is A or when X 154 is G; further provided X 158 is not G when X 154 is L or when X 153 is M; further provided X 153 is not S when X 154 is P or S; further provided X 154 is not S when X 157 is R; further provided X 153 is not P when X 156 is N; further provided X153 and X 154 are not both D; and further provided X 153 is not V when X 154 is Y.
  • the insertion sequence comprises a sequence of Formula XXIII wherein X 154 is E. In some embodiments, the insertion sequence comprises a sequence of Formula XXIII wherein X 159 is E. In some embodiments, the insertion sequence comprises a sequence of Formula XXIII wherein X 159 is S or T.
  • the insertion sequence as described in Table 23, is selected from QEGNLVS (SEQ ID NO: 227), PDNTTTS (SEQ ID NO: 228), WSGTLVH (SEQ ID NO: 229), MLHGHHL (SEQ ID NO: 230), VWHDQSA (SEQ ID NO: 231), IPFPGPE (SEQ ID NO: 232), SHHHPTT (SEQ ID NO: 233), RYDERNA (SEQ ID NO: 234), IGNRYPT (SEQ ID NO: 235) and DEDRSGE (SEQ ID NO: 236).
  • AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula XXIV X 160 -X 161 -T-T-K (XXIV) (SEQ ID NO: 25) wherein: X 160 is an amino acid selected from L, I, A, S, T and E; and X 161 is an amino acid selected from N and H.
  • the insertion sequence comprises a sequence of Formula XXIV wherein X 160 is L and X 161 is N.
  • AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula XXIVa
  • X 160 -X 161 -T-T-K-X 162 (XXIVa) (SEQ ID NO: 26) wherein X 160 is an amino acid selected from L, I, A, S, T and E; X 161 is an amino acid selected from N and H; and X 162 is an amino acid selected from P, L, M, N, R, S and D.
  • the insertion sequence comprises a sequence of Formula XXIVa wherein X 160 is L, X 161 is N and X 162 is S or P
  • AAV capsids comprising an AAV capsid protein comprising an insertion of Formula XXIVb
  • X 160 -X 161 -T-T-K-X 162 -X 163 (XXIVb) (SEQ ID NO: 27) wherein X 160 is an amino acid selected from L, I, A, S, T and E; X 161 is an amino acid selected from N and H; X 162 is an amino acid selected from M, P, N, R, S and D; and X 163 is an amino acid selected from P, I, Y, F, Q, E, S and L.
  • the insertion sequence comprises a sequence of Formula XXIVb wherein X 160 is L, X 161 is N, X 162 is S or P and X 163 is I.
  • the insertion sequence is selected from ANTTKDL (SEQ ID NO: 237), INTTKMY (SEQ ID NO: 238), TNTTKNF (SEQ ID NO: 239), ENTTKRE (SEQ ID NO: 240), LNTTKPI (SEQ ID NO: 241), SHTTKPQ (SEQ ID NO: 242) and GNTTKSS (SEQ ID NO: 243).
  • AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula XXV
  • X 164 is an amino acid selected from I, L, A, G, S, T, K and R;
  • X 165 is an amino acid selected from K, R, I, L, A and G;
  • X 166 is an amino acid selected from T, N, Q and S;
  • X 167 is an amino acid selected from I, L, A, G, E, D, S and T.
  • the AAV capsid protein comprises an insertion sequence of Formula
  • X 164 is an amino acid selected from I, L, A, G, S, T and R; X 165 is an amino acid selected from K, R and G; X 166 is an amino acid selected from T, N and S; and X 167 is an amino acid selected from I, A, E, D, S and T.
  • X 164 is an amino acid selected from I, T and R;
  • X 165 is an amino acid selected from K and R;
  • X 166 is an amino acid selected from T, N and S;
  • X 167 is an amino acid selected from I, D, S and T.
  • the insertion sequence is selected from ENHIKTI (SEQ ID NO: 244), ENHTRNS (SEQ ID NO: 245), ENHTKND (SEQ ID NO: 246) and ENHRGST (SEQ ID NO: 247).
  • AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula XXVI
  • X 168 -S-R-E-X 169 -X 170 -X 171 (SEQ ID NO: 29) wherein X 168 is an amino acid selected from D, H, I, K, M and N;
  • X 169 is an amino acid selected from F, S, W, A, L and R;
  • X 170 is an amino acid selected from K, N, S, Y, L, T, E and D;
  • X 171 is an amino acid selected from I, K, V, Y, A, T and S.
  • the AAV capsid protein comprises an insertion sequence of Formula
  • X 168 is an amino acid selected from D, I and K
  • X 169 is an amino acid selected from F, S, W, A and L
  • X 170 is an amino acid selected from K, N, Y, L, T, E and D
  • X 171 is an amino acid selected from I, K, Y, A and T.
  • the insertion sequence is selected from DSRESNK (SEQ ID NO: 248), HSREFSV (SEQ ID NO: 249), ISREFYK (SEQ ID NO: 38), ISRESLY (SEQ ID NO: 250), ISREWTA (SEQ ID NO: 251), KSREAEY (SEQ ID NO: 252), KSRELDT (SEQ ID NO: 253) and NSRESEA (SEQ ID NO: 254).
  • AAV capsids comprising an AAV capsid protein comprising an insertion sequence of Formula XXVII
  • X 172 -N-X 173 -X 174 -X 175 -X 176 -X 177 (XXVII) (SEQ ID NO: 30) wherein X 172 is an amino acid selected from G, T, D, L and E;
  • X 173 is an amino acid selected from T, S, M, N and H;
  • X 174 is an amino acid selected from V, T and I;
  • X 175 is an amino acid selected from R and K;
  • X 176 is an amino acid selected from D, Q, N, S and P;
  • X 177 is an amino acid selected from I, V, Y, L, T and S;
  • X 172 is not T when X 174 is T or X 173 is N; further provided X 175 is not R when X 176 is P or when X 171 is L; further provided X 171 is not E when X 173 is M.
  • the insertion sequence comprises a sequence of Formula XXVII wherein X 172 is G. In some embodiments, the insertion sequence comprises a sequence of Formula XXVII wherein X 173 is T. In some embodiments, the insertion sequence comprises a sequence of Formula XXVII wherein X 174 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXVII wherein X 176 is S.
  • the insertion sequence as described in Table 24 is selected from GNTTRDY (SEQ ID NO: 255), GNMVKQV (SEQ ID NO: 256), TNSVKNL (SEQ ID NO: 257), GNNVKSI (SEQ ID NO: 258), DNSTRSV (SEQ ID NO: 259), LNTTKPI (SEQ ID NO: 241), GNTTKSS (SEQ ID NO: 243), ENNIRSI (SEQ ID NO: 260), DNSIRNT (SEQ ID NO: 261) and ENHTRNS (SEQ ID NO: 245).
  • AAV capsids having the best expression in the BRAIN of the insertions expressed in the one spinal cord group comprising an AAV capsid protein comprising an insertion sequence of Formula XXVIII
  • X 178 -N-X 179 -X 180 -X 181 -X 182 -X 183 (XXVIII) (SEQ ID NO: 31) wherein X 178 is an amino acid selected from N, Q, A, S, G and E;
  • X 179 is an amino acid selected from R, V, S, N and T;
  • X 180 is an amino acid selected from R, I, T and V;
  • X 181 is an amino acid selected from M, P, R and K;
  • X 182 is an amino acid selected from D, L, N, R, A and P; and X 183 is an amino acid selected from D, T, I, M, L, N and V;
  • X 178 is not S when X 181 is K or when X 179 is R; further provided X 181 is not R when X 180 is I or when X 182 is L or when X 183 is T; and further X180 is not T when X182 is A.
  • the insertion sequence comprises a sequence of Formula XXVIII wherein X 178 is N, and X 183 is L. In some embodiments, the insertion sequence comprises a sequence of Formula XXVIII wherein X 179 is T, and X 183 is L. In some embodiments, the insertion sequence comprises a sequence of Formula XXVIII wherein X 179 is T, X 182 is N, and X 183 is L.
  • the insertion sequence as described in Table 27, is selected from NNRRPDD (SEQ ID NO: 262), QNVIKPT (SEQ ID NO: 263), QNSTKLI (SEQ ID NO: 264), ANNTRNM (SEQ ID NO: 265), SNTTRNL (SEQ ID NO: 266), ENSVRNN (SEQ ID NO: 267), NNSTKLL (SEQ ID NO: 268), GNSVRAN (SEQ ID NO: 269), SNSTRPL (SEQ ID NO: 270) and GNSTMRV (SEQ ID NO: 271).
  • AAV capsids having the best expression in the BRAIN of the insertions expressed in another spinal cord group comprising an AAV capsid protein comprising an insertion sequence of Formula XXIX
  • X 184 -X 185 -X 186 -X 187 -X 188 -X 189 -X 190 (XXIX) (SEQ ID NO: 32) wherein X 184 is an amino acid selected from G, T, M, S, A and Y;
  • X 185 is an amino acid selected from K, V, N and D;
  • X 186 is an amino acid selected from S, K, V, R, T and H;
  • X 187 is an amino acid selected from M, G, V, I, T and K;
  • X 188 is an amino acid selected from K, L, R, S and G;
  • X 189 is an amino acid selected from N, S, D, I and L;
  • X 190 is an amino acid selected from F, M, T, Y, N, G, V and Q; provided X 185 is not N when X 184 is M or A or when X 186 is H; further provided X 185 is not V when X 184 is T; further provided X 185 is not D when X 184 is Y ; further provided X 186 is not S when X 190 is V; further provided X 186 and X 190 are not both T; further provided X 186 is not R when X 188 is S; and further provided X 187 is not V when X 188 is R.
  • the insertion sequence comprises a sequence of Formula XXIX wherein X 185 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXIX wherein X 186 is S. In some embodiments, the insertion sequence comprises a sequence of Formula XXIX wherein X 189 is N.
  • the insertion sequence as described in Table 28 is selected from GNSTKIG (SEQ ID NO: 272), TNTTKNF (SEQ ID NO: 239), MKSGLSM (SEQ ID NO: 273), SNKMGNT (SEQ ID NO: 274), SNSVKDY (SEQ ID NO: 275), AVHKSDF (SEQ ID NO: 276), SNSIRNN (SEQ ID NO: 277), TDRMGLT (SEQ ID NO: 278), SNVIKNV (SEQ ID NO: 279) and YNSTRNQ (SEQ ID NO: 280).
  • AAV capsids having the best expression in the BRAIN of the insertions expressed in the brain and the spinal cord comprising an AAV capsid protein comprising an insertion sequence of Formula XXX
  • X 191 -X 192 -X 193 -X 194 -X 195 -X 196 -X 197 (SEQ ID NO: 33) wherein X 191 is an amino acid selected from G, D, N, T, L, S, I, Q and F; X 192 is an amino acid selected from G, S, V, N and R;
  • X 193 is an amino acid selected from E, V, R, T, S, N and H;
  • X 194 is an amino acid selected from I, D, L, N, S, V, R and T;
  • X 195 is an amino acid selected from L, P, R, I, K and V;
  • X 196 is an amino acid selected from R, P, N, A, T, S, V, M and K;
  • X 197 is an amino acid selected from D, T, L, N, E, I and G;
  • X 191 is not S when X 192 is G or when X 193 is N; further provided X 192 is not V when X 191 is L or T; further provided X 192 is not R when X 193 is S or when X 196 is A; further provided X 192 is not G when X 191 is Q or when X 195 is L; further provided X 192 is not S when X 195 is L or R; further provided X 193 is not T when X 191 is T or when X 194 is T or V; further provided X 192 is not N when X 193 is N or when X 194 is T or when X 197 is G; further provided X 193 is not S when X 191 is L or N; further provided X 194 is not N when X 191 is S or F; further provided X 195 is not P when X 191 is L or when X 193 is V; further provided X 197 is not T when X 193 is S or when X
  • the insertion sequence comprises a sequence of Formula XXX wherein X 192 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXX wherein X 195 is R. In some embodiments, the insertion sequence as described in Table 26, is selected from GNEVRRD (SEQ ID NO: 281), DNVIRPT (SEQ ID NO: 282), NVRDLNL (SEQ ID NO: 283), TSRLPAL (SEQ ID NO: 284), LNTNRTN (SEQ ID NO: 285), SRTSISE (SEQ ID NO: 286), SNSVRND (SEQ ID NO: 287), IGNRPVI (SEQ ID NO: 288), QNTIKMT (SEQ ID NO: 289) and FSHTVKG (SEQ ID NO: 290).
  • GNEVRRD SEQ ID NO: 281
  • DNVIRPT SEQ ID NO: 282
  • NVRDLNL SEQ ID NO: 283
  • TSRLPAL SEQ ID NO: 284
  • AAV capsids having greater expression in the BRAIN and low expression in the spinal cord comprising an AAV capsid protein comprising an insertion sequence of Formula XXXI
  • X 198 -X 199 -X 200 -X 201 -X 202 -X 203 -X 204 (XXXI) (SEQ ID NO: 34) wherein X 198 is an amino acid selected from R, E, M, S, N, L, T and G; X 199 is an amino acid selected from N, S and R;
  • X 200 is an amino acid selected from D, S, N and A;
  • X 201 is an amino acid selected from M, S, K, V and T;
  • X 202 is an amino acid selected from D, R, A and K;
  • X 203 is an amino acid selected from P, Y, Q, R, M, A and G;
  • X 204 is an amino acid selected from F, T, L, Y, I and S;
  • X 199 is not S when X 201 is S; further provided X 198 is not S when X 200 is S; further provided X 200 is not N when X 198 is T or G; further provided X 198 is not N when X 204 is T; further provided X 202 is not R when X 203 is Q; and further provided X 198 is not T when X 200 is D.
  • the insertion sequence comprises a sequence of Formula XXXI wherein X 199 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXXI wherein X 200 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXXI wherein X 201 is T. In some embodiments, the insertion sequence comprises a sequence of Formula XXXI wherein X 202 is R.
  • the insertion sequence as described in Table 25, is selected from RRDMDPT (SEQ ID NO: 291), ENSTRYT (SEQ ID NO: 292), MNSTRPF (SEQ ID NO: 293), SNNVKQT (SEQ ID NO: 294), SNNSRPY (SEQ ID NO: 295), NNSTARI (SEQ ID NO: 296), LSNKAML (SEQ ID NO: 297), TNATRPL (SEQ ID NO: 298), GNAVRGT (SEQ ID NO: 299) and GNSTKAS (SEQ ID NO: 300).
  • RRDMDPT SEQ ID NO: 291
  • ENSTRYT SEQ ID NO: 292
  • MNSTRPF SEQ ID NO: 293
  • SNNVKQT SEQ ID NO: 294
  • SNNSRPY SEQ ID NO: 295
  • NNSTARI SEQ ID NO: 296
  • LSNKAML SEQ ID NO: 297
  • TNATRPL SEQ ID NO: 298
  • X 207 is an amino acid selected from S, R, G, K and N;
  • X 208 is an amino acid selected from H, D, N, Q, S, E and T;
  • X 209 is an amino acid selected from G, S, R, I, N, A and Q;
  • X 210 is an amino acid selected from S, N, R, E, T, M and Q;
  • X 211 is an amino acid selected from K, N, V, R, S, and F;
  • X 206 is not L when X 205 is N or when X 208 is T or when X 210 is S; further provided X 206 is not S when X 205 is G or when X 209 is N; further provided X 207 is not G when X 205 is L or N; further provided X 207 is not S when X 208 is S or when X 210 is T; further provided X 211 is not S when X 207 is R or when X 209 is G or when X 210 is S; further provided X 205 is not S when X 207 is N; further provided X 206 is not N when X 208 is S; further provided X 206 is not T when X 211 is V; and further provided X 209 is not A when X 210 is Q.
  • the insertion sequence comprises a sequence of Formula XXXII wherein X 211 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXXII wherein X 205 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXXII wherein X 208 is S.
  • the insertion sequence as described in Table 6, is selected from EQSHGSK (SEQ ID NO: 301), LLRDSNN (SEQ ID NO: 302), ILGNSRV (SEQ ID NO: 303), VDKQREN (SEQ ID NO: 304), NDNQITR (SEQ ID NO: 305), GTNSSTS (SEQ ID NO: 306), LIKENRF (SEQ ID NO: 307), SSSTAMS (SEQ ID NO: 308), FQNSQTR (SEQ ID NO: 309) and NTSQSQK (SEQ ID NO: 310).
  • AAV capsids having greater enrichment in both SPINAL CORD and BRAIN over that found in the LIVER comprising an AAV capsid protein comprising an insertion sequence of Formula XXXIII X 212_ X 213_ X 214_ X 215_ X 216_ X 217_ X 218 ( XXXIII ) (SEQ ID N0: 36) wherein X 212 is an amino acid selected from T, A, S, E, N, L and F;
  • X 213 is an amino acid selected from Q, L, E, N, P and S;
  • X 214 is an amino acid selected from P, V, Y, M, H, E, D and L;
  • X 215 is an amino acid selected from T, S, G, I, T, V and H;
  • X 216 is an amino acid selected from M, G, T, K, Q, P, N, L and T;
  • X 217 is an amino acid selected from E, D, K, N, T, S, N and Y;
  • X 218 is an amino acid selected from N, V, H, I, R, S, and A;
  • X 212 is not A when X 213 is S or when X 215 is T; further provided X 212 is not T when X 214 is H or when X 218 is V; further provided X 218 is not S when X 215 is T or when X 217 is S; further provided X 212 is not L when X 214 is P; further provided X 212 is not S when X 213 is L; further provided X 213 is not N when X 218 is A; further provided X 214 is not V when X 218 is R; further provided X 214 is not L when X 218 is N; further provided X 214 is not D when X 216 is M; further provided X 215 is not S when X 216 is L; and further provided X 216 is not T when X 217 is T.
  • the insertion sequence comprises a sequence of Formula XXXIII wherein X 213 is N. In some embodiments, the insertion sequence comprises a sequence of Formula XXXIII wherein X 215 is T, In some embodiments, the insertion sequence comprises a sequence of Formula XXXIII wherein X 216 is T.
  • the insertion sequence as described in Table 13, is selected from TQPTMEN (SEQ ID NO: 311), ALVSGDV (SEQ ID NO: 312), SEYGTKH (SEQ ID NO: 313), ENMTKNI (SEQ ID NO: 314), ENHIKTI (SEQ ID NO: 244), NNVSQEI (SEQ ID NO: 315), TPEGPSN (SEQ ID NO: 316), LNDTNER (SEQ ID NO: 317), NSLVLNS (SEQ ID NO: 318) and FEPHTYA (SEQ ID NO: 319).
  • the insertion sequence is represented by the peptide sequences listed in Table 1.
  • the insertion amino acid sequence is at least 71.4% identical to the amino acid sequence provided in Tables 1 and 4-30, Figure 5 and/or Formulas I- XXXIII. In some aspects, the insertion amino acid sequence is at least 86.7% identical to the amino acid sequence provided in Tables 1 and 4-30, Figure 5 and/or Formulas I- XXXIII.
  • Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid (e.g., a transgene, therapeutic nucleic acid).
  • an episomal heterologous nucleic acid e.g., a transgene, therapeutic nucleic acid.
  • a rAAV Upon delivery to a host in vivo environment, a rAAV will (1) bind or attach to cellular surface receptors on the target cell, (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid , (5) convert of the heterologous nucleic acid from single- stranded to double- stranded DNA as a template for transcription in the nucleus, and (6) transcribe of the episomal heterologous nucleic acid in the nucleus of the host cell (“transduction”).
  • An rAAV comprises an AAV capsid that can be engineered to encapsidate a heterologous nucleic acid (e.g., therapeutic nucleic acid, gene editing machinery).
  • the AAV capsid is made up of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1:1: 10 ratio to form the viral capsid.
  • VP1 covers the whole of VP2 protein in addition to a -137 amino acid N-terminal region (VPlu), VP2 covers the whole of VP3 in addition to -65 amino acid N-terminal region (VP1/2 common region).
  • the three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736).
  • a parent AAV capsid sequence comprises a VP1 region.
  • a parent AAV capsid sequence comprises a VP1, VP2 and/or VP3 region, or any combination thereof.
  • a parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.
  • the AAV VP3 structure contains highly conserved regions that are common to all serotypes, a core eight-stranded P-barrel motif (
  • the loop regions inserted between the P-strands consist of the distinctive HI loop between P-strands H and I, the DE loop between P-strands D and E, and nine variable regions (VRs), which form the top of the loops.
  • VRs such as the AA588 loop, are found on the capsid surface and can be associated with specific functional roles in the AAV life cycle including receptor binding, transduction and antigenic specificity.
  • AAV capsids comprising AAV capsid proteins with a substitution at the 588 loop that confer a desired tropism characterized by a higher efficiency and specificity for transduction in specific cell-types, including for e.g., CNS, brain cell types (e.g., brain endothelial cells, neurons, astrocytes).
  • the AAV capsid proteins disclosed herein enable rAAV-mediated transduction of a heterologous nucleic acid (e.g., transgene) in the CNS of a subject.
  • the AAV capsids of the present disclosure, or the AAV capsid proteins may be formulated as a pharmaceutical composition.
  • the AAV capsids or the AAV capsid proteins can be isolated and purified to be used for a variety of applications.
  • rAAV capsids which comprise AAV capsid proteins that are engineered with a modified capsid protein (e.g., VP1, VP2, VP3).
  • the rAAV capsid proteins of the present disclosure are generated using the methods disclosed herein.
  • the AAV capsid proteins are used in the methods of delivering a therapeutic nucleic acid (e.g., a transgene) to a subject.
  • the rAAV capsid proteins have desired AAV tropisms rendering them particularly suitable for certain therapeutic applications, e.g., the treatment of a disease or disorder in a subject such as those disclosed herein.
  • the rAAV capsid proteins are engineered for optimized expression in the CNS, for example the brain, of a subject upon systemic administration of the rAAV to the subject, such as those insertions provided in Tables 1 and 4-30, FIG. 5 and/or Formulas I-XXXIII.
  • the rAAV capsid proteins provided in Tables 1 and 4-30, FIG. 5 and/or Formulas I-XXXIII are engineered to have tropisms that eliminate the need for intracranial injection, while also achieving widespread and efficient transduction of an encapsidated transgene.
  • the tropisms comprise at least one of an increased specificity and efficiency (e.g., of viral transduction) in the CNS of a subject, as compared to a reference AAV.
  • the engineered AAV capsid proteins described herein have, in some cases, an insertion of an amino acid that is heterologous to the parental AAV capsid protein at the amino acid position in the 588 loop.
  • the amino acid is not endogenous to the parental AAV capsid protein at the amino acid position of the insertion.
  • the amino acid may be a naturally occurring amino acid in the same or equivalent amino acid position as the insertion of the substitution in a different AAV capsid protein.
  • rAAVs with engineered capsid proteins that are optimized for targeting specific organ or tissue within a subject.
  • the rAAVs of the present embodiment have increased specificity and transduction in the CNS .
  • the insertion comprises a five-, six-, or seven-amino acid sequence (5-mer, 6- mer, or 7-mer, respectively) that is inserted or substituted at the 588 loop in a parental AAV capsid protein.
  • amino acid insertions comprising seven amino acid polymer (7-mer) inserted at AA588-589, and may additionally include a substitution of one or two amino acids at amino acid positions flanking the 7-mer sequence (e.g., AA587-588 and/or AA589-590) to produce an eleven amino acid polymer (11-mer) at the 588 loop of a parental AAV capsid protein.
  • the 7-mers described herein were advantageously generated using polymerase chain reaction (PCR) with degenerate primers, where each of the seven amino acids is encoded by a deoxyribose nucleic acid (DNA) sequence N-N-K.
  • N is any of the four DNA nucleotides and K is guanine (G) or thymine (T). This method of generating random 7-mer amino acid sequences enables 1.28 billion possible combinations at the protein level.
  • Peptide insertion sequences of the disclosure include sequences that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) alter binding affinities, and (3) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., equivalent, conservative or non-conservative substitutions, deletions or additions) may be made in a sequence.
  • a conservative amino acid substitution refers to the substitution of an amino acid in an insertion sequence with a functionally similar amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and/or aromaticity.
  • the following six groups each contain amino acids that are conservative substitutions for one another are found in Table 2.
  • one amino acid may be substituted for another, in one embodiment, within the groups of amino acids indicated herein below:
  • Amino acids with polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,)
  • sequence relationships between two or more nucleic acids or nucleic acids or polypeptides are used to describe the sequence relationships between two or more nucleic acids or nucleic acids or polypeptides: (a)“reference sequence,” (b) “comparison window,” (c)“sequence identity,” (d)“percentage of sequence identity,” and (e)“substantial identity.”
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • the reference sequence can be a nucleic acid sequence.
  • a reference sequence may be a subset or the entirety of a specified sequence.
  • a reference sequence may be a segment of a full-length cDNA or of a genomic DNA sequence, or the complete cDNA or complete genomic DNA sequence, or a domain of a polypeptide sequence.
  • comparison window refers to a contiguous and specified segment of a nucleic acid or an amino acid sequence, wherein the nucleic acid/amino acid sequence can be compared to a reference sequence and wherein the portion of the nucleic acid/amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window can vary for nucleic acid and polypeptide sequences. Generally, for nucleic acids, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or more nucleotides.
  • the comparison window is at least about 10 amino acids, and can optionally be 15, 20, 30, 40, 50, 100 or more amino acids.
  • a gap penalty is typically introduced and is subtracted from the number of matches.
  • Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art.
  • the local homology algorithm (BESTFIT) of Smith and Waterman (1981) Adv. Appl. Math 2:482, may permit optimal alignment of compared sequences; by the homology alignment algorithm (GAP) of Needleman and Wunsch (1970) J. Mol. Biol.
  • the BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-53, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP makes a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or
  • GAP presents one member of the family of best alignments. There may be many members of this family. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity.
  • the Quality is the metric maximized in order to align the sequences.
  • Ratio is the quality divided by the number of bases in the shorter segment.
  • Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • the scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see: Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915).
  • Sequence identity/similarity values provided herein can refer to the value obtained using the BLAST+ 2.5.0 suite of programs using default settings (blast.ncbi.nlm.nih.gov) (Camacho, C., et al. (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
  • a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17: 149-63) and XNU (Ci- ayerie and States (1993) Comput. Chem. 17: 191-201) low-complexity filters can be employed alone or in combination.
  • substantially identical indicates that a polypeptide or nucleic acid comprises a sequence with between 55-100% sequence identity to a reference sequence, with at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity or any percentage of value within the range of 55-100% sequence identity relative to the reference sequence.
  • the percent sequence identity may occur over a specified comparison window.
  • Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, supra.
  • the insertion sequences may include, but are not limited to, sequences that are not exactly the same as the sequences disclosed herein, but which have, in addition to the substitutions explicitly described for various sequences listed herein, additional substitutions of amino acid residues which substantially do not impair the activity or properties of the sequences described herein, such as those predicted by homology software e.g. BLOSUM62 matrices.
  • additional substitutions of amino acid residues which substantially do not impair the activity or properties of the sequences described herein, such as those predicted by homology software e.g. BLOSUM62 matrices.
  • conservative amino acid substitutions may include, but are not limited to the sequences of Formulas I- III.
  • Recombinant AAVs were generated, each with a unique 7-mer at the 588 loop and each encapsidating a reporter gene that, when administered systemically in NHPs, enabled the selective amplification and recovery of sequences that effectively transduced the reporter gene in a target in vivo environment of the transgenic animal.
  • 7-mers that were found to be positively enriched in the target in vivo environment e.g, central nervous system, liver
  • “A subset of the rAAVs with desired expression profiles were tested individually in vivo to determine exact systemic expression (e.g., specificity and transduction efficiency).
  • rAAVs from this subset exhibiting a desired tropism comprising increased specificity of viral transduction, and in some cases, transduction efficiency are considered to be uniquely suited for targeted rAAV-mediated transgene delivery useful for a wide variety of purposes (e.g., therapeutic, diagnostic, scientific discovery).
  • the rAAV particles with the insertion sequences described herein have an increased transduction efficiency in a target in vivo environment (e.g., tissue or cell type).
  • the increased transduction efficiency comprises a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold increase, or more, relative to a reference AAV.
  • the increased transduction efficiency is at least 2-fold.
  • the increased transduction efficiency is at least 10-fold.
  • the increased transduction efficiency is at least 20-fold.
  • the rAAV particles with the insertion sequences described herein have an increased expression efficiency or specificity in a target in vivo environment (e.g., tissue or cell type).
  • the increased specificity is correlated with increased viral genomes or an increased expression in the target in vivo environment, which in some cases is represented with expression values provided herein in Tables 1 and 4-30 and/or FIG. 5.
  • the reference AAV may have a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof.
  • the rAAV capsid proteins of the present disclosure comprise an insertion of an amino acid in an amino acid sequence of an AAV capsid protein.
  • the AAV capsid, from which an engineered AAV capsid protein of the present disclosure is produced, is referred to as a “parental” AAV capsid.
  • the parental AAV has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.
  • the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J.
  • the parental AAV is derived from an AAV with a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.
  • the AAV capsid protein that is “derived” from another may be a variant AAV capsid protein.
  • a variant may include, for example, a heterologous amino acid in an amino acid sequence of the AAV capsid protein.
  • the heterologous amino acid may be non-naturally occurring in the AAV capsid protein.
  • the heterologous amino acid may be naturally occurring in a different AAV capsid protein.
  • the parental AAV capsid is described in US Pat Publication 2020/0165576 and U.S. Pat. App. Ser. No. 62/832,826 and PCT/US20/20778; the content of each of which is incorporated herein..
  • the parental AAV is AAV9.
  • the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1.
  • the parental AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 1.
  • AAV capsid proteins from native AAV serotypes, such as AAV9, with tropisms including the liver activate the innate immune response, which is come cases causes a severe inflammatory response in a subject, which can lead to multi-organ failure.
  • the rAAV particles of the present disclosure reduce the immunogenic properties of AAV-mediated transgene delivery and prevent activation of the innate immune response.
  • the parental AAV is AAV9.
  • the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1.
  • the parental AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 1, or part of SEQ ID NO: 1.
  • the parental AAV capsid protein comprises the entire VP1 region provided in SEQ ID NO: 1 (e.g., amino acids 1-736).
  • the parental AAV capsid protein comprises amino acids 217-736 in SEQ ID NO: 1, which is the common region found in VP1, VP2 and VP3 AAV9 capsid proteins.
  • the AAV capsid protein comprises amino acids 64- 736 in SEQ ID NO: 1, which is the common region found in VP1 and VP2.
  • the parental AAV capsid protein sequence may comprise amino acids selected from 1-736, 10-736, 20-736, 30-736, 40-736, 50-736, 60-736, 70-736, 80-736, 90-736, 100-736, 110-736, 120-736, 130-736, 140-736, 150-736, 160-736, 170-736, 180-736, 190-736, 200-736, 210-736, 220-736, 230-736, 240-736,
  • insertions of an amino acid sequence in an AAV capsid protein are disclosed herein. Where the sequence numbering designation “588-589” is noted for AAV9, for example AAV VP1, the invention also includes insertions in similar locations in the other AAV serotypes. As used herein, “AA588-589” indicates that the insertion of the amino acid (or amino acid sequence) is immediately after an amino acid (AA) at position 588 and immediately before an AA at position 589 within an amino acid sequence of a parental AAV VP capsid protein (VP1 numbering). Amino acids 587-591 include a motif comprising “AQAQA” as set forth in SEQ ID NO: 1. Exemplary AAV capsid protein sequences are provided in Table 3.
  • GNTTRDY (SEQ ID NO: 255) is inserted at AA588-589 in an AAV9 capsid amino acid sequence and provides variant C (SEQ ID NO: 376). It is envisioned that the insertions disclosed herein (Tables 1 and 4-30, FIG. 5 and/or Formulas I-XXXIII) may be inserted at AA588-589 in an amino acid sequence of a parental AAV9 capsid protein, a variant thereof, or equivalent amino acid position a parental AAV of a different serotype (e.g., AAV1, AAV2, AAV3, and the like).
  • a parental AAV9 capsid protein e.g., AAV1, AAV2, AAV3, and the like.
  • the rAAV capsid proteins described herein may be isolated and purified.
  • the AAV may be isolated and purified by methods standard in the art such as by column chromatography, iodixanol gradients or cesium chloride gradients. Methods for purifying AAV from helper virus are known in the art and may include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98/09657.
  • the rAAV capsid protein can be conjugated to a nanoparticle, a second molecule, or a viral capsid protein.
  • the nanoparticle or viral capsid protein would encapsidate the therapeutic nucleic acid described herein.
  • the second molecule is a therapeutic agent, e.g., a small molecule, antibody, antigen-binding fragment, peptide, or protein, such as those described herein.
  • AAV capsid proteins with an insertion of at least one amino acid at an amino acid position described above in a parental AAV capsid protein that confers an increased efficiency or specificity for the CNS in a subject, even when delivered systemically.
  • AAV capsid proteins described herein is their ability to target tissue and cells within the CNS.
  • the tissue can be the brain or the spinal cord.
  • Non-limiting examples of CNS cells include a neuron and a glial cell.
  • Glial cells can be selected from an oligodendrocyte, an ependymal cell, an astrocyte and a microglia.
  • the in vivo environment can be a tissue.
  • the tissue can be the brain, or the spinal cord.
  • the tissue can be a region of an organ, for example, the cerebrum, the cerebellum, the brainstem, the cortex, the striatum, the thalamus, the lateral ventricles, the putamen, the hypothalamus, the medulla, the pons, the hippocampus, the amygdala, the motor cortex, or a combination thereof.
  • AAV capsid proteins with at least one amino acid insertion in a parental AAV capsid protein.
  • the insertion can be of at least five, six, or seven amino acids, or more. In some instances, the amino acids are contiguous. In some instances, the amino acids are not contiguous.
  • the insertion is of at least five amino acids provided in any one of the sequences provided in any one of Tables 1 and 4-30, FIG. 5 and/or Formulas I- XXXIII. In some instances, the insertion is of at least six amino acids provided in any one of Tables 1 and 4-30, FIG. 5 and/or Formulas I- XXXIII. In some instances, the insertion is of at least seven amino acids provided in any one of Tables 1 and 4-30, FIG. 5 and/or Formulas I- XXXIII.
  • the AAV capsid protein comprises an insertion of at least or about five, six, or seven amino acids of an amino acid sequence of Tables 1 and 4-30, FIG. 5 and/or Formulas I- XXXIII at an amino acid position 588-589 in a parental AAV9 capsid protein (SEQ ID NO: 1).
  • the AAV capsid protein has an increased specificity for viral transduction in brain cortex
  • the rAAV capsid proteins of the present disclosure may also have a substitution of an amino acid sequence at amino acid position 452- 458 in a parental AAV9 capsid protein, or variant thereof, as described in W02020068990.
  • the substitution of the amino acid sequence comprises KDNTPGR (SEQ ID NO: 367) at amino acid position 452- 458 in the parental AAV9 capsid protein.
  • the substitution of the amino acid sequence comprises DGAATKN (SEQ ID NO: 368) at amino acid position 452- 458 in the parental AAV9 capsid protein
  • the AAV capsids and AAV capsid proteins disclosed herein are isolated. In some instances, the AAV capsids and AAV capsid proteins disclosed herein are isolated and purified. In addition, the AAV capsids and AAV capsid proteins disclosed herein, either isolated and purified, or not, may be formulated into a pharmaceutical formulation, which in some cases, further comprises a pharmaceutically acceptable carrier.
  • An AAV vector can comprise a viral genome comprising a nucleic acid encoding the recombinant AAV (rAAV) capsid protein described herein.
  • the viral genome can comprise a Replication (Rep) gene encoding a Rep protein, and Capsid (Cap) gene encoding an AAP protein in the first open reading frame (ORF1) or a Cap protein in the second open reading frame (ORF2).
  • the Rep protein is selected from Rep78, Rep68, Rep52, and Rep40.
  • the Cap gene is modified encoding a modified AAV capsid protein described herein.
  • a wild-type Cap gene encodes three proteins, VP1, VP2, and VP3.
  • VP1 is modified.
  • VP2 is modified.
  • VP3 is modified.
  • all three VP1-VP3 are modified.
  • the AAV vector can comprise nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins.
  • the AAV9 VP1 gene provided in SEQ ID NO: 384 is modified to include any one of SEQ ID NOS: 385-654.
  • the AAV vector described herein may be used to produce a variant AAV capsid by the methods described herein.
  • the nucleic acid sequence of the AAV9 VP1 (AAV9 >AY530579.1 Adeno-associated virus 9 isolate hu.14 capsid protein VP1 (cap) gene, complete cds) gene is provided below (SEQ ID NO: 384):
  • the 5' ITR and the 3' ITR are derived from an AAV2 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV5 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV9 serotype.
  • methods of the invention may include administering to a patient suspected of having mucopolysaccharidosis type II a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) such as those discussed above and containing an expression vector encoding IDS.
  • delivery of the vector comprises administering to the subject the composition using any one of the routes of administration described herein.
  • methods of increasing transduction of an encoded gene in a target in vivo environment comprise delivering a rAAV particle described herein, the rAAV engineered to have an increased transduction efficiency in a target in vivo environment (e.g., tissue or cell type).
  • the increased transduction efficiency comprises a 1-fold, 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or 100-fold increase, or more, relative to a reference AAV.
  • the increased transduction efficiency is at least 2-fold.
  • the increased transduction efficiency is at least 10-fold.
  • the increased transduction efficiency is at least 20-fold.
  • Methods of delivering a heterologous nucleic acid to a target in vivo environment comprising delivering the rAAV particle described herein that has been engineered to have an increased expression or specificity in an in vivo environment (e.g., tissue or cell type), as compared to a reference AAV.
  • Methods in some cases, comprise detecting whether a rAAV possesses more specificity for an in vivo environment, includes measuring a level of gene expression product (e.g., IDS) expressed from the vector encapsidated by the rAAV in a tissue sample obtained from the in vivo environment in a subject;
  • a level of gene expression product e.g., IDS
  • the reference AAV has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof.
  • the rAAV particle encapsidating the heterologous nucleic acid comprises a rAAV capsid protein engineered with an increased specificity and, in some cases, transduction efficiency when measured in the CNS of the subject, even when administered to the subject systemically.
  • Methods may comprise delivering a rAAV particle comprising an rAAV capsid protein with increased specificity and/or transduction efficiency when measured in the CNS in the subject.
  • delivery is systemic.
  • delivery is direct (e.g., into the affected area of the CNS).
  • the rAAV capsid protein may comprise an insertion of five, six, or seven, amino acids provided in an amino acid sequence provided in any one of Tables 1 and 4-30, FIG. 5 and/or Formulas I- XXXIII, at an amino acid position 588-589 in a parental AAV capsid protein [AAV9 numbering] .
  • a subject is treated with a pharmaceutical composition comprising the rAAV particle and a pharmaceutically acceptable carrier.
  • the one or more compositions are administered to the subject alone (e.g., stand alone therapy).
  • the one or more compositions are administered in combination with an additional agent.
  • the composition is a first-line therapy for the disease or condition.
  • the composition is a second-line, third-line, or fourth-line therapy, for the disease or condition.
  • a disease or a condition associated with an aberrant expression or activity of a target gene e.g., IDS
  • gene expression product thereof e.g., I2S
  • the method comprising modulating the expression or the activity of a target gene or gene expression product in a subject by administering a rAAV encapsidating a heterologous nucleic acid of the present disclosure.
  • administration is systemic administration.
  • the expression or the activity of the target gene or gene expression product is decreased, relative to that in a normal (non-diseased) individual; and administering the rAAV to the subject is sufficient to increase the expression of the activity of the target gene or gene expression product.
  • kits for treating mucopolysaccharidosis type II, or a symptom of mucopolysaccharidosis type II comprising: (a) diagnosing a subject with mucopolysaccharidosis type II affecting a target in vivo environment; and (b) treating mucopolysaccharidosis type II by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particle, AAV vector, pharmaceutical composition), wherein the composition is engineered with an increased efficiency or specificity for the target in vivo environment.
  • a composition disclosed herein e.g., rAAV particle, AAV vector, pharmaceutical composition
  • methods disclosed herein comprise administering a therapeutic rAAV composition by systemic administration.
  • methods comprise administering a therapeutic rAAV composition by intraperitoneal injection.
  • methods comprise administering a therapeutic rAAV composition by intravenous (“i.v.”) administration.
  • i.v. intravenous
  • routes such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection percutaneous administration, intranasal administration, intralymphatic injection, rectal administration intragastric administration, intraocular administration, intracerebroventricular administration, intrathecally, or any other suitable parenteral administration.
  • routes, dosage, time points, and duration of administrating therapeutics may be adjusted.
  • administration of therapeutics is prior to, or after, onset of either, or both, acute and chronic symptoms of the disease or condition.
  • CNS central nervous system
  • central nervous system means a tissue selected from brain, thalamus, cortex, putamen, lateral ventricles, medulla, the pons, the amygdala, the motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, cerebrum, cerebellum, brain stem, and spinal cord.
  • the brain includes a variety of cortical and subcortical areas, including the frontal, temporal, occipital and parietal lobes.
  • systemic delivery is defined as a route of administration of medication or other substance into a circulatory system so that the entire body is affected. Administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation). “Circulatory system” includes both blood or cerebrospinal fluid circulatory systems. Examples of systemic administration for the CNS include intraarterial, intravenous or intrathecal injection. Other examples include administration to the cerebrospinal fluid at any location, in the spine (i.e. but not limited to lumbar) or brain (i.e. but not limited to cisterna magna). The terms “systemic administration” and “systemic delivery” are used interchangeably.
  • routes for administration includes administration into the CSF, for example via a intracerebroventricular [ICV], intrathecal cisternal, or intrathecal lumbar route.
  • ICV intracerebroventricular
  • Particular embodiments result in delivery to neurons and glial cells of the brain.
  • Other routes of delivery to the CNS/brain include, but are not limited to intracranial administration, lateral cerebroventricular administration, intranasal administration, endovascular administration, and intraparenchymal administration.
  • An effective dose and dosage of pharmaceutical compositions to prevent or treat the disease or condition disclosed herein is defined by an observed beneficial response related to the disease or condition, or symptom of the disease or condition.
  • Beneficial response comprises preventing, alleviating, arresting, or curing the disease or condition, or symptom of the disease or condition.
  • the beneficial response may be measured by detecting a measurable improvement in the presence, level, or activity, of biomarkers, transcriptomic risk profile, or intestinal microbiome in the subject.
  • An “improvement,” as used herein refers to shift in the presence, level, or activity towards a presence, level, or activity, observed in normal individuals (e.g. individuals who do not suffer from the disease or condition).
  • the dosage amount and/or route of administration may be changed, or an additional agent may be administered to the subject, along with the therapeutic rAAV composition.
  • the patient is also weaned off (e.g., step-wise decrease in dose) a second treatment regimen.
  • a dose of the pharmaceutical composition may comprise a concentration of infectious particles of at least or about 10 7 , 10 8 , 10 9 , IO 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or 10 17 .
  • the concentration of infectious particles is 2xl0 7 , 2xl0 8 , 2xl0 9 , 2xlO 10 , 2xlO n , 2xl0 12 , 2xl0 13 , 2xl0 14 , 2xl0 15 , 2xl0 16 , or 2xl0 17 .
  • the concentration of the infectious particles is 3xl0 7 , 3xl0 8 , 3xl0 9 , 3xlO 10 , 3xl0 n , 3xl0 12 , 3xl0 13 , 3xl0 14 , 3xl0 15 , 3xl0 16 , or 3xl0 17 .
  • the concentration of the infectious particles is 4xl0 7 , 4xl0 8 , 4xl0 9 , 4xlO 10 , 4xlO n , 4xl0 12 , 4xl0 13 , 4xl0 14 , 4xl0 15 , 4xl0 16 , or 4xl0 17 .
  • the concentration of the infectious particles is 5xl0 7 , 5xl0 8 , 5xl0 9 , 5xlO 10 , 5xl0 n , 5xl0 12 , 5xl0 13 , 5xl0 14 , 5xl0 15 , 5xl0 16 , or 5xl0 17 .
  • the concentration of the infectious particles is 6xl0 7 , 6xl0 8 , 6xl0 9 , 6xlO 10 , 6xlO n , 6xl0 12 , 6xl0 13 , 6xl0 14 , 6xl0 15 , 6xl0 16 , or 6xl0 17 .
  • the concentration of the infectious particles is 7xl0 7 , 7xl0 8 , 7xl0 9 , 7xlO 10 , 7xlO n , 7xl0 12 , 7xl0 13 , 7xl0 14 , 7xl0 15 , 7xl0 16 , or 7xl0 17 .
  • the concentration of the infectious particles is 8xl0 7 , 8xl0 8 , 8xl0 9 , 8xlO 10 , 8xl0 n , 8xl0 12 , 8xl0 13 , 8xl0 14 , 8xl0 15 , 8xl0 16 , or 8xl0 17 .
  • the concentration of the infectious particles is 9xl0 7 , 9xl0 8 , 9xl0 9 , 9xlO 10 , 9xlO n , 9xl0 12 , 9xl0 13 , 9xl0 14 , 9xl0 15 , 9xl0 16 , or 9xl0 17 .
  • the amount of therapeutic gene expression product in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • the rAAV compositions are suitably formulated pharmaceutical compositions disclosed herein, to be delivered either intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, intraperitoneally, by nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.
  • the pharmaceutical forms of the AAV-based viral compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.
  • sterile injectable solutions comprising the rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • Injectable solutions may be advantageous for systemic administration, for example by intravenous administration.
  • compositions in a neutral or salt form include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • Suitable dose and dosage administrated to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, disease condition and its severity, the identity (e.g., weight, sex, age) of the subject in need of treatment, and can be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.
  • AAV compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, obviating the need for multiple or chronic dosing.
  • target cells e.g., neurons
  • the number of infectious particles administered to a mammal may be on the order of about 10 7 , 10 8 , 10 9 , IO 10 , 10 11 , 10 12 , 10 13 , 10 14 ,or even higher, infectious particles/ml given either as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated.
  • the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the therapeutic rAAV composition used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.
  • the effective dosage ranges may be adjusted based on subject’s response to the treatment. Some routes of administration will require higher concentrations of effective amount of therapeutics than other routes.
  • the daily dosage range and/or the unit dosage amount varies within this range depending upon the dosage form employed and the route of administration utilized.
  • AAVs engineered adeno-associated viruses
  • NHPs non-human primates
  • Insertion of peptides between positions 588 and 589 has been studied in the past by us, and others, and has resulted in novel receptor binding (AAV-PHP.B/AAV-PHP.eB binding of Ly6a on rodent brain endothelium to facilitate blood-brain barrier crossing and high transduction of the brain) and drastically altered capsid tropism.
  • a library of viral capsid was created by performing a random 7 amino acid insertion at this site within AAV9, hoping for novel tropism toward the NHP central nervous system (CNS).
  • Plasmids The first-round viral DNA library was generated by amplification of a section of the AAV9 capsid genome between amino acids 450-599 using NNK degenerate primers (Integrated DNA Technologies, Inc., IDT) to insert seven random amino acids between amino acids 588 and 589 with all possible variations. The resulting library inserts were then introduced into the rAAV-ACap-in-cis-Lox plasmid via Gibson assembly as previously described (Deverman et al., Nat Biotechnol. 2016 Feb; 34(2): 204-209). The resulting capsid DNA library, rAAV-Cap-in-cis-Lox, contained a diversity of -1.28 billion variants at the amino acid level.
  • the second round viral DNA library was generated similarly to the first round, but instead of NNK degenerate primers inserted at the 588 position, a synthesized oligo pool (Twist Biosicence) was used to generate only selected variants.
  • This second-round DNA library contained a diversity of 33,287 variants at the amino acid level, and 66,574 variants at the DNA level (the 33,287 pulled out of the first round and a codon-modified version of each).
  • the AAV2/9 REP-AAP- CAP plasmid transfected into HEK293T cells to provide the Rep gene for library viral production prevents production of a wild-type AAV9 capsid during viral library production after a plausible recombination event between this plasmid co-transfected with rAAV-ACap-in-cis-Lox containing the library inserts.
  • AAVs were generated according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were quadruple transfected with four vectors using polyethylenimine (PEI). The first vector was the rAAV-Cap-in-cis-Lox library flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus. The second vector was the AAV2/9 REP-AAP-ACAP plasmid. The third vector contains nucleic acids encoding helper virus proteins needed for viral assembly and packaging of the heterologous nucleic acid into the modified capsid structure. The fourth is a pUC-18 plasmid included to achieve the right PEI/DNA ratio for optimal transfection efficiency.
  • ITR inverted terminal repeat
  • rAAV-Cap-in-cis-Lox library DNA was transfected (per 150 mm plate) to decrease the likelihood of multiple library DNAs entering the same cell.
  • Viral particles are harvested from the cells and media after 60 h post transfection. Virus present in the media is concentrated by precipitation with 8% polyethylene glycol and 500mM sodium chloride and the precipitated virus is added to the lysates prepared from the collected cells. The viruses are purified over iodixanol (Optiprep, Sigma) step gradients (15%, 25%, 40%, and 60%). Viruses are concentrated and formulated in PBS. Virus titers are determined by measuring the number of DNasel-resistant vector genome copies (VGs) using qPCR and the linearized genome plasmid as a control.
  • VGs DNasel-resistant vector genome copies
  • Marmoset (Callithrix jacchus) procedures were approved by ACUC of the National Institutes of Mental Health. Marmosets were born and raised in NIMH colonies and housed in family groups under standard conditions of 27oC temperature and 50% humidity. They were fed ad libitum and received enrichment as part of the primate enrichment program for NHPs at the NIH. For AAV infusions, animals were screened for endogenous neutralizing antibodies (Nab). None of the animals that were screened showed any detectible blocking reaction at 1:5 dilution of serum (Penn Vector Core, University of Pennsylvania). They were then housed individually for several days and acclimated to a new room before injections.
  • Round 1 and round 2 viral libraries were injected into marmosets at a dose of 2x1012 vg/animal and rAAV genomes were recovered four weeks post injection. Animals were euthanized and brain (both round 1 and round 2), spinal cord (round 2 only) and liver (round 2 only) were recovered, snap frozen, and place into long-term storage at -80oC.
  • Brain was separated into four coronal sections, and for round 2, six coronal sections. lOOmg of each brain section, spinal cord, and liver was homogenized in Trizol (Life Technologies, 15596) using a BeadBug (Benchmark Scientific, D1036) and viral DNA was isolated according to the manufacturers recommended protocol.
  • Recovered viral DNA was treated with RNase, underwent restriction digestion with Smal (found within the ITRs) to improve later rAAV genome recovery by PCR, and purified with a Zymo DNA Clean and Concentrator kit (D4033).
  • Viral genomes were enriched by 25 cycles of PCR amplification with primers flanking the 588-589 insertion site in the capsid genome using 50% of the total extracted viral DNA as a template. After Zymo DNA purification, samples were diluted 1:100 and each dilution further amplified around the library variable region with 10 cycles of PCR.
  • samples were further amplified using NEBNext Dual Index Primers for Illumina sequencing (New England Biolabs, E7600) for 10 more cycles.
  • the amplification products were run on a 2% low-melting point agarose gel (ThermoFisher Scientific, 16520050) for better separation and recovery of the 210 bp band.
  • packaged viral library DNA was isolated from the injected viral library by digestion of the viral capsid and purification of the contained ssDNA. These viral genomes were amplified by two PCR amplification steps, like the viral DNA extracted from tissue, to add adapters and indices for Illumina next-generation sequencing, and purified after gel electrophoresis. This viral library DNA, along with the viral DNA extracted from tissue, was sent for deep sequencing using an Illumina HiSeq 2500 system (Millard and Muriel Jacobs Genetics and Genomics Laboratory, Caltech).
  • NGS data alignment and processing Raw fastq files from NGS runs were processed with custom-built scripts (https://github.com/GradinaruLab/protfarm).
  • the pipeline to process these datasets involved filtering to remove low-quality reads, utilizing a quality score for each sequence, and eliminating bias from PCR-induced mutations or high GC- content.
  • the filtered dataset was then aligned by a perfect string match algorithm and trimmed to improve the alignment quality.
  • Read counts for each sequence were pulled out and displayed by tissue, at which point all sequences found in the brain were compiled for formation of the second round library.
  • Plasmids One rAAV genome was used in this study.
  • pAAV-CAG-hFXN-HA utilizes an ssAAV genome containing an HA-tagged human frataxin (hFXN) protein under control of the synthetic CAG promoter and harboring a unique 12bp sequence in the 3’UTR to differentiate different capsids packaging the same transgene.
  • hFXN human frataxin
  • AAVs were generated according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were triple transfected with three vectors using polyethylenimine (PEI).
  • the first vector contains a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus.
  • the transgene cassette has a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of the target cell.
  • the second vector contains nucleic acids encoding the AAV Rep gene as well as the modified Cap gene for the variant being produced.
  • the modified Cap gene comprises any one of SEQ ID NOS: 37-366, which are the DNA sequences encoding the modified AAV capsid proteins of the present disclosure.
  • the modified CAP gene in some cases, comprises any one of SEQ ID NOS: 385-654, which are the DNA sequences encoding the full-length VP1 protein with the insertions at amino acid positions 588-589.
  • the third vector contains nucleic acids encoding helper virus proteins needed for viral assembly and packaging of the heterologous nucleic acid into the modified capsid structure.
  • Viral particles are harvested from the media after 72 h post transfection and from the cells and media at 120 h post transfection. Virus present in the media is concentrated by precipitation with 8% polyethylene glycol and 500mM sodium chloride and the precipitated virus is added to the lysates prepared from the collected cells.
  • viruses are purified over iodixanol (Optiprep, Sigma) step gradients (15%, 25%, 40%, and 60%). Viruses are concentrated and formulated in PBS. Virus titers are determined by measuring the number of DNasel-resistant vector genome copies (VGs) using qPCR and the linearized genome plasmid as a control.
  • VGs DNasel-resistant vector genome copies
  • the monkeys were placed in the prone position and the needle of the injection assembly introduced between L4-L5 and slowly advanced until cerebrospinal fluid (CSF) was aspirated.
  • CSF cerebrospinal fluid
  • Pooled virus (0.5mL) formulated in sterile PBS was injected followed by a sterile saline flush immediately afterward. After dosing, the monkeys were placed in the ventral recumbency position while recovering from anesthesia. General wellbeing was confirmed twice daily throughout the extent of the study.
  • DNA/RNA recovery and sequencing A pool of viruses (AAV9, AAV-PHP.eB, AAV.CAP-A4, AAV.CAP-B2, AAV.CAP-B 10, AAV.CAP-B22, and variant of the current invention) packaging CAG-hFXN-HA with unique 12 bp barcodes were injected into two 5.5 mo old macaques. After four weeks, animals were euthanized, one hemisphere of the brain was split into eight even thickness coronal sections, and along with samples of the spinal cord and liver were snap frozen.
  • viruses AAV9, AAV-PHP.eB, AAV.CAP-A4, AAV.CAP-B2, AAV.CAP-B 10, AAV.CAP-B22, and variant of the current invention
  • lOOmg slices from each coronal brain section as well as from the spinal cord and liver were homogenized in Trizol (Life Technologies, 15596) using a BeadBug (Benchmark Scientific, D1036) and total DNA and RNA were recovered according to the manufacturer’s recommended protocol.
  • Recovered DNA was treated with RNase, underwent restriction digestion with Smal, and purified with a Zymo DNA Clean and Concentrator Kit (D4033).
  • Recovered RNA was treated with DNase, and cDNA was generated from the mRNA using Superscript III (Thermo Fisher Scientific, 18080093) and oligo(dT) primers according to the manufacturer’s recommended protocol.
  • Barcoded FXN transcripts were recovered from both the DNA and cDNA libraries, as well as the injected pool, using primers that bound around the barcoded region on the 3’UTR of the transcripts and Q5 DNA polymerase in five reactions using 50ng of DNA, cDNA or viral DNA, each, as a template. After Zymo DNA purification, samples were diluted 1:100 and further amplified around the barcode region using porimers to attach adapters for Illumina next-generation sequencing. After cleanup, these products were further amplified using NEBNext Dual Index Primers for Illumina sequencing (New England Biolabs, E7600) for ten cycles. The amplification products were run on a 2% low-melting point agarose gel (ThermoFisher Scientific, 16520050) for better separation and recovery of the 210 bp band. All indexed samples were sent for deep sequencing similar to previous.
  • NGS data alignment and processing Raw fastq files from NGS runs were processed with custom-built scripts (https://github.com/GradinaruLab/protfarm).
  • the pipeline to process the NGS results was similar to that of the first library experiment, with the difference that data was aligned to a hFXN-HA template containing the 12 bp unique barcodes.
  • Read counts for each sequence were pulled out and normalized to the respective contribution of that barcode to the initial, injected pooled virus to account for small inequalities in the amount of each member of the pool that was injected into the monkeys.
  • the distribution of the unique barcodes found within the DNA and RNA was averaged across the eight brain regions and represented as a single value for the entire brain.
  • the DNA and RNA values for each of the variants, read out by their unique barcodes, was then averaged across the two animals, normalized to the value of AAV9, and graphed as viral genomes or RNA transcripts, respectively (FIG. 6).
  • Tissue preparation and immunohistochemistry Macaques were euthanized (Euthanasia, VetOne) and perfused with IX PBS. Each hemisphere of the brain was cut into eight coronal blocks, with one hemisphere, along with a sample of spinal cord and liver being flash frozen in 2-methylbutane (Sigma Aldrich, M32631) chilled with dry ice. The other hemisphere and pieces of spinal cord and liver were removed and post- fixed with 4% PFA at 4oC for 48 hours. Each of the coronal sections of brain were sectioned at 100 pm with a vibratome. Immunohistochemistry (IHC) was performed on floating sections with primary and secondary antibodies in PBS containing 10% donkey serum and 0.1% Triton X-100.
  • IHC Immunohistochemistry
  • Two successive rounds of selection were performed on the viral library based on the marmoset data described in Example 1, focusing on ability to transduce the CNS after systemic administration through the vasculature.
  • the original library sized at 1.28 billion potential variants, was produced in HEK293 cells, which as a first pass removed many of the variants that were unable to produce functional viral capsids, and injected into a set of two adult marmosets.
  • a binary assessment was performed of whether or not the viral sequences were able to be recovered from the tissue of interest. Any sequence found present in the marmoset brains, 33,287 sequences in total, was passed along to the second round of screening.
  • each of the variants within the library was able to be produced.
  • each of the variants is present at a much higher titer than the original library, allowing for a much larger fraction of sequences to reach and transduce the tissue of interest, and thus a much more robust readout of the data.
  • Cpm counts per million
  • Such a distribution identifies additional subclasses of SpinalCord+ variants.
  • six additional variant groups were identified: SpinalCord+Low, SpinalCord+High, SpinalCord+LowBrain+, SpinalCord+LowBrain-, SpinalCord+HighBrain+, and SpinalCord+HighBrain-.
  • the appearance of this bimodal distribution of the SpinalCord+ variants is indicative to us of the potential for a different mechanism of action of these viral groups. Even though the end result, efficient transduction of cells within the Spinal Cord, is the same, there seem to be two different ways these groups achieve it.
  • Table 4 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • CPM is defined as counts per million.
  • Table 5 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 6 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in both the BRAIN and in the SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 7 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the BRAIN over that found in the LIVER after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 8 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the BRAIN over that found in the SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 9 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the BRAIN over that found in the LIVER and SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 10 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the SPINAL CORD over that found in the LIVER and BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 11 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the SPINAL CORD over that found in BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 12 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in the SPINAL CORD over that found in the LIVER after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 13 provides amino acid sequences of rAAV capsid protein insertions, having a greater enrichment in both SPINAL CORD and BRAIN over that found in the LIVER after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 14 provides other amino acid sequences of rAAV capsid protein insertions, having an improved enrichment in the SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 15 provides yet a third group of amino acid sequences of rAAV capsid protein insertions, having improved enrichment in the SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 16 provides other amino acid sequences of rAAV capsid protein insertions, having improved enrichment in both the BRAIN and in the SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 17 provides yet a third group of amino acid sequences of rAAV capsid protein insertions, having significant enrichment in both the BRAIN and in the SPINAL CORD after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 18 provides other amino acid sequences of rAAV capsid protein insertions, having improved enrichment in the SPINAL CORD over the LIVER after two rounds of in vivo selection, as well as the DNA sequences encoding them. TABLE 18
  • Table 19 provides yet a third group of amino acid sequences of rAAV capsid protein insertions, having significant enrichment in the SPINAL CORD over the LIVER after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 20 provides other amino acid sequences of rAAV capsid protein insertions, having a improved enrichment in the SPINAL CORD over that found in BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 21 provides yet a third group of amino acid sequences of rAAV capsid protein insertions, having significant enrichment in the SPINAL CORD over that found in BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 22 provides other amino acid sequences of rAAV capsid protein insertions, having improved enrichment in the SPINAL CORD over that found in the LIVER and BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 23 provides yet a third group amino acid sequences of rAAV capsid protein insertions, having significant enrichment in the SPINAL CORD over that found in the LIVER and BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 24 provides amino acid sequences of rAAV capsid protein insertions, having a maximum expression in the BRAIN after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 25 provides amino acid sequences of rAAV capsid protein insertions, having a greater expression in the BRAIN and low expression in the spinal cord after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 26 provides amino acid sequences of rAAV capsid protein insertions, having the best expression in the BRAIN of the insertions expressed in the brain and the spinal cord after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 27 provides amino acid sequences of rAAV capsid protein insertions, having the best expression in the BRAIN of the insertions expressed in the one spinal cord group after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 28 provides amino acid sequences of rAAV capsid protein insertions, having the best expression in the BRAIN of the insertions expressed in another spinal cord group after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 29 provides other amino acid sequences of rAAV capsid protein insertions, having improved enrichment in the SPINAL CORD AND BRAIN over that found in the LIVER after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • Table 30 provides yet a third group amino acid sequences of rAAV capsid protein insertions, having significant enrichment in the SPINAL CORD and BRAIN over that found in the LIVER after two rounds of in vivo selection, as well as the DNA sequences encoding them.
  • AAV9 a pooled virus experiment was performed in young Rhesus Macaques as described in Example 2.
  • a pool of viruses was produced [AAV9 and AAV-PHP.eB as controls, AAV.CAP-A4, AAV.CAP-B2, AAV.CAP-B10 and AAV.CAP-B22 as variants pulled out of previous rodent engineering efforts that shouldn’t translate well to NHPS, and AAV variants of the present invention, selected from the round 2 library analysis].
  • Each virus packaged an HA-tagged human frataxin (hFXN-HA) with a unique molecular barcode under control of the ubiquitous CAG promoter.
  • hFXN was used because it is an endogenous protein expressed throughout the body.
  • Each packaged hFXN contained a separate 12-base barcode on the 3’UTR to differentiate the contribution of each virus from the rest after NGS.
  • the viruses were pooled at equal ratios and injected intrathecally in the CSF at the lumbar region of the spine into two young macaques, aged roughly 5.5 mo old, at a total dose of 1.5xl0 12 vg/kg (each virus injected at 1.875xl0 n vg/kg).
  • Intrathecal administration as opposed to intravenous administration, was used for this experiment to characterize the variants that performed better due to their ability to enter and express their cargo within cells of the CNS vs.
  • the variants of the present invention are an incredibly potent viral delivery vehicle for targeting the primate CNS after an intrathecal injection, with significant therapeutic potential for gene therapy applications today.
  • pooled variant testing in the macaques recapitulates the results of the library data analysis and validates the selection of top variants within each of the groups separated within the data.
  • Selected viral genomes comprising a nucleic acid encoding I2S are designed and packaged into one or more of the rAAVs described above.
  • the viral genome from ITR to ITR comprises an ITR, a promoter; a human iduronate-2- sulfatase (IDS) sequence; an optional microRNA sequence; a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); a polyadenylation signal, and an ITR.
  • ITR human iduronate-2- sulfatase
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • polyadenylation signal and an ITR.
  • the viral genomes are packaged into one of the capsids described above, purified and formulated in phosphate buffered saline (PBS) with 0.001 % F-68.
  • PBS phosphate buffered saline
  • the concentration of I2S enzyme was measured in the brain, spinal cord, dorsal root ganglia and liver after intravenous injection of 7.5E+13 vg/kg of a variant [E] or saline control in three 8-month old cynomolgus macaques. The animals were sacrificed after 4 weeks in-life. Averages were generated from two animals for the saline treated group and three animals for the AAV treated group.
  • the I2S enzyme concentration was measured by Western Blot and quantified against a standard curve of I2S protein input. Brain values were generated by averaging the values in ten different brain areas, cortical and subcortical.
  • I2S enzyme activity was measured from homogenized tissue using a 2-step assay. Tissue samples were homogenized, then incubated with an I2S substrate. Recombinant human IDUA was added and the activities were then quantified on a plate reader compared against a standard curve. Brain values were generated by averaging the values in ten different brain areas, cortical and subcortical. Spinal cord values were generated by averaging the values from cervical, thoracic and lumbar regions of the spinal cord. DRG values were generated by averaging the values from cervical, thoracic and lumbar regions. Individual points on the graph indicate biological replicates. See Fig 7B.
  • the animals were sacrificed after 3 months in-life.
  • I2S enzyme activity was measured in the cortex, spinal cord, liver, spleen, kidney, heart, lungs and plasma after intra-cisterna magna injection of 5E+10 vg of a variant [E] in 8-week old IDS-KO mice, compared to uninjected WT and KO mouse controls.
  • I2S enzyme activity was measured from homogenized tissue using a 2- step assay. Tissue samples were homogenized, then incubated with an I2S substrate. Recombinant human IDUA was added and the activities were then quantified on a plate reader compared against a standard curve. Averages were generated from two animals for the WT group and three animals for the KO and variant groups. In many tissues, supranormal levels of enzyme activity were achieved. See Figure 8.
  • GAG normalized glucosaminoglycan
  • GAGs both heparan and dermatan sulfate, were measured in the cortex, spinal cord, liver, spleen, kidney, heart, and lungs after intra-cisterna magna injection of 5E+10 vg of a Variant [E] in 8-week old IDS-KO mice, compared to uninjected WT and KO mouse controls.
  • GAGs were measured from homogenized tissue using the Blyscan GAG assay. Averages were generated from two animals for the WT group and three animals for the KO and variant groups.
  • GAGs were reduced to or below WT levels.
  • a recombinant adeno-associated virus comprising a capsid containing an AAV vector comprising: a promoter; a sequence encoding human iduronate 2-sulfatase (hIDS) and comprising SEQ ID NO: 655; a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and a polyadenylation signal.
  • EMBODIMENT 2 The rAAV of claim 1 wherein the promoter is selected from the group consisting a CAG synthetic promoter, a CBh synthetic promoter, and a human synapsin I promoter.
  • EMBODIMENT 3 The rAAV of claim 2 wherein the promoter is a CAG synthetic promoter comprising SEQ ID NO: 656.
  • EMBODIMENT 4 The rAAV of claim 2 wherein the promoter is a CBh synthetic promoter comprising SEQ ID NO: 657.
  • EMBODIMENT 5. The rAAV of claim 2 wherein the promoter is a human synapsin I promoter comprising SEQ ID NO: 658.
  • EMBODIMENT 6 The rAAV of claim 1 wherein the WPRE comprises SEQ ID NO: 659.
  • EMBODIMENT 7 The rAAV of claim 1 wherein the poly adenylation signal is selected from the group consisting of a human growth hormone polyadenylation signal (hGH Poly A) and a simian virus 40 polyadenylation signal (SV40 Poly A).
  • hGH Poly A human growth hormone polyadenylation signal
  • SV40 Poly A simian virus 40 polyadenylation signal
  • EMBODIMENT 8 The rAAV of claim 7 wherein the poly adenylation signal is hGH Poly A comprising SEQ ID NO: 660.
  • EMBODIMENT 9 The rAAV of claim 7 wherein the polyadenylation signal is SV40 PolyA comprising SEQ ID NO: 661.
  • EMBODIMENT 10 The rAAV of claim 1 wherein the capsid comprises an amino acid sequence at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1.
  • EMBODIMENT 11 The rAAV of claim 10 wherein the capsid comprises an insertion at amino acid position 588-589.
  • EMBODIMENT 12 The rAAV of claim 11 wherein the insertion comprises a sequence set forth in any one of Tables 1 and 4-30, Figure 5 and/or Formulas I-XXXIII.
  • EMBODIMENT 13 The rAAV of claim 11 wherein the insertion comprises SEQ ID NO: 38, SEQ ID NO: 77, SEQ ID NO: 84, SEQ ID NO: 241, SEQ ID NO: 246, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO:264, SEQ ID NO: 279 or SEQ ID NO: 283.
  • EMBODIMENT 14 The rAAV of claim 13 wherein the capsid further comprises a substitution at amino acid positions 452-458.
  • EMBODIMENT 15 The rAAV of claim 1 wherein the AAV vector further comprises a microRNA signal.
  • EMBODIMENT 16 The rAAV of claim 15 wherein the microRNA signal is miR-183 comprising SEQ ID NO: 662.
  • EMBODIMENT 17 A method for treating mucopolysaccharidosis type II in a subject, the method comprising administering to said subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) comprising a capsid containing an AAV vector comprising: a promoter; a sequence encoding human iduronate 2-sulfatase (hIDS) and comprising SEQ ID NO:
  • rAAV recombinant adeno-associated virus
  • hIDS human iduronate 2-sulfatase
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • EMBODIMENT 18 The method of claim 17 wherein the promoter is selected from the group consisting a CAG synthetic promoter, a CBh synthetic promoter, and a human synapsin I promoter.
  • EMBODIMENT 19 The method of claim 18 wherein the promoter is a CAG synthetic promoter comprising SEQ ID NO: 656.
  • EMBODIMENT 20 The method of claim 18 wherein the promoter is a CBh synthetic promoter comprising SEQ ID NO: 657.
  • EMBODIMENT 21 The method of claim 18 wherein the promoter is a human synapsin I promoter comprising SEQ ID NO: 658.
  • EMBODIMENT 22 The method of claim 17 wherein the WPRE comprises SEQ ID NO: 659.
  • EMBODIMENT 23 The method of claim 17 wherein the poly adenylation signal is selected from the group consisting of a human growth hormone polyadenylation signal (hGH Poly A) and a simian virus 40 polyadenylation signal (SV40 Poly A).
  • hGH Poly A human growth hormone polyadenylation signal
  • SV40 Poly A simian virus 40 polyadenylation signal
  • EMBODIMENT 24 The method of claim 23 wherein the poly adenylation signal is hGH Poly A comprising SEQ ID NO: 660.
  • EMBODIMENT 25 The method of claim 23 wherein the polyadenylation signal is SV40 PolyA comprising SEQ ID NO: 661.
  • EMBODIMENT 26 The method of claim 17 wherein the capsid comprises an amino acid sequence at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1.
  • EMBODIMENT 27 The method of claim 26 wherein the capsid comprises an insertion at amino acid position 588-589.
  • EMBODIMENT 28 The rAAV of claim 27 wherein the insertion comprises a sequence set forth in any one of Tables 1 and 4-30, Figure 5 and/or Formulas I-XXXIII.
  • EMBODIMENT 29 The method of claim 28 wherein the insertion comprises SEQ ID NO: 38, SEQ ID NO: 77, SEQ ID NO: 84, SEQ ID NO: 241, SEQ ID NO: 246, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO:264, SEQ ID NO: 279 or SEQ ID NO: 283.
  • EMBODIMENT 30 The method of claim 29 wherein the capsid further comprises a substitution at amino acid positions 452-458.
  • EMBODIMENT 31 The method of claim 17 wherein the AAV vector further comprises a microRNA signal.
  • EMBODIMENT 32 The method of claim 31 wherein the microRNA signal is miR-183 comprising SEQ ID NO: 662.
  • a recombinant adeno-associated virus comprising a capsid comprising any one of SEQ ID NO: 38, SEQ ID NO: 77, SEQ ID NO: 84, SEQ ID NO: 241, SEQ ID NO: 246, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO:264, SEQ ID NO: 279 or SEQ ID NO: 283 and containing an AAV vector comprising a sequence encoding human iduronate 2-sulfatase (hIDS).
  • rAAV recombinant adeno-associated virus
  • EMBODIMENT 34 The rAAV of claim 33 wherein the sequence encoding hIDS comprises SEQ ID NO:
  • EMBODIMENT 35 The rAAV of claim 33, wherein the AAV vector further comprises one or more of: a promoter; a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and a polyadenylation signal.
  • a promoter a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and a polyadenylation signal.
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • EMBODIMENT 36 The rAAV of claim 35, wherein the AAV vector further comprises a microRNA signal.
  • EMBODIMENT 37 The rAAV of claim 36, wherein the microRNA signal is miR-183 comprising SEQ ID NO: 662.
  • EMBODIMENT 38 The rAAV of claim 35 wherein the promoter is selected from the group consisting a CAG synthetic promoter, a CBh synthetic promoter, and a human synapsin I promoter.
  • EMBODIMENT 39 The rAAV of claim 38 wherein the promoter is a CAG synthetic promoter comprising SEQ ID NO: 656.
  • EMBODIMENT 40 The rAAV of claim 38 wherein the promoter is a CBh synthetic promoter comprising SEQ ID NO: 657.
  • EMBODIMENT 41 The rAAV of claim 38 wherein the promoter is a human synapsin I promoter comprising SEQ ID NO: 658.
  • EMBODIMENT 42 The rAAV of claim 35 wherein the WPRE comprises SEQ ID NO: 659.
  • the rAAV of claim 35 wherein the poly adenylation signal is selected from the group consisting of a human growth hormone polyadenylation signal (hGH Poly A) and a simian virus 40 polyadenylation signal (SV40 Poly A).
  • EMBODIMENT 44 The rAAV of claim 43 wherein the poly adenylation signal is hGH Poly A comprising SEQ ID NO: 660.
  • EMBODIMENT 45 The rAAV of claim 43 wherein the polyadenylation signal is SV40 PolyA comprising SEQ ID NO: 661.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des compositions de virus pour traiter la mucopolysaccharidose II.
PCT/US2021/046907 2020-08-21 2021-08-20 Compositions de virus pour traiter la mucopolysaccharidose ii Ceased WO2022040530A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063068606P 2020-08-21 2020-08-21
US63/068,606 2020-08-21

Publications (2)

Publication Number Publication Date
WO2022040530A2 true WO2022040530A2 (fr) 2022-02-24
WO2022040530A3 WO2022040530A3 (fr) 2022-03-24

Family

ID=80323344

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/046907 Ceased WO2022040530A2 (fr) 2020-08-21 2021-08-20 Compositions de virus pour traiter la mucopolysaccharidose ii

Country Status (1)

Country Link
WO (1) WO2022040530A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022221400A3 (fr) * 2021-04-13 2022-11-24 Capsida, Inc. Compositions aav ayant des niveaux d'expression élevés dans le cerveau
WO2023244919A1 (fr) * 2022-06-16 2023-12-21 Capsida, Inc. Compositions de virus adéno-associé ayant un enrichissement cérébral accru et un enrichissement hépatique réduit
WO2024008949A1 (fr) * 2022-07-08 2024-01-11 Sensorion Séquences régulatrices comprenant des sites cibles de micro-arn

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040142325A1 (en) * 2001-09-14 2004-07-22 Liat Mintz Methods and systems for annotating biomolecular sequences
CA2554818A1 (fr) * 2004-02-09 2005-08-25 Thomas Jefferson University Diagnostic et traitement de cancers a l'aide de microarn present dans ou au voisinage de caracteristiques chromosomiennes liees aux cancers
US20060041961A1 (en) * 2004-03-25 2006-02-23 Abad Mark S Genes and uses for pant improvement
WO2017137585A1 (fr) * 2016-02-12 2017-08-17 Combigene Ab Vecteur
SG10201912761UA (en) * 2016-04-15 2020-02-27 The Trustees Of The Univ Of Pennsyvania Gene therapy for treating mucopolysaccharidosis type ii
MX2020011386A (es) * 2018-04-27 2021-01-29 Spacecraft Seven Llc Terapia genica para la degeneracion del snc.
CN113966399A (zh) * 2018-09-26 2022-01-21 加州理工学院 用于靶向基因疗法的腺相关病毒组合物

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022221400A3 (fr) * 2021-04-13 2022-11-24 Capsida, Inc. Compositions aav ayant des niveaux d'expression élevés dans le cerveau
WO2023244919A1 (fr) * 2022-06-16 2023-12-21 Capsida, Inc. Compositions de virus adéno-associé ayant un enrichissement cérébral accru et un enrichissement hépatique réduit
WO2024008949A1 (fr) * 2022-07-08 2024-01-11 Sensorion Séquences régulatrices comprenant des sites cibles de micro-arn

Also Published As

Publication number Publication date
WO2022040530A3 (fr) 2022-03-24

Similar Documents

Publication Publication Date Title
JP7746437B2 (ja) ウイルソン病を処置するための遺伝子治療構築物
EP3068889B1 (fr) Compositions de thérapie génique destinées à être utilisées dans la prévention et/ou le traitement d'une stéatose hépatique non-alcoolique
US20230303630A1 (en) Adeno-associated virus compositions having preferred expression levels
WO2022221421A2 (fr) Compositions d'aav ayant une expression cérébrale élevée pour le traitement de la mucopolysaccharidose ii
WO2022040530A2 (fr) Compositions de virus pour traiter la mucopolysaccharidose ii
US20250188128A1 (en) Adeno-associated virus compositions having increased heart enrichment
CN119487053A (zh) 具有优选脑、脊髓和/或心脏表达水平的选定腺相关病毒组合物
JP2025509971A (ja) 好ましい脳エンリッチメントを有する選択されたａａｖ組成物
JP2025535374A (ja) 好ましい脳エンリッチメントおよび低い肝臓エンリッチメントを有するアデノ随伴ウイルス組成物
JP2024505885A (ja) リソソーム酸リパーゼ欠損症（ｌａｌ－ｄ）の治療のための材料及び方法
JP7641911B2 (ja) 新型の酵素組成物
US20240342309A1 (en) Gene therapy vectors for use in parkinson's disease
US20240269328A1 (en) Recombinant adeno-associated viruses for lesch-nyhan disorders and uses thereof
US20230233708A1 (en) Enhancing aav-mediated delivery and transduction with polyvinyl alcohol
WO2025213064A1 (fr) Thérapie génique pour encéphalopathie stxbp1
WO2025213066A1 (fr) Compositions de virus adéno-associés pour le traitement de la maladie de parkinson (pd)
CA3169548A1 (fr) Vecteurs aav-naglu pour le traitement de la mucopolysaccharidose iiib
HK40063644B (en) Gene therapy constructs for treating wilson disease
HK40063644A (en) Gene therapy constructs for treating wilson disease

Legal Events

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

Ref document number: 21859202

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21859202

Country of ref document: EP

Kind code of ref document: A2