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WO2024206831A1 - Compositions comprenant des aav tropiques du cerveau et leurs méthodes d'utilisation - Google Patents

Compositions comprenant des aav tropiques du cerveau et leurs méthodes d'utilisation Download PDF

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Publication number
WO2024206831A1
WO2024206831A1 PCT/US2024/022244 US2024022244W WO2024206831A1 WO 2024206831 A1 WO2024206831 A1 WO 2024206831A1 US 2024022244 W US2024022244 W US 2024022244W WO 2024206831 A1 WO2024206831 A1 WO 2024206831A1
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disclosed
seq
capsid protein
aav
aav capsid
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Derek SOUTHWELL
Aravind Asokan
Aaron MITCHELL-DICK
Michael ADOFF
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Duke University
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Duke University
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors

Definitions

  • Adeno- Associated Virus (AAV) vectors have become a leading platform for gene delivery for the treatment of a variety of diseases. Although there has been clinical success using AAV gene therapies, limitations and challenges associated with use of this gene delivery platform remain. For example, AAV as a gene delivery vehicle providing direct access to the brain or parts thereof, by central nervous system (CNS) injection, peritoneal injection, or direct administration, has remained an unaddressed challenge. Moreover, there are very limited therapeutic options for the numerous neurological diseases and neurological disorders (many of which have underlying genetic etiologies) that may be prevented or treated by gene therapy. Thus, there is an undeniable need to develop AAV-based gene therapies that can selectively and specifically target cells and/or parts of the brain.
  • CNS central nervous system
  • compositions and methods disclosed herein demonstrate enhanced gene transfer to human CNS tissue and the peripheral nervous system (PNS), especially via direct delivery to the CNS (by, for example, peritoneal or direct CNS injection) thereby representing an inventive contribution to the state of the art.
  • PNS peripheral nervous system
  • AAV capsid protein Disclosed herein is a AAV capsid protein. Disclosed herein is an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:01 with one or more substitutions in variable region IV (VR-IV).
  • an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:02 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:03 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO: 04 - SEQ ID NO: 10.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • positions 585 - 590 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 142 - SEQ ID NO: 154, wherein the positions 585 - 590 of the AAV capsid protein is numbered with reference to SEQ ID NO:01.
  • an AAV capsid protein wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113, wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • an AAV capsid protein wherein positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein the positions 452 - 458 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, wherein the positions 586 - 592 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:41.
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:42 - SEQ ID NO:69.
  • an AAV capsid protein comprising the sequence set forth in SEQ ID NO:04.
  • an AAV capsid protein comprising the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • an AAV capsid protein comprising the sequence set forth in SEQ ID NO:08, SEQ ID NO:09, or SEQ ID NO: 10.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:10.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 585 - 590 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 142 - SEQ ID NO: 154, wherein the positions 585 - 590 of the AAV capsid protein is numbered with reference to SEQ ID NO:01.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113, wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein the positions 452 - 458 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, wherein the positions 586 - 592 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:11 - SEQ ID NO:41.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:42 - SEQ ID NO:69.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:41.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:42 - SEQ ID NO:69.
  • an AAV particle comprising (i) an AAV capsid comprising at least one disclosed AAV capsid protein and (ii) a vector genome.
  • AAV particles comprising delivering to one or more cells a nucleic acid molecule comprising a nucleic acid sequence encoding a AAV capsid protein; culturing the one or more cells; and harvesting the AAV particles from the one or more producer cells.
  • Disclosed herein is a method of delivering a payload, the method comprising contacting one or more target cells with a disclosed AAV particle; and expressing the encoded payload.
  • a method of delivering a payload comprising contacting one or more target cells with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.
  • a method of delivering a payload comprising contacting one or more target cells in a subject in need thereof with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.
  • a method of treating a subject comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid.
  • a method of treating a subject comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid, wherein the contacting step allows for expression of the encoded payload, the encoded gene of interest, or the encoded transgene in the one or more cells.
  • FIG. 1A - FIG. IB show the method of enrichment of capsid variants selected via enhanced tropism on human brain slice culture.
  • FIG. 1A shows how capsid variant libraries containing NNS substitutions for a length of 6 or 7 amino acids were generated. Cortical explants donated from humans with intractable epilepsy were sliced into 300 pm thick cortical slice cultures and were cultured long-term.
  • FIG. IB shows that on the day of surgery, human slice cultures were generated and 3-5 hours after plating the slice cultures, AAV libraries were applied to slices. AAV libraries were used to transduce human cultures for three hours, then two to three rounds of washes were performed to remove any unbound AAV capsids.
  • AAV capsid sequences were amplified, and re-cloned.
  • AAV was made from the selected library pool for a subsequent round of selection, and for deep sequencing the library. From the deep sequencing, enrichment of each capsid variant was determined.
  • FIG. 2A - FIG. 2F shows the enrichment of capsid variants selected via enhanced tropism on human brain slice culture.
  • FIG. 2A shows the 3-D locations of VR4 and VR8 within the VP3 monomer in either AAV6, AAV8, or AAV9 serotypes.
  • Capsid variant libraries containing NNS substitutions for a length of 6 or 7 amino acids were selected on live human brain tissue in slice culture.
  • FIG. 2B - FIG. 2F show that 3 rounds of selection were performed and resulted in many capsid sequences enriched significantly above the parental amino acid sequence and were significantly enriched compared to their original representation within the respective parental library.
  • FIG. 2B displays the enrichment of each capsid variant compared to its starting representation within the library for VR4 in AAV8 following three rounds of directed evolution / directed selection within human CNS tissue explants (with the amino acid residues from the topmost capsids represented in the WebLogo. This is also true for figures 2C-2E) while FIG. 2C displays the enrichment of each capsid variant compared to its starting representation within the library for VR8 in AAV8 following three rounds of directed evolution / directed selection within human CNS tissue explants.
  • FIG. 2D displays the enrichment of each capsid variant compared to its starting representation within the library for VR4 in AAV9 following three rounds of directed evolution / directed selection within human CNS tissue explants while FIG.
  • FIG. 2E displays the enrichment of each capsid variant compared to its starting representation within the library for VR8 in AAV9 following three rounds of directed evolution / directed selection within human CNS tissue explants.
  • FIG. 2F displays the enrichment of each capsid variant compared to its starting representation within the library for AAV6 VR8 evolution following three rounds of directed evolution / directed selection within human CNS tissue explants.
  • each grey dot represents a different capsid protein variant
  • the black dot represents the wild type AAV6 (SEQ ID NO:01), AAV8 (SEQ ID NO:02), or AAV9 (SEQ ID NO:03).
  • FIG. 3A - FIG. 3E show that AAV8 derived capsids transduced human glioma U87 cells of neuronal origin more efficiently than did the WT AAV8 capsid.
  • FIG. 3A shows AAV capsids packaging a single-stranded CBA-TdTomato TR-flanked cassette were titer-matched and U87 cells were transduced at 10k MOI 24 hours after plating.
  • FIG. 3B shows AAV8-derived capsids packaging a single-stranded scCBH-GFP TR flanked cassette were titer matched and U87 cells were transduced at 10K MOI 24 hours after plating.
  • FIG. 3A shows AAV capsids packaging a single-stranded CBA-TdTomato TR-flanked cassette were titer-matched and U87 cells were transduced at 10k MOI 24 hours after plating.
  • FIG. 3B shows AAV8-derived capsids packaging a
  • FIG. 3C shows AAV9-derived capsids packaging CBA-TdTomato TR-flanked cassette were titer-matched and U87 cells were transduced at 10k MOI 24 hours after plating.
  • FIG. 3D shows U87 cells imaged 5 days after transduction, U87 cells were imaged, while FIG. 3E shows FAC sorted U87 cells for expression of GFP and corresponding mean fluorescence intensity measurements of U87 cells following transduction with AAV9 compared to novel capsids.
  • FIG. 4A - FIG. 4F show ICV delivery of enriched, novel AAV capsids resulted in higher transduction of the mouse cortex compared to AAV8 and/or AAV9.
  • FIG. 4A shows the experimental schematic.
  • AAV capsids packaging a self-complimentary CBh-scGFP TR-flanked cassette (FIG. 4B) or a CBA-tdTomato (human codon optimized) TR-flanked cassette (FIG. 4C) were titer-matched and injected into the lateral ventricle of P0-P1 mouse pups at lelO total vg.
  • FIG. 4D AAV8 and hb3
  • FIG. 4E AAV9, hb32, hb33, and hb42
  • FIG. 4F AAV9, hb32, hb33, and hb42.
  • Total cell number was measured in 150 pm x 500 pm or 150 pm x 200 pm cortical windows.
  • N > 2 animals per capsid, average of 2 sections per animal.
  • FIG. 5A - FIG. 5C show AAV8 or AAV9 VR4- and VR8-derived capsids in capsid pools exhibit higher transduction in human cortical brain slices than their parental capsids, either AAV8 or AAV9.
  • FIG. 5A shows the study design. AAV capsids packaging a self-complementary CBh- NLS-scGFP TR-flanked cassette with a barcode in the 3’UTR were pooled and the AAV pool was used to transduce human cortical slices. After 8 days in vitro, the slices were flash-frozen, and RNA, and gDNA were extracted.
  • FIG. 5B shows the enrichment of AAV genomes within human CNS tissue or expression of a recombinant transgene following transduction by novel capsids in human brain tissue.
  • N > 2 replicates, graphed and normalized to AAV8 expression levels.
  • Variants AAV.hbl7, AAV.hb21, AAV.hb22, AAV.hb23, and AAV.hb25 show markedly increased expression compared to AAV8.
  • 5C shows the enrichment of AAV genomes within human CNS tissue or expression of a recombinant transgene following transduction by novel capsids in human brain tissue.
  • N > 2 replicates, graphed and normalized to AAV9 expression levels.
  • Variants AAV.hb32, AAV.hb33, AAV.hb35, and AAV.hb39 showed a 5x to 12x increase in uptake, transduction efficiency, and expression compared to AAV9.
  • AAV.hb32 and other capsids show increases in uptake, transduction, and expression, compared to AAV9.
  • FIG. 5D shows the enrichment of AAV genomes within human CNS tissue or expression of a recombinant transgene following transduction by novel capsids in human brain tissue.
  • N > 2 replicates, graphed and normalized to AAV9 expression levels.
  • Variants AAV.hb27, AAV.hb28, and AAV.hb29 show increased expression compared to both AAV8 and AAV9.
  • the terms “AAV.hb27” or “hb27” are synonymous. The same applies to all other AAV capsid proteins shown in this figure, and the same applies for all capsids discussed in the drawings and the application. “AAV.hb##” and “hb##” are synonymous.
  • FIG. 6A - FIG. 6E show novel capsid AAV.hb21 cell-type transduction in human cortical slices.
  • FIG. 6A - FIG. 6B show the number of cells transduced within human cortical slices, on a graph where the distribution of transduced cells within the full cross-section of each human cortical slice are separated into 5 bins.
  • Human cortical slice cultures were generated by serial sectioning, and pairwise transduced with AAV.hb21 or AAV8 packaging CBh-eGFP. Slices were fixed at 11 days. GFP, NeuN, and GFAP were assessed in 1000 pm cortical windows.
  • FIG. 7A - FIG. 71 show capsid barcode pool transduction per organ following IV delivery in mice.
  • Novel capsid variants are grouped by variable region (VR4 or VR8). Barcodes in the 3’ UTR were quantified and evaluated via high-throughput sequencing, and log2 fold change graphed relative to the parental capsid AAV8 or AAV9.
  • FIG. 8A - FIG. 8D show digital re-sectioning of transduced human cortical explants.
  • FIG. 8A AAV8, hb21, hb25
  • FIG. 8B AAV9, hb32
  • FIG. 8C AAV8, hb21, hb25
  • FIG. 8D AAV9, hb32
  • MFI mean fluorescence intensity
  • FIG. 9A - FIG. 9G show that evolved capsid exhibited high transduction and expression in the human brain.
  • FIG. 9A - FIG. 9B show a schematic of AAV transduction in human cortical slice cultures.
  • FIG. 9C - FIG. 9D show expression of AAV.hb21 compared to wild-type AAV8 in human cortical slices following hydrophilic tissue clearing (FIG. 9C) or a standard fixation and imaging protocol (FIG. 9D).
  • FIG. 9E shows an inset of FIG. 9D.
  • FIG. 9F shows the proportion of GFP+ cells colocalized with NeuN in human ex vivo cortical cultures.
  • FIG. 9G - FIG. 9H show the expression of AAV.hb21 compared to wild-type AAV9 in human cortical slices following hydrophilic tissue clearing (FIG. 9G) or a standard fixation and imaging protocol (FIG. 9H).
  • Native GFP, maximum projection scale bar is 1 mm.
  • the present disclosure describes isolated nucleic acid molecules, viral vectors, viral capsid proteins, viral particles, plasmids, cells, kits, pharmaceutical formulations, and compositions thereof and methods of using the disclosed compositions. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value unless otherwise stated or otherwise evident from the context.
  • in vitro refers to events or experiments that occur in an artificial environment, e.g., in a petri dish, test tube, cell culture, etc., rather than within a multicellular organism.
  • in vivo refers to events or experiments that occur within a multicellular organism.
  • the term “comparable” in the context of a particular value and a reference value means that the particular value is consistent with the reference value, or that the deviation from the reference value (above or below) is at most 10%.
  • references in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed.
  • X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
  • a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.
  • the term “subject” refers to the target of administration.
  • a subject can be a human being.
  • the term “subject” includes domesticated animals (e.g, cats, dogs, etc.), livestock (e.g, cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent.
  • the term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered.
  • a subject can be a human patient.
  • a subject can have a disease, a disorder, an infection, a symptom, and/or a complication, be suspected of having a disease, a disease, a disorder, an infection, a symptom, and/or a complication, or be at risk of developing a disease, a disorder, an infection, a symptom, and/or a complication.
  • a subject can have risk factors for developing a disease, a disorder, an infection, a symptom, and/or a complication.
  • a subject can be at risk due to genetic predisposition, employment type (e.g., a health care worker, a miner), attendance at a specific location (e.g., school), attendance at social events (e.g., sporting events, concerns, religious services, political rallies and events, social justice rallies, marches, and events, etc.), by use of public transportation or public services, exposure to natural and man-made disasters (e.g., Chernobyl, 9/11 attacks, etc.).
  • employment type e.g., a health care worker, a miner
  • attendance at a specific location e.g., school
  • attendance at social events e.g., sporting events, concerns, religious services, political rallies and events, social justice rallies, marches, and events, etc.
  • social events e.g., sporting events, concerns, religious services, political rallies and events, social justice rallies, marches, and events, etc.
  • exposure to natural and man-made disasters e.g., Chernobyl
  • a subject can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • a subject can have a genetic disorder.
  • a subject can have a missing, deficient, and/or mutant protein or enzyme can be encoded by some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, LMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE
  • “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease, a disorder, an infection, a symptom, and/or a complication, or a suspected disease, disorder, infection, symptom, and/or complication (related to, for example, to one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof).
  • an effective amount can alleviate and/or improve one or more symptoms and/or complications associated with a neurological disease or disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, a neuro-oncological disorder, or any combination thereof).
  • a “therapeutically effective amount” refers to an amount (i.e., vector genome / body weight or vg/kg) that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.
  • “therapeutically effective amount” means an amount of a disclosed composition that (i) treats the particular disease, disorder, and/or infection, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, and/or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, and/or disorder described herein.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions, or methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions employed; the duration of the treatment; drugs used in combination or coincidental with a disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions employed, and other like factors well known in the medical arts.
  • a disclosed composition and/or a pharmaceutical preparation comprising one or more disclosed composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of a disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions, or methods can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease, a disorder, an infection, a symptom, and/or a complication.
  • “control” refers a standard or reference condition, against which results are compared.
  • a control is used at the same time as a test variable or subject to provide a comparison.
  • a control is a historical control that has been performed previously, a result or amount that has been previously known, or an otherwise existing record.
  • a control can be a positive or negative control.
  • the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a disease, a disorder, an infection, a symptom, and/or a complication (related to, for example a neurological disease or disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, a neuro-oncological disorder, or any combination thereof) that can be diagnosed or treated by one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, and/or the disclosed methods.
  • “suspected of having” can mean having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition (e.g., one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof) that can likely be treated by one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed AAV vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, and/or the disclosed methods.
  • a condition e.g., one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treatment may not necessarily result in the complete clearance of an infection but may reduce or minimize complications, the side effects, and/or the progression of a disease, a disorder, an infection, a symptom, and/or a complication.
  • the success or otherwise of treatment can be monitored by physical examination of the subject as well as cytopathological, DNA, and/or mRNA detection techniques.
  • treat or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease.
  • a mammal e.g., a human
  • treating an infection can reduce the severity of an established infection in a subject by 1%- 100% as compared to a control (such as, for example, a subject not having the disease, the disorder, the infection, the symptom, and/or the complication.
  • treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, disorder, infection, symptom, and/or complication.
  • treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of the disease, disorder, infection, symptom, and/or complication. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of the disease, disorder, infection, symptom, and/or complication.
  • Methods and techniques to monitor a subject’s response to a disclosed method can comprise qualitative (or subjective) means as well as quantitative (or objective) means.
  • qualitative means (or subjective means) can comprise a subject’s own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration or an intensification of one or more symptoms, or a combination thereof.
  • quantitative means can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject’s fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., blood analysis of a subject
  • a “patient” refers to a subject afflicted with a disease, disorder, infection, symptom, and/or complication (e.g., one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof).
  • a patient can refer to a subject that has been diagnosed with or is suspected of having a disease, disorder, infection, symptom, and/or complication (relating to, for example, (e.g., one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof)).
  • a patient can refer to a subject that has been diagnosed with or is suspected of having an established disease, disorder, infection, symptom, and/or complication and is seeking treatment or receiving treatment.
  • the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease, disorder, infection, symptom, and/or complication is intended.
  • prevent and “preventing” and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., having a neurological disease or disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, a neuro-oncological disorder, or any combination thereof) not having a given infection related complication from progressing to that complication.
  • a subject e.g., having a neurological disease or disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, a neuro-oncological disorder, or any combination thereof
  • Individuals in which prevention is required include those who have an infection.
  • administering and “administration” refer to any method of providing one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, and/or the disclosed methods to a subject.
  • Such methods include, but are not limited to, the following: retrograde ureteral infusion, renal arterial administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, inter utero administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV).
  • HPV hepatic portal vein
  • Administration of a disclosed AAV vector and/or a disclosed AAV particle can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventriular, inthrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.
  • modifying the method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • a method can be altered by changing the amount of one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration, or by changing the duration of time of administration or between administrations to a subject.
  • “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.
  • the term “contacting” can refer to bringing one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed AAV vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, or any combination thereof with a target area or intended target area (e.g., one or more parts and/or structures of the brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof)) in such a manner that the one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed AAV vectors, the disclosed
  • determining can refer to measuring or ascertaining the presence and severity of a disease, disorder, infection, symptom, and/or complication (e.g., one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof).
  • a disease, disorder, infection, symptom, and/or complication e.g., one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • Methods and techniques used to determining the presence and/or severity of a disease, disorder, infection, symptom, and/or complication are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease, disorder, infection, symptom, and/or complication.
  • the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • a pharmaceutical carrier employed can be a solid, liquid, or gas.
  • examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water.
  • examples of gaseous carriers can include carbon dioxide and nitrogen.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like
  • oral solid preparations such as powders, capsules and tablets.
  • tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
  • Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
  • the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (such as, e.g., a polypeptide having the sequence set forth in any of SEQ ID NO:01 - SEQ ID NO: 3 or a nucleic acid having the sequence set forth in any of SEQ ID NO: 155 - SEQ ID NO: 157) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.
  • a parent compound such as, e.g., a polypeptide having the sequence set forth in any of SEQ ID NO:01 - SEQ ID NO: 3 or a nucleic acid having the sequence set forth in any of SEQ ID NO: 155 - SEQ ID NO: 15
  • AAV genomes or AAV vectors according to the invention when present in a suitable producer cell and in the presence of AAV Rep and Cap proteins, can replicate and package into rAAV viral particles, particularly infectious viral particles.
  • “genome particles (gp),” “genome equivalents,” or “genome copies” can refer to a viral titer or the number of virions containing the rAAV DNA genome, regardless of infectivity or functionality.
  • AAV adeno-associated virus
  • AAV refers to a viral particle consisting of at least one AAV capsid protein VP1, VP2, and/or VP3, preferably all capsid proteins of a wild-type AAV, and an encapsidated polynucleotide AAV genome or AAV vector.
  • An AAV of the invention can typically be a recombinant AAV.
  • An AAV can be a non-naturally occurring AAV.
  • the AAV can comprise one or more heterologous polynucleotides, i.e., polynucleotides other than wild-type AAV polynucleotides, such as transgenes.
  • transgene is a therapeutic gene.
  • a “therapeutic gene” refers to a gene that, when expressed, produces a therapeutic gene product that confers a beneficial effect on the cell or tissue in which it is present, or on a mammal in which the gene is expressed.
  • beneficial effects include amelioration of a sign or symptom of a condition or disease, prevention or inhibition of a condition or disease, or conferral of a desired characteristic.
  • Therapeutic genes include, but are not limited to, genes that correct a genetic deficiency in a cell or mammal.
  • a therapeutic gene can comprise some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, SOD1, SPG11, SQSIM1, TARDBP, TBK1, TBP, TDP-43, TRPC6, TRPM
  • transfer plasmid or “pTransfer” contains the viral genome.
  • the pTransfer further comprises two ITRs, a transgene, gene of interest, heterologous nucleic acid, and/or payload, a promoter, and one or more cis-regulatory elements (e.g., Lox sites, WPRE, poly A, etc.).
  • cis-regulatory elements e.g., Lox sites, WPRE, poly A, etc.
  • an “AAV inverted terminal repeat (ITR)” sequence” or “ITR” can comprise an approximately 145-nucleotide sequence that is present at both termini of the native singlestranded AAV genome.
  • an ITR can be an AAV2 ITR.
  • a “transgene” is a polynucleotide encoding a gene that is delivered to a cell by a disclosed AAV vector.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product after being transcribed, and sometimes also translated.
  • the term “gene” or “coding sequence” refers to a nucleotide sequence in vitro or in vivo that encodes a gene product.
  • the gene consists or consists essentially of coding sequence, that is, sequence that encodes the gene product.
  • the gene comprises additional, non-coding, sequence that permits, facilitates or directs the cellular expression machinery to express the encoded product.
  • sequences can include, but are not limited to promoters, enhancers, transcriptional termination and/or poly(A) addition signals, and elements that affect transcript processing and/or stability.
  • a gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’ UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • a “gene product” is a molecule resulting from expression of a particular gene or fragment thereof.
  • Gene products can include, for example, a polypeptide, an aptamer, an interfering RNA, an mRNA, and the like.
  • a “gene product” can be a polypeptide, peptide, protein or interfering RNA including short interfering RNA (siRNA), miRNA or small hairpin RNA (shRNA).
  • a disclosed gene product can be a therapeutic gene product, e.g., a therapeutic protein or a therapeutic RNA (e.g., a trans-splicing molecule, an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA).
  • a therapeutic gene product e.g., a therapeutic protein or a therapeutic RNA (e.g., a trans-splicing molecule, an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA).
  • AAV can be replication competent or replication incompetent.
  • replication competent is meant that the virus or viral particle is infectious and capable of replication in a suitable infected cell.
  • the disclosed AAV can be replication-incompetent.
  • the term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral particle.
  • the viral vector can contain a nucleic acid (e.g., a transgene, a gene of interest, and/or a payload) encoding a polypeptide as described herein in place of non-essential viral genes.
  • the vector and/or particle can be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • an “AAV virus” or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein such as VP1 (typically by all of the capsid proteins of a wildtype AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a “recombinant AAV vector particle” or simply a “rAAV vector”. Thus, production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector is contained within a rAAV particle.
  • AAV capsid protein such as VP1 (typically by all of the capsid proteins of a wildtype AAV) and an encapsidated polynucleotide r
  • viral capsid polypeptide refers to the proteinaceous shell or coat of a viral particle.
  • a viral capsid polypeptide permits packaging or assembly of the capsid polypeptide into a viral particle that is competent for delivery of nucleic acid to the host cell.
  • Capsids function to encapsidate, protect, transport, and release into a host cell a viral genome.
  • Capsids are generally comprised of oligomeric structural subunits of a polypeptide of the viral capsid polypeptides.
  • “encapsidated” means enclosed within a viral capsid.
  • the AAV genome comprises three overlapping sequences which encode capsid proteins, VP1, VP2 and VP3, which start from one promoter, p40.
  • the AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of 1 : 1 : 10.
  • packing refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.
  • payload refers to a nucleic acid that is encapsidated within a viral vector, e.g., an AAV vector.
  • a payload nucleic acid can encode a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA.
  • a “payload” refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome or an expression product of such polynucleotide or polynucleotide region, e.g., a transgene, a polynucleotide encoding a polypeptide or multi-polypeptide or a modulatory nucleic acid or regulatory nucleic acid.
  • a disclosed payload can comprise any nucleic acid that is useful for modulating the expression in a target cell transduced or contacted with the AAV particle carrying the payload.
  • modulation can be by supplementation of the payload in a target cell or tissue.
  • modulation can be gene replacement of the payload in a target cell or tissue. In an aspect, modulation can be by inhibition using a modulatory nucleic acid of the payload in a target cell or tissue.
  • a disclosed payload can comprise a combination of coding and non-coding nucleic acid sequences, and can be codon-optimized.
  • a payload can comprise one or more regulatable elements.
  • a disclosed payload can encode a messenger RNA (mRNA) can be encoded by a disclosed payload.
  • a disclosed payload can encode a gene therapy product.
  • mRNA messenger RNA
  • a gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect.
  • a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated.
  • a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.
  • polypeptide refers to a polymer of amino acids.
  • protein and “polypeptide” are used interchangeably herein.
  • a peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length.
  • Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used.
  • One or more of the amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc.
  • polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a “polypeptide.”
  • exemplary modifications include glycosylation and palmitoylation.
  • Polypeptides can be purified from natural sources, produced using recombinant DNA technology or synthesized through chemical means such as conventional solid phase peptide synthesis, etc.
  • the term “polypeptide sequence” or “amino acid sequence” can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide.
  • a polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated.
  • a variant amino acid or DNA sequence can be at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, e.g., that are freely available on the world wide web (e.g., BLASTp or BLASTn with default settings).
  • Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide- directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are well established.
  • cysteine residues not involved in maintaining the proper conformation of a polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) can be added to a polypeptide to improve its stability or facilitate oligomerization.
  • polynucleotide sequence can refer to the polynucleotide material itself and/or to the sequence information (i.e., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid.
  • sequence information i.e., the succession of letters used as abbreviations for bases
  • a polynucleotide sequence presented herein is presented in a 5’ to 3’ direction unless otherwise indicated.
  • the term “corresponding to,” when used in reference to an amino acid or polynucleotide sequence means that a given amino acid or polynucleotide sequence in one polypeptide or polynucleotide molecule has structural properties, functional properties, or both that are similar relative to an amino acid or polynucleotide sequence in a similar location in another polypeptide or polynucleotide molecule.
  • Homologues of a given polypeptide in different species “correspond to” each other, as do regions or domains of homologous polypeptides from different species.
  • capsid polypeptides of different serotypes of viral vectors including but not limited to AAV vectors, “correspond to” each other, as do regions of such polypeptides, defined, for example by alignment of their amino acid sequences. While other alignment parameters can be used to define such regions, for the avoidance of doubt, alignment can be performed using BLAST® (Basic Local Alignment Search Tool) using default parameters.
  • BLAST® Basic Local Alignment Search Tool
  • promoter or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
  • tissue-specific promoters are known to the art and include, but are not limited to, neuron-specific promoters, brain specific promoters, kidney specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and heart-specific promoters.
  • a “ubiquitous/constitutive promoter” refer to a promoter that allows for continual transcription of its associated gene.
  • a ubiquitous/constitutive promoter is always active and can be used to express genes in a wide range of cells and tissues, including, but not limited to, the liver, kidney, skeletal muscle, cardiac muscle, smooth muscle, diaphragm muscle, brain, spinal cord, endothelial cells, intestinal cells, pulmonary cells (e.g., smooth muscle or epithelium), peritoneal epithelial cells, and fibroblasts.
  • Ubiquitous/constitutive promoters include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFla) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a P-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promote
  • an “inducible promoter” refers to a promoter that can be regulated by positive or negative control.
  • Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.
  • an “isolated” biological component such as a nucleic acid molecule, protein, or virus
  • nucleic acids, proteins, and/or viruses that have been “isolated” include nucleic acids, proteins, and viruses purified by standard purification methods.
  • the term also embraces nucleic acids, proteins, and viruses prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins.
  • isolated does not require absolute purity; rather, it is intended as a relative term.
  • an isolated or purified nucleic acid, protein, virus, or other active compound is one that is isolated in whole or in part from associated nucleic acids, proteins, and other contaminants.
  • substantially purified refers to a nucleic acid, protein, virus or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.
  • sequence identity and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity.
  • Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more.
  • Such sequences are also referred to as “variants” herein, e.g., other variants of AAV capsid proteins. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and/or 5’-side are 100% identical.
  • a “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species or group of species).
  • a nucleic acid sequence can be optimized for expression in mammalian cells or in a particular mammalian species (such as human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.
  • AAV capsid protein having one or more substitutions in variable region IV (VR-IV).
  • a AAV capsid protein having one or more substitutions in variable region VIII VR-VIII.
  • an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:01 with one or more substitutions in variable region IV (VR-IV).
  • an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:02 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:03 with one or more substitutions in variable region IV (VR- IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • a disclosed AAV capsid protein can be a variant of a parental wild-type capsid protein.
  • a disclosed parental wild-type capsid protein can be a capsid protein of AAV6, AAV8, or AAV9.
  • a disclosed parental wild-type capsid protein can be VP1 of AAV6, AAV8, or AAV9.
  • positions 585 - 590 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 142 - SEQ ID NO: 154, wherein the positions 585 - 590 of the AAV capsid protein is numbered with reference to SEQ ID NO:01.
  • an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:70 - SEQ ID NO:82.
  • positions 585 - 590 can comprise the sequence set forth in any one of SEQ ID NO: 142 - SEQ ID NO: 154.
  • positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:26.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 11 - SEQ ID NO:26.
  • positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO:113, wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO:27 - SEQ ID NO:41.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:27 - SEQ ID NO:41.
  • positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO: 02, and wherein positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113 wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98.
  • positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98 and positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • an AAV capsid protein wherein positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein the positions 452 - 458 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, wherein the positions 586 - 592 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, and wherein the positions 452 - 458 and positions 586 - 592 of the AAV capsid protein are numbered with reference to SEQ ID NO:03.
  • positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 114 - SEQ ID NO: 126.
  • positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 114 - SEQ ID NO: 126 and positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:11 - SEQ ID NO:41.
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:42 - SEQ ID NO:69.
  • an AAV capsid protein comprising the sequence set forth in SEQ ID NO:04.
  • an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:04.
  • an AAV capsid protein comprising the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • an AAV capsid protein comprising the sequence set forth in SEQ ID NO:08, SEQ ID NO:09, or SEQ ID NO: 10.
  • an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:08, SEQ ID NO:09, or SEQ ID NO: 10.
  • an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 of in SEQ ID NO:04, wherein the substitution at position 585 is any amino acid other than Q; wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than S; wherein the substitution at position 588 is any amino acid other than S; wherein the substitution at position 589 is any amino acid other than T; and/or wherein the substitution at position 590 is any amino acid other than D.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than QSSSTD.
  • each of positions 585 - 590 can have an amino acid substitution.
  • 1 or more of positions 585 - 590 can have an amino acid substitution.
  • 2 or more of positions 585 - 590 can have an amino acid substitution.
  • 3 or more of positions 585 - 590 can have an amino acid substitution.
  • 4 or more of positions 585 - 590 can have an amino acid substitution.
  • positions 585 - 590 can have an amino acid substitution.
  • 6 or more of positions 585 - 590 can have an amino acid substitution.
  • an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:05, wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than G; wherein the substitution at position 457 is any amino acid other than T; wherein the substitution at position 458 is any amino acid other than A; wherein the substitution at position 459 is any amino acid other than N; wherein the substitution at position 460 is any amino acid other than T, and/or wherein the substitution at position 461 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than GGTANTQ.
  • each of positions 455 - 461 can have an amino acid substitution.
  • 1 or more of positions 455 - 461 can have an amino acid substitution.
  • 2 or more of positions 455 - 461 can have an amino acid substitution.
  • 3 or more of positions 455 - 461 can have an amino acid substitution.
  • 4 or more of positions 455 - 461 can have an amino acid substitution.
  • positions 455 - 461 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 6 or more of positions 455 - 461 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 7 or more of positions 455 - 461 can have an amino acid substitution.
  • an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:06, wherein the substitution at position 586 is any amino acid other than L; wherein the substitution at position 587 is any amino acid other than Q; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than Q; wherein the substitution at position 590 is any amino acid other than N; and/or wherein the substitution at position 591 is any amino acid other than T.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than LQQQNT.
  • each of positions 586 - 591 can have an amino acid substitution.
  • positions 586 - 591 can have an amino acid substitution.
  • 2 or more of positions 586 - 591 can have an amino acid substitution.
  • 3 or more of positions 586 - 591 can have an amino acid substitution.
  • 4 or more of positions 586 - 591 can have an amino acid substitution.
  • 5 or more of positions 586 - 591 can have an amino acid substitution.
  • 6 or more of positions 586 - 591 can have an amino acid substitution.
  • an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:07, wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than G; wherein the substitution at position 457 is any amino acid other than T; wherein the substitution at position 458 is any amino acid other than A; wherein the substitution at position 459 is any amino acid other than N; wherein the substitution at position 460 is any amino acid other than T, wherein the substitution at position 461 is any amino acid other than Q, wherein the substitution at position 586 is any amino acid other than L; wherein the substitution at position 587 is any amino acid other than Q; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than Q; wherein the substitution at position 590 is any amino acid other than N; and/or wherein the substitution at position 591 is any amino acid other than T.
  • the substitution at position 455 is any amino acid other than G
  • an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:08, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N, and/or wherein the substitution at position 458 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than NGSGQNQ.
  • each of positions 452 - 458 can have an amino acid substitution.
  • 1 or more of positions 452 - 458 can have an amino acid substitution.
  • 2 or more of positions 452 - 458 can have an amino acid substitution.
  • 3 or more of positions 452 - 458 can have an amino acid substitution.
  • 4 or more of positions 452 - 458 can have an amino acid substitution.
  • positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 6 or more of positions 452 - 458 can have an amino acid substitution.
  • an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:09, wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than A; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position
  • the one or more disclosed amino acid substitutions can comprise substitutions other than SAQAQAQ.
  • each of positions 586 - 592 can have an amino acid substitution.
  • 1 or more of positions 586 - 592 can have an amino acid substitution.
  • 2 or more of positions 586 - 592 can have an amino acid substitution.
  • 3 or more of positions 586 - 592 can have an amino acid substitution.
  • 4 or more of positions 586 - 592 can have an amino acid substitution.
  • positions 586 - 592 can have an amino acid substitution.
  • 6 or more of positions 586 - 592 can have an amino acid substitution.
  • 7 or more of positions 586 - 592 can have an amino acid substitution.
  • an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO: 10, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 459 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N, wherein the substitution at position 458 is any amino acid other than Q, wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than A; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than A; wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 4
  • substitutions at positions 452 - 458 can comprise substitutions other than NGSGQNQ, and wherein the substitutions at 586 - 592 can comprise substitutions other than SAQAQAQ.
  • a disclosed AAV capsid protein can comprise the sequence set forth in SEQ ID NO 35, SEQ ID NO:38, SEQ ID NO:31, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:49.
  • a disclosed wild-type sequence (as presented below in Table 1) has one or more substitutions in one or more regions of the protein.
  • the disclosed one or more substitutions can comprise 1 substitution, 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, or 10 substitutions. In an aspect, the disclosed one or more substitutions can comprise 6 substitutions or 7 substitutions.
  • a disclosed AAV capsid protein can be used to improve and/or enhance gene transfer and/or expression in one or more regions and/or structures of the human brain when compared to a disclosed parental wild-type capsid protein.
  • gene transfer and/or expression can be improved in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least
  • a disclosed AAV capsid protein can be used to improve and/or enhance gene transfer and/or expression to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • gene transfer and/or expression can be improved and/or enhanced in one or more cells types in the CNS and/or the PNS (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • cells types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle
  • a disclosed AAV capsid protein can be used to effect widespread transduction of cells in the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells of the human brain (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV capsid protein can be used to effect widespread transduction in one or more regions and/or structures of the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least
  • tropism for one or more cell types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof
  • a disclosed AAV capsid protein can demonstrate improved tropism for one or more cell types and/or one or more tissue types (such as, for example, one or more disclosed cell types in the human CNS and/or in the human PNS).
  • a disclosed AAV capsid protein can exhibit improved transduction efficiency and/or properties when introduced to one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV capsid variant can efficiently transduce one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV capsid protein can outperform the parental wild-type AAV in one or more disclosed cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV capsid protein can be used to increase and/or enhance the level of viral genomes delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of viral genomes delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV capsid protein can be used to increase and/or enhance the level of a payload delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of a payload delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV capsid protein can be used to increase penetrance through the BBB when compared to the wild-type capsid protein.
  • penetrance through the BBB can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV capsid protein can be used to increase and/or widen distribution to one or more regions and/or parts of the human brain.
  • distribution to one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or widened by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3
  • a disclosed AAV capsid protein can be used to increase and/or enhance payload expression in one or more regions and/or parts of the human brain.
  • payload expression in one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least
  • a disclosed AAV capsid protein can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed AAV capsid protein can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.
  • a disclosed AAV capsid protein can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder.
  • a disclosed AAV capsid protein can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject.
  • a disclosed AAV capsid protein can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject.
  • a disclosed AAV capsid protein can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.
  • a disclosed AAV capsid protein can be used to improve neurological function and/or neuromuscular function in the subject. In an aspect, a disclosed AAV capsid protein can be used to decrease the risk for a progressive neurological disease or disorder or the progress of a neurological disease or disorder in the subject. In an aspect, a disclosed AAV capsid protein can be used to decrease the risk for a progressive neuromuscular disease or disorder or the progress of a neuromuscular disease or disorder in the subject. In an aspect, a disclosed AAV capsid protein can be used to decrease the risk for a progressive neuro-oncologic disease or disorder or the progress of a neuro-oncologic disease or disorder in the subject.
  • a disclosed AAV capsid protein can be used to reduce the risk of infection in one or more CNS or PNS cell types in the subject. In an aspect, a disclosed AAV capsid protein can be used to reduce the risk of developing inflammation in one or more regions and/or structures of the human brain and/or spinal cord. in the subject.
  • inflammation can affect the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, the spinal cord, or any combination thereof.
  • a disclosed AAV capsid protein can be used to repair diseased and/or dysfunctional cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV capsid protein can be used to treat a subject in need thereof.
  • a disclosed AAV capsid protein can be used in a method of delivering gene therapy to a subject in need thereof.
  • a subject in need thereof can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • a disclosed neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder can comprise the absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD). Adie's Pupil.
  • Adie's Syndrome Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS- Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Bechet'
  • Disclosed herein is a library of disclosed AAV capsid proteins. Disclosed herein is a library of AAV capsid proteins. Disclosed herein is a library of AAV capsid proteins having one or more substitutions in variable region IV (VR-IV). Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO:04. Disclosed herein is a library of AAV capsid proteins comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:04. Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO:05.
  • Disclosed herein is a library of AAV capsid proteins comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:05. Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO:06. Disclosed herein is a library of AAV capsid proteins comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:06. Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO:07. Disclosed herein is a library of AAV capsid proteins comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:07.
  • Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO:08. Disclosed herein is a library of AAV capsid proteins comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:08. Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO:09. Disclosed herein is a library of AAV capsid proteins comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:09. Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO: 10.
  • a library of AAV capsid proteins comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 10.
  • a library comprising one or more AAV capsid proteins comprising the sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:82.
  • a library comprising one or more AAV capsid proteins comprising the sequence having at least 85% identity to the sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:82.
  • Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed AAV capsid protein.
  • an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein.
  • an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region VIII (VR-VIII).
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region IV (VR-IV) and one or more substitutions in variable region VIII (VR-VIII).
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:01 with one or more substitutions in variable region IV (VR-IV).
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:02 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:03 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • amino acid sequence of SEQ ID NO:01 is encoded by the nucleotide sequence of SEQ ID NO: 155.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • the amino acid sequence of SEQ ID NO:02 is encoded by the nucleotide sequence of SEQ ID NO: 156.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • the amino acid sequence of SEQ ID NO:03 is encoded by the nucleotide sequence of SEQ ID NO: 157.
  • a disclosed encoded AAV capsid protein can be a variant of a parental wildtype capsid protein.
  • a disclosed parental wild-type capsid protein can be a capsid protein of AAV6, AAV8, or AAV9.
  • a disclosed parental wild-type capsid protein can be VP1 of AAV6, AAV8, or AAV9.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 585 - 590 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 142 - SEQ ID NO: 154, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:70 - SEQ ID NO:82.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:26.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 11 - SEQ ID NO:26.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113, wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO:27 - SEQ ID NO:41.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:27 - SEQ ID NO:41.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02, and wherein positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113 wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98.
  • positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98 and positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO: 99 - SEQ ID NO: 113.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein the positions 452 - 458 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, wherein the positions 586 - 592 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, and wherein the positions 452 - 458 and positions 586 - 592 of the AAV capsid protein are numbered with reference to SEQ ID NO:03.
  • positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 114 - SEQ ID NO: 126.
  • positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 114 - SEQ ID NO: 126 and positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:11 - SEQ ID NO:41.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:42 - SEQ ID NO:69.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:04.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:04.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:08, SEQ ID NO:09, or SEQ ID NO: 10.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:08, SEQ ID NO:09, or SEQ ID NO: 10.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:04, wherein the substitution at position 585 is any amino acid other than Q; wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than S; wherein the substitution at position 588 is any amino acid other than S; wherein the substitution at position 589 is any amino acid other than T; and/or wherein the substitution at position 590 is any amino acid other than D.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than QSSSTD.
  • each of positions 585 - 590 can have an amino acid substitution.
  • 1 or more of positions 585 - 590 can have an amino acid substitution.
  • 2 or more of positions 585 - 590 can have an amino acid substitution.
  • 3 or more of positions 585 - 590 can have an amino acid substitution.
  • 4 or more of positions 585 - 590 can have an amino acid substitution.
  • 5 or more of positions 585 - 590 can have an amino acid substitution.
  • 6 or more of positions 585 - 590 can have an amino acid substitution.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:05, wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than G; wherein the substitution at position 457 is any amino acid other than T; wherein the substitution at position 458 is any amino acid other than A; wherein the substitution at position 459 is any amino acid other than N; wherein the substitution at position 460 is any amino acid other than T, and/or wherein the substitution at position 461 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than GGTANTQ.
  • each of positions 455 - 461 can have an amino acid substitution.
  • 1 or more of positions 455 - 461 can have an amino acid substitution.
  • 2 or more of positions 455 - 461 can have an amino acid substitution.
  • 3 or more of positions 455 - 461 can have an amino acid substitution.
  • 4 or more of positions 455 - 461 can have an amino acid substitution.
  • positions 455 - 461 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 6 or more of positions 455 - 461 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 7 or more of positions 455 - 461 can have an amino acid substitution.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:06, wherein the substitution at position 586 is any amino acid other than L; wherein the substitution at position 587 is any amino acid other than Q; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than Q; wherein the substitution at position 590 is any amino acid other than N; and/or wherein the substitution at position 591 is any amino acid other than T.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than LQQQNT.
  • each of positions 586 - 591 can have an amino acid substitution.
  • 1 or more of positions 586 - 591 can have an amino acid substitution.
  • 2 or more of positions 586 - 591 can have an amino acid substitution.
  • 3 or more of positions 586 - 591 can have an amino acid substitution.
  • 4 or more of positions 586 - 591 can have an amino acid substitution.
  • 5 or more of positions 586 - 591 can have an amino acid substitution.
  • 6 or more of positions 586 - 591 can have an amino acid substitution.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:07, wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than G; wherein the substitution at position 457 is any amino acid other than T; wherein the substitution at position 458 is any amino acid other than A; wherein the substitution at position 459 is any amino acid other than N; wherein the substitution at position 460 is any amino acid other than T, wherein the substitution at position 461 is any amino acid other than Q, wherein the substitution at position 586 is any amino acid other than L; wherein the substitution at position 587 is any amino acid other than Q; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than Q; wherein the substitution at position 590 is any amino acid other than N; and/or wherein the substitution at position 455 is any amino acid other than G; where
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:08, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N, and/or wherein the substitution at position 458 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than NGSGQNQ.
  • each of positions 452 - 458 can have an amino acid substitution.
  • 1 or more of positions 452 - 458 can have an amino acid substitution.
  • 2 or more of positions 452 - 458 can have an amino acid substitution.
  • 3 or more of positions 452 - 458 can have an amino acid substitution.
  • 4 or more of positions 452 - 458 can have an amino acid substitution.
  • positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 6 or more of positions 452 - 458 can have an amino acid substitution.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:09, wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than A; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than A; wherein the substitution at position 590 is any amino acid other than Q; wherein the substitution at position 591 is any amino acid other than A; and/or wherein the substitution at position 592 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than SAQAQAQ.
  • each of positions 586 - 592 can have an amino acid substitution.
  • 1 or more of positions 586 - 592 can have an amino acid substitution.
  • 2 or more of positions 586 - 592 can have an amino acid substitution.
  • 3 or more of positions 586 - 592 can have an amino acid substitution.
  • 4 or more of positions 586 - 592 can have an amino acid substitution.
  • positions 586 - 592 can have an amino acid substitution.
  • 6 or more of positions 586 - 592 can have an amino acid substitution.
  • 7 or more of positions 586 - 592 can have an amino acid substitution.
  • nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO: 10, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 459 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N, wherein the substitution at position 458 is any amino acid other than Q, wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than A; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than A; wherein the substitution at position 590 is any amino acid other than Q; wherein the substitution at position 591 is any
  • a disclosed isolated nucleic acid molecule can comprise the nucleotide sequence for one or more regulatory elements.
  • a disclosed regulatory element can comprise a promoter operably linked to a disclosed isolated nucleic acid molecule, wherein the promoter drives the expression of a disclosed capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.
  • a disclosed promoter can comprise a brain cell specific promoter.
  • Brain cell specific promoter are known to the art and can comprise a synapsin 1 (Synl) promoter, a calmodulin/calcium dependent kinase II (CAMKII) promoter, a glial fibrillary acidic protein (GFAP) promoter, a Rgs5 promoter, a S100 beta promoter, a neuronspecific enolase (NSE) promoter, a Thyl promoter, or any combination thereof.
  • Synl synapsin 1
  • CAMKII calmodulin/calcium dependent kinase II
  • GFAP glial fibrillary acidic protein
  • Rgs5 promoter a Rgs5 promoter
  • S100 beta promoter a S100 beta promoter
  • NSE neuronspecific enolase
  • a disclosed isolated nucleic acid molecule can comprise the sequence for at least one of polypeptide, a glycopeptide, a polysaccharide, a glycolipid, a lipid, or a nucleic acid polymer, or a combination thereof.
  • a disclosed isolated nucleic acid molecule can comprise the sequence for at least one therapeutic agent.
  • a disclosed therapeutic agent can be an oligonucleotide therapeutic agent.
  • a disclosed oligonucleotide therapeutic agent can be a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), a trans-splicing molecule, an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof.
  • a disclosed therapeutic agent can be an ASO or an RNAi.
  • a disclosed nucleic acidbased molecule can comprise one or more modifications at any position applicable.
  • a disclosed therapeutic agent can comprise one or more elements of a gene editing system.
  • a disclosed gene editing system can be a CRISPR gene editing system (e.g., a CRISPR-Cas of any class, type, or subtype), a prime editing system, a base editing system, a zinc- finger nuclease gene editing system, a TALN gene editing system, an ARCUS nuclease gene editing system, a meganuclease gene editing system, a recombinase gene editing system, a transposase gene editing system, an integrase gene editing system, or a homologous recombination gene editing system.
  • CRISPR gene editing system e.g., a CRISPR-Cas of any class, type, or subtype
  • a prime editing system e.g., a CRISPR-Cas of any class, type, or subtype
  • a prime editing system e.g., a CRISPR-Ca
  • a disclosed element of a gene editing system can a CRISPR- based endonuclease.
  • a disclosed CRISPR-based endonuclease can be derived from a CRISPR/Cas type I, type II, or type III system.
  • a disclosed endonuclease can be Cas9 or a Cas 12.
  • a disclosed Cas9 can be that of Staphylococcus aureus or Streptococcus pyogenes.
  • a disclosed Cas9 can be derived from Staphylococcus aureus or Streptococcus pyogenes.
  • a disclosed Cas9 can be that of or derived from a species other than S. aureus or S. Pyogenes.
  • a disclosed Cas9 can be any known Cas9.
  • a disclosed Casl2 can be any known Casl2.
  • a disclosed Cas9 can be any Cas9 analog.
  • Cas9 and Cas 12 are well known to the art and the skilled person can identify and employ a Cas9 or a Cas 12 from one or more species without undue experimentation.
  • a disclosed element of a gene editing system can comprise a sgRNA. The art is familiar with sgRNAs and the skilled person can identify and employ a sgRNA without undue experimentation.
  • a disclosed sgRNA can be directed at any functional domain of a target sequence including, but not limited to those genes causing and/or related to a disclosed neurological disease or disorder, a disclosed neurodegenerative disorder, a disclosed muscular disorder, a disclosed neuromuscular disorder, and/or a disclosed neuro-oncological disorder.
  • a disclosed sgRNA can target some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, SOD1, SPG11, SQSIM1, TARDBP, TBK1, TBP, TDP-43, TRPC
  • a gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect.
  • a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated.
  • a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.
  • a disclosed transgene can comprise a neurodegenerative disease (NDD)-relevant gene.
  • NDD neurodegenerative disease
  • NDDs can comprise Parkinson’s disease (PD), Dementia with Lewy Bodies (DLB), related synucleinopathies, Alzheimer’s disease (AD), Amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), or any combination thereof.
  • PD Parkinson’s disease
  • DLB Dementia with Lewy Bodies
  • AD Alzheimer’s disease
  • ALS Amyotrophic lateral sclerosis
  • HD Huntington’s disease
  • transgene can comprise some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, S0D1, SPG11, SQSIM1, TARDBP, TBK1, TBP, TDP-43, TRPC6, TRPM
  • a disclosed isolated nucleic acid molecule can encode an AAV capsid protein can be incorporated into a disclosed AAV capsid.
  • a disclosed encoded AAV capsid protein can be used to improve and/or enhance gene transfer and/or expression in one or more regions and/or structures of the human brain when compared to a disclosed parental wild-type capsid protein.
  • gene transfer and/or expression can be improved in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at
  • a disclosed encoded AAV capsid protein can be used to improve and/or enhance gene transfer and/or expression to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • gene transfer and/or expression can be improved and/or enhanced in one or more cells types in the CNS and/or the PNS (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • cells types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle
  • a disclosed encoded AAV capsid protein can be used to effect widespread transduction of cells in the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells of the human brain (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed encoded AAV capsid protein can be used to effect widespread transduction in one or more regions and/or structures of the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times
  • a disclosed encoded AAV capsid protein can be used to increase and/or enhance tropism for one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • tropism for one or more cell types in the CNS and/or the PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed encoded AAV capsid protein can be incorporated into a disclosed AAV capsid.
  • a disclosed AAV capsid protein can demonstrate improved tropism for one or more cell types and/or one or more tissue types (such as, for example, one or more disclosed cell types in the human CNS and/or in the human PNS).
  • a disclosed AAV capsid protein can exhibit improved transduction efficiency and/or properties when introduced to one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV capsid variant can efficiently transduce one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV capsid protein can outperform the parental wild-type AAV in one or more disclosed cell types in the human CNS and/or in the human PNS.
  • a disclosed encoded AAV capsid protein can be used to increase and/or enhance the level of viral genomes delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of viral genomes delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed encoded AAV capsid protein can be used to increase and/or enhance the level of a payload delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of a payload delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed encoded AAV capsid protein can be used to increase penetrance through the blood brain barrier (BBB) when compared to the wild-type capsid protein.
  • penetrance through the BBB can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed encoded AAV capsid protein can be used to increase and/or widen distribution to one or more regions and/or parts of the human brain.
  • distribution to one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or widened by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%
  • a disclosed encoded AAV capsid protein can be used to increase and/or enhance payload expression in one or more regions and/or parts of the human brain.
  • payload expression in one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least
  • a disclosed encoded AAV capsid protein can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level.
  • a disclosed encoded AAV capsid protein can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.
  • a disclosed encoded AAV capsid protein can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder.
  • a disclosed encoded AAV capsid protein can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject.
  • a disclosed encoded AAV capsid protein can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject.
  • a disclosed encoded AAV capsid protein can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing disease regression in the subject.
  • a disclosed encoded AAV capsid protein can be used to improve neurological function and/or neuromuscular function in the subject.
  • a disclosed encoded AAV capsid protein can be used to decrease the risk for a progressive neurological disease or disorder or the progress of a neurological disease or disorder in the subject.
  • a disclosed encoded AAV capsid protein can be used to decrease the risk for a progressive neuromuscular disease or disorder or the progress of a neuromuscular disease or disorder in the subject.
  • a disclosed encoded AAV capsid protein can be used to decrease the risk for a progressive neuro-oncologic disease or disorder or the progress of a neuro-oncologic disease or disorder in the subject.
  • a disclosed encoded AAV capsid protein can be used to reduce the risk of infection in one or more CNS or PNS cell types in the subject.
  • a disclosed encoded AAV capsid protein can be used to reduce the risk of developing inflammation in one or more regions and/or structures of the human brain and/or spinal cord. in the subject.
  • inflammation can affect the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, the spinal cord, or any combination thereof.
  • a disclosed encoded AAV capsid protein can be used to repair diseased and/or dysfunctional cell types in the human CNS and/or in the human PNS.
  • a disclosed encoded AAV capsid protein can be used to treat a subject in need thereof.
  • a disclosed AAV capsid protein can be used in a method of delivering gene therapy to a subject in need thereof.
  • a subject in need thereof can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof. 3.
  • Disclosed herein is a cell comprising a disclosed nucleic acid molecule.
  • a cell comprising a disclosed AAV having a capsid comprising a capsid protein Disclosed herein is a cell comprising a disclosed capsid having a disclosed variant.
  • a cell comprising a disclosed viral vector Disclosed herein is a cell comprising a rAAV vector particle.
  • producer cells capable of generating AAV having a disclosed AAV capsid variant Disclosed herein are cells used to perform and/or effect a disclosed method.
  • cells used to perform and/or effect a disclosed method of directed evolution of the AAV capsid protein Disclosed herein is a cell comprising a disclosed nucleic acid molecule.
  • Disclosed herein are cells used to perform and/or effect a disclosed method of generating AAV particles. Disclosed herein are cells used to perform and/or effect a disclosed method of delivering a payload. Disclosed herein are cells used to perform and/or effect a disclosed method of treating a subject.
  • a vector comprising a disclosed isolated nucleic acid molecule.
  • a vector comprising a disclosed isolated nucleic acid molecule encoding a disclosed AAV capsid protein.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region IV (VR-IV).
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region VIII (VR-VIII).
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region IV (VR-IV) and one or more substitutions in variable region VIII (VR-VIII).
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:01 with one or more substitutions in variable region IV (VR-IV).
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:02 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:03 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 04 - SEQ ID NO: 10.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • a disclosed encoded AAV capsid protein can be a variant of a parental wildtype capsid protein.
  • a disclosed parental wild-type capsid protein can be a capsid protein of AAV6, AAV8, or AAV9.
  • a disclosed parental wild-type capsid protein can be VP1 of AAV6, AAV8, or AAV9.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 585 - 590 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 142 - SEQ ID NO: 154, wherein the positions 585 - 590 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:70 - SEQ ID NO:82.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:26.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:11 - SEQ ID NO:26.
  • AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions
  • 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113, wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, comprising the amino acid sequence set forth in any one of SEQ ID NO:27 - SEQ ID NO:41.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:27 - SEQ ID NO:41.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02, and wherein positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113 wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98.
  • positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98 and positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO: 99 - SEQ ID NO: 113.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein the positions 452 - 458 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, wherein the positions 586 - 592 of the AAV capsid protein is numbered with reference to SEQ ID NO:03.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 452 - 458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, wherein positions 586 - 592 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141, and wherein the positions 452 - 458 and positions 586 - 592 of the AAV capsid protein are numbered with reference to SEQ ID NO:03.
  • positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO:114 - SEQ ID NO: 126.
  • positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 114 - SEQ ID NO: 126 and positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:42 - SEQ ID NO:69.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:11 - SEQ ID NO:41.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:04.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:04.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:08, SEQ ID NO:09, or SEQ ID NO: 10.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 08, SEQ ID NO: 09, or SEQ ID NO: 10.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:04, wherein the substitution at position 585 is any amino acid other than Q; wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than S; wherein the substitution at position 588 is any amino acid other than S; wherein the substitution at position 589 is any amino acid other than T; and/or wherein the substitution at position 590 is any amino acid other than D.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than QSSSTD.
  • each of positions 585 - 590 can have an amino acid substitution.
  • 1 or more of positions 585 - 590 can have an amino acid substitution.
  • 2 or more of positions 585 - 590 can have an amino acid substitution.
  • 3 or more of positions 585 - 590 can have an amino acid substitution.
  • 4 or more of positions 585 - 590 can have an amino acid substitution.
  • 5 or more of positions 585 - 590 can have an amino acid substitution.
  • 6 or more of positions 585 - 590 can have an amino acid substitution.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:05, wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than G; wherein the substitution at position 457 is any amino acid other than T; wherein the substitution at position 458 is any amino acid other than A; wherein the substitution at position 459 is any amino acid other than N; wherein the substitution at position 460 is any amino acid other than T, and/or wherein the substitution at position 461 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than GGTANTQ.
  • each of positions 455 - 461 can have an amino acid substitution.
  • 1 or more of positions 455 - 461 can have an amino acid substitution.
  • 2 or more of positions 455 - 461 can have an amino acid substitution.
  • 3 or more of positions 455 - 461 can have an amino acid substitution.
  • 4 or more of positions 455 - 461 can have an amino acid substitution.
  • positions 455 - 461 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 6 or more of positions 455 - 461 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 7 or more of positions 455 - 461 can have an amino acid substitution.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:06, wherein the substitution at position 586 is any amino acid other than L; wherein the substitution at position 587 is any amino acid other than Q; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than Q; wherein the substitution at position 590 is any amino acid other than N; and/or wherein the substitution at position 591 is any amino acid other than T.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than LQQQNT.
  • each of positions 586 - 591 can have an amino acid substitution.
  • 1 or more of positions 586 - 591 can have an amino acid substitution.
  • 2 or more of positions 586 - 591 can have an amino acid substitution.
  • 3 or more of positions 586 - 591 can have an amino acid substitution.
  • 4 or more of positions 586 - 591 can have an amino acid substitution.
  • 5 or more of positions 586 - 591 can have an amino acid substitution.
  • 6 or more of positions 586 - 591 can have an amino acid substitution.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:07, wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than G; wherein the substitution at position 457 is any amino acid other than T; wherein the substitution at position 458 is any amino acid other than A; wherein the substitution at position 459 is any amino acid other than N; wherein the substitution at position 460 is any amino acid other than T, wherein the substitution at position 461 is any amino acid other than Q, wherein the substitution at position 586 is any amino acid other than L; wherein the substitution at position 587 is any amino acid other than Q; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than Q; wherein the substitution at position 590 is any amino acid other
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:08, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N, and/or wherein the substitution at position 458 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than NGSGQNQ.
  • each of positions 452 - 458 can have an amino acid substitution.
  • 1 or more of positions 452 - 458 can have an amino acid substitution.
  • 2 or more of positions 452 - 458 can have an amino acid substitution.
  • 3 or more of positions 452 - 458 can have an amino acid substitution.
  • 4 or more of positions 452 - 458 can have an amino acid substitution.
  • positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 6 or more of positions 452 - 458 can have an amino acid substitution.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO:09, wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than A; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than A; wherein the substitution at position 590 is any amino acid other than Q; wherein the substitution at position 591 is any amino acid other than A; and/or wherein the substitution at position 592 is any amino acid other than Q.
  • the one or more disclosed amino acid substitutions can comprise substitutions other than SAQAQAQ.
  • each of positions 586 - 592 can have an amino acid substitution.
  • 1 or more of positions 586 - 592 can have an amino acid substitution.
  • 2 or more of positions 586 - 592 can have an amino acid substitution.
  • 3 or more of positions 586 - 592 can have an amino acid substitution.
  • 4 or more of positions 586 - 592 can have an amino acid substitution.
  • positions 586 - 592 can have an amino acid substitution.
  • 6 or more of positions 586 - 592 can have an amino acid substitution.
  • 7 or more of positions 586 - 592 can have an amino acid substitution.
  • an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions in SEQ ID NO: 10, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 459 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N, wherein the substitution at position 458 is any amino acid other than Q, wherein the substitution at position 586 is any amino acid other than S; wherein the substitution at position 587 is any amino acid other than A; wherein the substitution at position 588 is any amino acid other than Q; wherein the substitution at position 589 is any amino acid other than A; wherein the substitution at position 590 is any amino acid other than
  • a rAAV vector comprising a disclosed isolated nucleic acid molecule encoding a disclosed AAV capsid protein.
  • a rAAV vector comprising at least one heterologous nucleic acid.
  • a rAAV vector comprising a vector genome.
  • a disclosed vector genome can comprise a first inverted terminal repeat (ITR) and a second ITR.
  • a disclosed vector genome can comprise a nucleic acid sequence encoding a transgene or a payload between the first ITR and the second ITR.
  • a rAAV vector comprising at least one heterologous nucleic acid and at least one inverted terminal repeat (ITR).
  • the at least one ITR can be AAV2 ITR.
  • a rAAV vector comprising a heterologous nucleic acid for a therapeutic protein and/or a therapeutic RNA.
  • a rAAV vector comprising a heterologous nucleic acid for treating a subject having a neurological disease or disorder, a neurodegenerative disorder, muscular disorder, a neuromuscular disorder, and/or a neuro-oncological disorder.
  • a rAAV vector comprising a nucleic acid sequence encoding a transgene.
  • a rAAV vector comprising a nucleic acid sequence encoding a transgene for treating a subject having a neurological disease or disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, and/or a neuro-oncological disorder.
  • an rAAV particle comprising a rAAV capsid comprising a disclosed AAV capsid protein.
  • an rAAV particle comprising (i) an AAV capsid comprising at least one disclosed AAV capsid protein and (ii) a vector genome.
  • a rAAV particle for use in a disclosed method Disclosed herein is a rAAV particle for use in a disclosed method.
  • the rAAV capsid can comprise an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:01 with one or more substitutions in variable region IV (VR-IV).
  • the rAAV capsid can comprise an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:02 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • the rAAV capsid can comprise an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:03 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • the rAAV capsid can comprise an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • the rAAV capsid can comprise an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02. In an aspect of a disclosed rAAV particle, the rAAV capsid can comprise an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02. In an aspect of a disclosed rAAV particle, the rAAV capsid can comprise an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • the rAAV capsid can comprise AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03. In an aspect of a disclosed rAAV particle, the rAAV capsid can comprise an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03. In an aspect of a disclosed rAAV particle, the rAAV capsid can comprise an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • the rAAV capsid can comprise an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • the rAAV capsid can comprise an AAV capsid protein having a sequence having at least 90% identity to the sequence set forth in in any one of SEQ ID NO: 04 - SEQ ID NO: 10.
  • the rAAV capsid can comprise an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:85.
  • the rAAV can capsid comprise an AAV capsid protein having a sequence having at least 90% identity to the sequence set forth in in any one of SEQ ID NO: 11 - SEQ ID NO:82.
  • a disclosed nucleic acid sequence encoding a transgene or heterologous nucleic acid can be operably linked to one or more transcription regulatory elements.
  • a disclosed transcription regulatory element can comprise a ubiquitous promoter operably linked to a disclosed transgene or heterologous nucleic acid, wherein the ubiquitous promoter drives the expression of a disclosed transgene or heterologous nucleic acid.
  • a disclosed transcription regulatory element can comprise a ubiquitous promoter operably linked to a disclosed transgene or heterologous nucleic acid, wherein the tissue specific promoter drives the expression of a disclosed transgene or heterologous nucleic acid.
  • a disclosed transcription regulatory element can comprise a brain-specific promoter and/or a brain-specific enhancer.
  • Brain-specific promoters and/or enhancers are known to the art and can comprise a synapsin 1 (Synl) promoter, a calmodulin/calcium dependent kinase II (CAMKII) promoter, a glial fibrillary acidic protein (GFAP) promoter, a Rgs5 promoter, a S100 beta promoter, a neuronspecific enolase (NSE) promoter, a Thyl promoter, a MeCP2 promoter, a mDLX promoter/enhancer, a mDLX5/6 enhancer, a calmodulin-dependent kinase II (CaMKII or Camk2a) promoter, or any combination thereof.
  • Synl synapsin 1
  • CAMKII calmodulin/calcium dependent kinase II
  • GFAP glial fibr
  • a disclosed payload can encode a therapeutic RNA or a therapeutic protein.
  • a disclosed transgene or a heterologous nucleic acid can encode a therapeutic RNA or a therapeutic protein.
  • a disclosed therapeutic RNA can be a trans-splicing molecule, antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA.
  • a disclosed transgene or disclosed payload can encode a missing, deficient, and/or mutant protein or enzyme.
  • a disclosed heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme.
  • a gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell (such as in one or more cells in the CNS and/or PNS), provides a desired therapeutic effect.
  • a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated.
  • a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.
  • a disclosed transgene can comprise a neurodegenerative disease (NDD)-relevant gene.
  • NDD neurodegenerative disease
  • NDDs can comprise Parkinson’s disease (PD), Dementia with Lewy Bodies (DLB), related synucleinopathies, Alzheimer’s disease (AD), Amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), or any combination thereof.
  • PD Parkinson’s disease
  • DLB Dementia with Lewy Bodies
  • AD Alzheimer’s disease
  • ALS Amyotrophic lateral sclerosis
  • HD Huntington’s disease
  • transgene can comprise some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, LMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, SOD1, SPG11, SQSTM1, TARDBP, TBK1, TBP, TDP-43, TRPC6, TRPM7
  • a disclosed transgene or disclosed heterologous nucleic acid can encode a gene-editing molecule.
  • a disclosed gene-editing molecule can comprise a nuclease or a single guide RNA (sgRNA).
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to improve and/or enhance gene transfer and/or expression in one or more regions and/or structures of the human brain when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising a parental wild-type capsid protein.
  • gene transfer and/or expression can be improved in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to improve and/or enhance gene transfer and/or expression to one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising the wild-type capsid protein.
  • gene transfer and/or expression can be improved and/or enhanced in one or more cells types in the CNS and/or the PNS (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • cells types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to effect widespread transduction of cells in the human brain when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising the wild-type capsid protein.
  • transduction can be increased and/or improved in cells of the human brain (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • cells of the human brain e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to effect widespread transduction in one or more regions and/or structures of the human brain when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising the wild-type capsid protein.
  • transduction can be increased and/or improved in cells in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to increase and/or enhance tropism for one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising the wild-type capsid protein.
  • tropism for one or more cell types in the CNS and/or the PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV vector and/or a disclosed AAV particle can be incorporated into a disclosed AAV capsid.
  • a disclosed AAV capsid protein can demonstrate improved tropism for one or more cell types and/or one or more tissue types (such as, for example, one or more disclosed cell types in the human CNS and/or in the human PNS).
  • a disclosed AAV capsid protein can exhibit improved transduction efficiency and/or properties when introduced to one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV capsid variant can efficiently transduce one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV vector and/or a disclosed AAV particle can outperform a disclosed AAV vector and/or a disclosed AAV particle comprising the parental wild-type AAV capsid in one or more disclosed cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to increase and/or enhance the level of viral genomes delivered to one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising the wild-type capsid protein.
  • the level of viral genomes delivered to one or more cell types in the CNS and/or PNS e.g.
  • excitatory neurons can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to increase and/or enhance the level of a payload delivered to one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising the wild-type capsid protein.
  • the level of a payload delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to increase penetrance through the BBB when compared to a disclosed AAV vector and/or a disclosed AAV particle comprising the wild-type capsid protein.
  • penetrance through the BBB can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to increase and/or widen distribution to one or more regions and/or parts of the human brain.
  • distribution to one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or widened by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to increase and/or enhance payload expression in one or more regions and/or parts of the human brain.
  • payload expression in one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pretreatment level.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pretreatment quality of life.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to improve neurological function and/or neuromuscular function in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to decrease the risk for a progressive neurological disease or disorder or the progress of a neurological disease or disorder in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to decrease the risk for a progressive neuromuscular disease or disorder or the progress of a neuromuscular disease or disorder in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to decrease the risk for a progressive neuro-oncologic disease or disorder or the progress of a neuro-oncologic disease or disorder in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to reduce the risk of infection in one or more CNS or PNS cell types in the subject.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to reduce the risk of developing inflammation in one or more regions and/or structures of the human brain and/or spinal cord. in the subject.
  • inflammation can affect the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, the spinal cord, or any combination thereof.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to repair diseased and/or dysfunctional cell types in the human CNS and/or in the human PNS.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used to treat a subject in need thereof.
  • a disclosed AAV vector and/or a disclosed AAV particle can be used in a method of delivering gene therapy to a subject in need thereof.
  • a subject in need thereof can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof (as discussed above).
  • a disclosed AAV vector and/or a disclosed AAV particle the expression of a disclosed payload can be restored and/or returned to a wild-type, normal, or control expression level.
  • a disclosed AAV vector and/or a disclosed AAV particle can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.
  • a disclosed method can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi -systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof.
  • restoring one or more aspects of cellular homeostasis can comprise
  • a disclosed AAV capsid protein can be incorporated into a disclosed AAV vector and/or a disclosed AAV particle.
  • a disclosed rAAV particle or a disclosed rAAV vector can be AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, or AAVcy.7.
  • a disclosed AAV vector can be bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, or non-primate AAV.
  • a disclosed AAV vector can be AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A- String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV- PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81.
  • a disclosed rAAV particle and/or a disclosed rAAV vector can be formulated for administration or can be administered via one or more routes.
  • routes include, but are not limited to, the following routes: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration.
  • Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV).
  • Administration of a disclosed AAV vector and/or a disclosed AAV particle can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS.
  • Administration can be continuous or intermittent.
  • a pharmaceutical formulation comprising a disclosed AAV particle or a disclosed AAV vector in a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising a disclosed rAAV particle or a disclosed rAAV vector in a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising a disclosed nucleic acid molecule in a pharmaceutically acceptable carrier.
  • a pharmaceutical formulation comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed AAV capsid protein.
  • a disclosed AAV capsid variant can have one or more substitutions in variable region IV (VR-IV).
  • a disclosed AAV capsid variant can have one or more substitutions in variable region VIII (VR-VIII).
  • a disclosed AAV capsid variant can have one or more substitutions in variable region IV (VR-IV) and one or more substitutions in variable region VIII (VR-VIII).
  • a disclosed AAV capsid variant can have a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:01 with one or more substitutions in variable region IV (VR-IV).
  • a disclosed AAV capsid variant can have a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:02 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • a disclosed AAV capsid variant can have a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:03 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII).
  • a disclosed AAV capsid variant can comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10, or can have a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • a disclosed AAV capsid variant can comprise one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01. In an aspect of a disclosed pharmaceutical formulation, a disclosed AAV capsid variant can comprise one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02. In an aspect of a disclosed pharmaceutical formulation, a disclosed AAV capsid variant can have one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02. In an aspect of a disclosed pharmaceutical formulation, a disclosed AAV capsid variant can have one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • a disclosed AAV capsid variant can have one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03. In an aspect of a disclosed pharmaceutical formulation, a disclosed AAV capsid variant can have one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03. In an aspect of a disclosed pharmaceutical formulation, a disclosed AAV capsid variant can have one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • a disclosed isolated nucleic acid molecule can comprise a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:03.
  • a disclosed isolated nucleic acid molecule can comprise a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03.
  • rAAV particles rAAV particles, rAAV vectors, AAV capsid proteins, and nucleic acid molecules are disclosed herein.
  • a disclosed pharmaceutical formulation can comprise about 1 x 10 6 DRP/mL to about 1 x 10 14 DRP/mL.
  • a disclosed pharmaceutical formulation can comprise about 1 x 10 6 DRP/mL, 1 x 10 7 DRP/mL, 1 x 10 8 DRP/mL, 1 x 10 9 DRP/mL, 1 x IO 10 DRP/mL, 1 x 10 11 DRP/mL, 1 x 10 12 DRP/mL, 1 x 10 13 DRP/mL, or 1 x 10 14 DRP/mL.
  • a disclosed pharmaceutical formulation can be administered to a subject in need thereof.
  • a disclosed pharmaceutical formulation can be formulated for administration and/or can be administered via one or more routes.
  • routes include, but are not limited to, the following routes: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration.
  • Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV).
  • Administration of a disclosed pharmaceutical formulation can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.
  • a disclosed pharmaceutical formulation can be used to improve and/or enhance gene transfer and/or expression in one or more regions and/or structures of the human brain when compared to a disclosed pharmaceutical formulation comprising the parental wildtype capsid protein.
  • gene transfer and/or expression can be improved in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least
  • a disclosed pharmaceutical formulation can be used to improve and/or enhance gene transfer and/or expression to one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed pharmaceutical formulation comprising the wild-type capsid protein.
  • gene transfer and/or expression can be improved and/or enhanced in one or more cells types in the CNS and/or the PNS (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • cells types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle
  • a disclosed pharmaceutical formulation can be used to effect widespread transduction of cells in the human brain when compared to a pharmaceutical formulation comprising the wild-type capsid protein.
  • transduction can be increased and/or improved in cells of the human brain (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed pharmaceutical formulation can be used to effect widespread transduction in one or more regions and/or structures of the human brain when compared to a disclosed pharmaceutical formulation comprising the wild-type capsid protein.
  • transduction can be increased and/or improved in cells in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%
  • a disclosed pharmaceutical formulation can be used to increase and/or enhance tropism for one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed pharmaceutical formulation comprising the wild-type capsid protein.
  • tropism for one or more cell types in the CNS and/or the PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed pharmaceutical formulation can be incorporated into a disclosed AAV capsid.
  • a disclosed pharmaceutical formulation can demonstrate improved tropism for one or more cell types and/or one or more tissue types (such as, for example, one or more disclosed cell types in the human CNS and/or in the human PNS).
  • a disclosed AAV capsid protein can exhibit improved transduction efficiency and/or properties when introduced to one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed pharmaceutical formulation can efficiently transduce one or more cell types in the human CNS and/or in the human PNS.
  • a disclosed pharmaceutical formulation can outperform the parental wild-type AAV in one or more disclosed cell types in the human CNS and/or in the human PNS.
  • a disclosed pharmaceutical formulation can be used to increase and/or enhance the level of viral genomes delivered to one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed pharmaceutical formulation comprising the wildtype capsid protein.
  • the level of viral genomes delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed pharmaceutical formulation can be used to increase and/or enhance the level of a payload delivered to one or more cell types in the human CNS and/or in the human PNS when compared to a disclosed pharmaceutical formulation comprising the wild-type capsid protein.
  • the level of a payload delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed pharmaceutical formulation can be used to increase penetrance through the BBB when compared to a disclosed pharmaceutical formulation comprising the wildtype capsid protein.
  • penetrance through the BBB can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed pharmaceutical formulation can be used to increase and/or widen distribution to one or more regions and/or parts of the human brain.
  • distribution to one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or widened by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least
  • a disclosed pharmaceutical formulation can be used to increase and/or enhance payload expression in one or more regions and/or parts of the human brain.
  • payload expression in one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at
  • a disclosed pharmaceutical formulation can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed pharmaceutical formulation can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.
  • a disclosed pharmaceutical formulation can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder.
  • a disclosed pharmaceutical formulation can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject.
  • a disclosed pharmaceutical formulation can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject.
  • a disclosed pharmaceutical formulation can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.
  • a disclosed pharmaceutical formulation can be used to improve neurological function and/or neuromuscular function in the subject.
  • a disclosed pharmaceutical formulation can be used to decrease the risk for a progressive neurological disease or disorder or the progress of a neurological disease or disorder in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to decrease the risk for a progressive neuromuscular disease or disorder or the progress of a neuromuscular disease or disorder in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to decrease the risk for a progressive neuro-oncologic disease or disorder or the progress of a neuro-oncologic disease or disorder in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to reduce the risk of infection in one or more CNS or PNS cell types in the subject.
  • a disclosed pharmaceutical formulation can be used to reduce the risk of developing inflammation in one or more regions and/or structures of the human brain and/or spinal cord. in the subject.
  • inflammation can affect the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, the spinal cord, or any combination thereof.
  • a disclosed pharmaceutical formulation can be used to repair diseased and/or dysfunctional cell types in the human CNS and/or in the human PNS.
  • a disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.
  • a disclosed method can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi -systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof.
  • restoring one or more aspects of cellular homeostasis can comprise
  • a disclosed pharmaceutical formulation can be used to treat a subject in need thereof, or can be used in a method of delivering gene therapy to a subject in need thereof.
  • a subject in need thereof can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof (as discussed above).
  • kits comprising one or more disclosed compositions.
  • a composition of a disclosed kit can comprise one or more disclosed isolated nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed rAAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type AAVs, or any combination thereof.
  • a disclosed kit can comprise a combination of one or more active agents.
  • a disclosed kit can comprise at least two components constituting the kit.
  • the components constitute a functional unit for a given purpose (such as, for example, performing directed evolution on AAV capsid proteins, generating AAV particles, or delivering a payload).
  • Individual member components can be physically packaged together or separately.
  • a disclosed kit comprising an instruction for using the kit can be physically included with instructions for other individual member components.
  • the instruction can be supplied as a separate member component, either in a paper form or an electronic form that can be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
  • a disclosed kit for use in a disclosed method can comprise one or more containers holding a disclosed composition (i.e., disclosed isolated nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed rAAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type AAVs, or any combination thereof) and a label or package insert with instructions for use.
  • a disclosed composition i.e., disclosed isolated nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed rAAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type AAVs, or any combination thereof
  • a disclosed composition i.e., disclosed isolated nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed rAAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsi
  • a disclosed kit can contain one or more additional agents (e.g., excipients, buffers, active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof).
  • one or more active agents can treat, inhibit, and/or ameliorate one or more comorbidities in a subject.
  • one or more active agents can treat, inhibit, and/or ameliorate a disease or a disorder (such as a neurological disease or disorder, a neurodegenerative disorder, muscular disorder, a neuromuscular disorder, and/or a neuro-oncological disorder), an infection, a symptom, a complication, or a combination thereof.
  • suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
  • the containers can be formed from a variety of materials such as glass or plastic.
  • the container can hold a disclosed composition (e.g., for example, disclosed isolated nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed rAAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type AAVs), or a disclosed pharmaceutical formulation and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • a disclosed composition e.g., for example, disclosed isolated nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed rAAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type
  • the label or package insert can indicate that a disclosed composition or a pharmaceutical formulation comprising a disclosed composition can be used for treating, preventing, inhibiting, and/or ameliorating a disease and/or a disorder (such as a neurological disease or disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, and/or a neuro- oncological disorder), an infection, a symptom, a complication, or a combination thereof.
  • a disclosed kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes.
  • a disclosed kit can comprise some or all the components necessary to practice and/or perform one or more disclosed methods.
  • a disclosed kit can be used in a method of delivering a payload.
  • a disclosed kit can be used in a method of treating a subject having a neurological disease or disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, and/or a neuro-oncological disorder.
  • AAV particles comprising delivering to one or more cells a nucleic acid molecule comprising a nucleic acid sequence encoding a AAV capsid protein; culturing the one or more cells; and harvesting the AAV particles from the one or more producer cells.
  • Disclosed herein is a method of generating AAV particles, the method comprising delivering to one or more cells three plasmids, wherein the first plasmid is a helper plasmid, wherein the second plasmid is RepCap plasmid, wherein the second plasmid comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a AAV capsid protein, and wherein the third plasmid is the cis-plasmid or transfer plasmid; culturing the one or more cells; and harvesting the AAV particles from the one or more cells.
  • a method of making an AAV particle the method comprising providing one or more cells comprising a disclosed AAV vector and culturing the one or more cells under conditions allowing for forming an AAV particle.
  • a disclosed method can comprise expressing a disclosed encoded AAV capsid protein.
  • a disclosed method can comprise culturing the one or more cells in a media.
  • a disclosed method can comprise harvesting the AAV particles.
  • a disclosed method can comprise purifying the harvested AAV particles.
  • a disclosed method can comprise using the purified AAV particles in gene therapy.
  • a disclosed method can comprise delivering to the one or more cells a helper plasmid.
  • a disclosed method can comprise delivering to the one or more cells a cis-plasmid or a transfer plasmid encoding a gene of interest or a transgene.
  • disclosed secreted AAV particles can comprise the gene of interest or the transgene.
  • disclosed secreted AAV particles can comprise one or more base-editing components and/or one or more gRNAs.
  • a disclosed method can comprise harvesting from AAV particles and/or rAAV particles by lysis of the host cells of the production culture or by harvest of the spent media from the production culture. Suitable methods of lysing cells are known to the skilled person and can include multiple freeze/thaw cycles, sonication, micro-fluidization, and treatment with chemicals (e.g., detergents and/or proteases), or any combination thereof.
  • a disclosed method can further comprise purifying the rAAV particles.
  • purified can comprise preparing rAAV particles devoid of at least some of the other components that can also be present where the rAAV particles naturally occur or are initially prepared from.
  • isolated rAAV particles can be prepared using a purification technique to enrich it from a source mixture (e.g., culture lysate or production culture supernatant).
  • a source mixture e.g., culture lysate or production culture supernatant.
  • rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anionic exchange chromatography, cationic exchange chromatography, affinity chromatography, or any combination thereof.
  • FFF tangential flow filtration
  • enrichment can be measured in a variety of ways known to the art, including by the proportion of DNase- resistant particles (DRPs) or genome copies (gc) present in a solution, or by infectivity.
  • enrichment can be measured in relation to a second, potentially interfering substance present in the source mixture (e.g., contaminants, including production culture contaminants or in-process contaminants, including helper virus, media components, etc.).
  • a disclosed method of generating and/or making AAV particles can comprise validating the purity of the AAV particles and/or the functionality of the AAV particles.
  • AAV capsid proteins comprising performing multiple rounds of evolution in one or more cortical tissue explants obtained from a singular individual.
  • a method of generating AAV capsid proteins comprising performing multiple rounds of evolution in one or more cortical tissue explants obtained from multiple individuals.
  • evolution can comprise sequence cycling.
  • generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region VIII (corresponding to amino acids 585-590) of SEQ ID NO:01.
  • generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region IV (corresponding to amino acids 455- 461) of SEQ ID NO:02.
  • generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region VIII (corresponding to amino acids 586-591) of SEQ ID NO:02.
  • generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region IV (corresponding to amino acids 455-461) and/or variable region VIII (corresponding to amino acids 586-591) of SEQ ID NO:02.
  • generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region IV (corresponding to amino acids 452- 458) of SEQ ID NO:03.
  • generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region VIII (corresponding to amino acids 586-592) of SEQ ID NO:03.
  • generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region IV (corresponding to amino acids 452-458) and/or variable region VIII (corresponding to amino acids 586-592) of SEQ ID NO: 03.
  • a disclosed method can further comprise generating an initial library of capsid proteins identified via serial evolution.
  • a disclosed parental or a disclosed wild-type capsid protein can comprise the sequence set forth in SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03.
  • a disclosed method can further comprise packaging the initial library of capsid proteins into an AAV vector using triple plasmid transfection.
  • Triple plasmid transfection is known to the art and discussed supra.
  • a disclosed first round of evolution can comprise contacting one or more cortical explants with a disclosed AAV vector comprising a variant capsid library.
  • a disclosed second round of evolution can comprise contacting one or more cortical explants with an AAV vector comprising the variant capsid library generated in the first round of evolution.
  • a disclosed third round of evolution can comprise contacting one or more cortical explants with an AAV vector comprising the variant capsid library generated in the second round of evolution.
  • a disclosed fourth round of evolution can comprise contacting one or more cortical explants with an AAV vector comprising the variant capsid library generated in the third round of evolution.
  • a disclosed first round of evolution can comprise contacting one or more cells in the CNS and/or PNS with a disclosed AAV vector comprising a variant capsid library.
  • a disclosed second round of evolution can comprise contacting one or more one or more cells of the CNS and/or PNS with an AAV vector comprising the variant capsid library generated in the first round of evolution.
  • a disclosed third round of evolution can comprise contacting one or more cells of the CNS and/or PNS with an AAV vector comprising the variant capsid library generated in the second round of evolution.
  • a disclosed fourth round of evolution can comprise contacting one or more cells of the CNS and/or PNS with an AAV vector comprising the variant capsid library generated in the third round of evolution.
  • the one or more cells in the CNS and/or PNS can comprise neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof.
  • the one or more disclosed cells of the CNS and/or PNS are human cells.
  • a disclosed first round of evolution can comprise contacting cells obtained from one or more regions and/or parts of the CNS and/or PNS with a disclosed AAV vector comprising a variant capsid library.
  • a disclosed second round of evolution can comprise contacting cells obtained from one or more regions and/or structures of the CNS and/or PNS with an AAV vector comprising the variant capsid library generated in the first round of evolution.
  • a disclosed third round of evolution can comprise contacting cells obtained from one or more regions and/or structures of the CNS and/or PNS with an AAV vector comprising the variant capsid library generated in the second round of evolution.
  • a disclosed fourth round of evolution can comprise contacting cells obtained from one or more regions and/or structures of the CNS and/or PNS with an AAV vector comprising the variant capsid library generated in the third round of evolution.
  • the one or more regions and/or structures of the CNS and/or PNS can comprise the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, or any combination thereof.
  • the one or more regions and/or structures of the CNS and/or PNS can comprise the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, the spinal cord, or any combination thereof.
  • a disclosed method can further comprise assessing sequence diversity of the variant capsid library generated in the one or more rounds of evolution. In an aspect, a disclosed method can further comprise calculating the percent representation and fold enrichment of each evolved variant capsid library when compared to the parental capsid library. In an aspect, a disclosed method can further comprise ranking the amino acid sequences based on percent representation and fold enrichment to identify one or more candidate capsid proteins. In an aspect, a disclosed method can further comprise characterizing the one or more candidate capsid proteins. [0272] In an aspect, the disclosed one or more cells can be obtained from male and/or female subjects.
  • the disclosed one or more cells can be obtained from subjects of any age (e.g., geriatric subjects, adult subject, adolescent subjects, child subject, infant subjects, and any combination thereof).
  • the disclosed one or more cells can be obtained from a living subject (e.g., via biopsy or tissue extraction) and/or from a deceased subject.
  • a disclosed method can comprise generating a disclosed AAV capsid protein including, for example, the AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • a disclosed method can comprise generating a disclosed AAV capsid protein including, for example, the AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:11 - SEQ ID NO:82.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01, wherein positions 585 - 590 can comprise the sequence set forth in any one of SEQ ID NO: 142 - SEQ ID NO: 154.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01, wherein positions 585 - 590 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 142 - SEQ ID NO: 154.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02, wherein positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02, wherein positions 455 - 461 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02, wherein positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02, wherein positions 586 -591 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • a disclosed method can comprise generating an AAV capsid protein comprising (i) one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02, wherein positions 455 - 461 can comprise the sequence set forth in any one of SEQ ID NO:83 - SEQ ID NO:98, and (ii) one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02, wherein positions 586 - 591 can comprise the sequence set forth in any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • a disclosed method can comprise generating an AAV capsid protein comprising (i) one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02, wherein positions 455 - 461 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, and (ii) one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02, wherein positions 586 -591 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03, wherein positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 114 — SEQ ID NO: 126.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03, wherein positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03, wherein positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03, wherein positions 586 -592 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • a disclosed method can comprise generating an AAV capsid protein comprising (i) one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03, wherein positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 114 - SEQ ID NO: 126, and (ii) one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03, wherein positions 586 - 592 can comprise the sequence set forth in any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • a disclosed method can comprise generating an AAV capsid protein comprising (i) one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03, wherein positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 114 - SEQ ID NO: 126, and (ii) one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03, wherein positions 586 -592 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 127 - SEQ ID NO: 141.
  • METHODS OF DELIVERING A PAYLOAD, TRANSGENE, OR HETEROLOGOUS NUCLEIC ACID Disclosed herein is a method of delivering a payload, the method comprising contacting one or more target cells with a disclosed AAV particle; and expressing the encoded payload.
  • a method of delivering a payload comprising contacting one or more target cells with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.
  • Disclosed herein is a method of delivering a payload, the method comprising contacting one or more target cells in a subject in need thereof with a disclosed AAV particle; and expressing the encoded payload.
  • a method of delivering a payload comprising contacting one or more target cells in a subject in need thereof with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.
  • a disclosed payload can comprise a nucleic acid that is encapsidated in the AAV particle.
  • a disclosed payload can encode a therapeutic RNA (e.g., a trans-splicing molecule, an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA) or a therapeutic protein.
  • a disclosed payload nucleic acid can encode a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA.
  • a disclosed payload can encode a messenger RNA (mRNA) can be encoded by a disclosed payload.
  • a disclosed payload can encode a gene therapy product.
  • a gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect.
  • a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated.
  • a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.
  • a disclosed transgene or a heterologous nucleic acid can encode a therapeutic RNA or a therapeutic protein.
  • a disclosed therapeutic RNA can be a trans-splicing molecule, an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA.
  • a disclosed gene therapy product can be used to treat and/or prevent one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • a disclosed payload can encode one or more a base-editing components and/or one or more gRNA targeting the region to be edited.
  • a disclosed transgene or disclosed heterologous nucleic acid can encode a gene-editing molecule.
  • a disclosed gene-editing molecule can comprise a nuclease or a single guide RNA (sgRNA).
  • a disclosed transgene or a heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed transgene or heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme.
  • a disclosed missing, deficient, and/or mutant protein or enzyme can be encoded by some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, LMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, SOD1, SPG11, SQSTM1, TARDBP, TBK
  • a gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect.
  • a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated.
  • a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.
  • a disclosed transgene can comprise a neurodegenerative disease (NDD)-relevant gene.
  • NDD neurodegenerative disease
  • NDDs can comprise Parkinson’s disease (PD), Dementia with Lewy Bodies (DLB), related synucleinopathies, Alzheimer’s disease (AD), Amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), or any combination thereof.
  • PD Parkinson’s disease
  • DLB Dementia with Lewy Bodies
  • AD Alzheimer’s disease
  • ALS Amyotrophic lateral sclerosis
  • HD Huntington’s disease
  • a disclosed method of delivering a payload can be used to improve and/or enhance gene transfer and/or expression in one or more regions and/or structures of the human brain when compared to a disclosed parental wild-type capsid protein.
  • gene transfer and/or expression can be improved in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at
  • a disclosed method of delivering a payload can be used to improve and/or enhance gene transfer and/or expression to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • gene transfer and/or expression can be improved and/or enhanced in one or more cells types in the CNS and/or the PNS (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • cells types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle
  • a disclosed method of delivering a payload can be used to effect widespread transduction of cells in the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells of the human brain (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed method of delivering a payload can be used to effect widespread transduction in one or more regions and/or structures of the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
  • a disclosed method of delivering a payload can be used to increase and/or enhance tropism for one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • tropism for one or more cell types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof
  • a disclosed method of delivering a payload can be used to increase and/or enhance the level of viral genomes delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of viral genomes delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed method of delivering a payload can be used to increase and/or enhance the level of a payload delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of a payload delivered to one or more cell types in the CNS and/or PNS can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed method of delivering a payload can be used to increase penetrance through the BBB when compared to the wild-type capsid protein.
  • penetrance through the BBB can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV capsid protein can be used to increase and/or widen distribution to one or more regions and/or parts of the human brain.
  • distribution to one or more regions and/or parts of the human brain can be increased and/or widened by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed method of delivering a payload can be used to increase and/or enhance payload expression in one or more regions and/or parts of the human brain.
  • payload expression in one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times,
  • a disclosed method of delivering a payload can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level.
  • a disclosed AAV capsid protein can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.
  • a disclosed method can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder.
  • a disclosed AAV capsid protein can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject.
  • a disclosed AAV capsid protein can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject.
  • a disclosed AAV capsid protein can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.
  • a disclosed method of delivering a payload can be used to improve and/or restore neurological function and/or neuromuscular function in the subject.
  • a disclosed method can be used to decrease the risk for a progressive neurological disease or disorder or the progress of a neurological disease or disorder in the subject and/or to decrease the risk for a progressive neuromuscular disease or disorder or the progress of a neuromuscular disease or disorder in the subject and/or to decrease the risk for a progressive neuro-oncologic disease or disorder or the progress of a neuro-oncologic disease or disorder in the subject.
  • a disclosed method of delivering a payload can be used to reduce the risk of infection in one or more CNS or PNS cell types in the subject. In an aspect, a disclosed method can be used to reduce the risk of developing inflammation in one or more regions and/or structures of the human brain and/or spinal cord.
  • inflammation can affect the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, the spinal cord, or any combination thereof.
  • a disclosed method of delivering a payload can be used to repair diseased and/or dysfunctional cell types in the human CNS and/or in the human PNS.
  • a disclosed method of delivering a payload can be used to treat a subject in need thereof.
  • a subject can be any age and can be male or female.
  • a subject can be treatment-naive.
  • a subject can have received treatment prior to the contacting step and/or administering step.
  • a subject in need thereof can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • a disclosed method can further comprise diagnosing the subject.
  • a disclosed method can comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art.
  • a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof.
  • a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.
  • a disclosed method of delivering a payload can be used in a method of delivering gene therapy to a subject in need thereof.
  • the contacting step allows for expression of the payload in the one or more target cells.
  • the contacting step allows for expression of the transgene or the heterologous nucleic acid in the one or more target cells.
  • a disclosed AAV particle can comprise a disclosed AAV capsid protein.
  • a disclosed AAV capsid protein can comprise any AAV capsid protein disclosed herein.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 10 vg/kg to about 2 x 10 14 vg/kg.
  • a disclosed AAV particle or disclosed vector can be administered at a dose of about 1 x 10 11 to about 8 x 10 13 vg/kg or about 1 x 10 12 to about 8 x 10 13 vg/kg or about 1 x 10 13 to about 6 x 10 13 vg/kg.
  • a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of at least about 1 x 10 10 , at least about 5 x 10 10 , at least about 1 x 10 11 , at least about 5 x 10 11 , at least about 1 x 10 12 , at least about 5 x 10 12 , at least about 1 x 10 13 , at least about 5 x 10 13 , or at least about 1 x 10 14 vg/kg.
  • a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of no more than about 1 x 10 10 , no more than about 5 x 10 10 , no more than about 1 x 10 11 , no more than about 5 x 10 11 , no more than about 1 x 10 12 , no more than about 5 x 10 12 , no more than about 1 x 10 13 , no more than about 5 x 10 13 , or no more than about 1 x 10 14 vg/kg.
  • a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of about 1 x 10 12 vg/kg.
  • a disclosed AAV particle or a disclosed vector can be administered at a dose of about 1 x 10 11 vg/kg.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total to about 1 x 10 17 vg per subject total.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total, about 1 x 10 13 vg per subject total, about 1 x 10 14 vg per subject total, about 1 x 10 15 vg per subject total, about 1 x 10 16 vg per subject total, or about 1 x 10 17 vg per subject total.
  • a disclosed AAV particle or a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range determined by a skilled person.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total to about 1 x 10 17 vg per subject total.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total, about 1 x 10 13 vg per subject total, about 1 x 10 14 vg per subject total, about 1 x 10 15 vg per subject total, about 1 x 10 16 vg per subject total, or about 1 x 10 17 vg per subject total.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can be by delivered retrograde ureteral infusion and/or renal arterial administration and can comprise a range of about 1 x 10 12 vg per subject total to about 1 x 10 17 vg per subject total.
  • a missing, deficient, and/or mutant protein or enzyme e.g., some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, LMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SM
  • the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level.
  • restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme).
  • restoration can be a partial or incomplete restoration.
  • restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.
  • techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.
  • a disclosed method of delivering a payload can further comprise monitoring the subject’s metabolic and/or physiologic improvement following the administering step and/or following the administering steps.
  • a clinician can measure and/or determine the subject’s metabolic and/or physiologic status over time to identify one or more improvements and/or one or more dimini shments.
  • a clinician can use the subject’s metabolic and/or physiologic status and/or the trend of the subject’s metabolic and/or physiological status and/or trend to make a treatment decision and/or to modify an aspect of a disclosed method and/or to continue treating the subject and/or continue to administer a disclosed vector, a disclosed composition, a disclosed therapeutic agent, and/or a disclosed immune modulator, or any combination thereof.
  • metabolic and/or physiologic data can inform the clinician.
  • a disclosed method the expression of a disclosed payload can be restored and/or returned to a wild-type, normal, or control expression level.
  • a disclosed method can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.
  • a disclosed method can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi -systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof.
  • restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.
  • contacting a cell can comprising methods known to the art.
  • contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • administering can comprise a variety of different routes.
  • routes include, but are not limited to, the following routes: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration.
  • Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV).
  • HPV hepatic portal vein
  • Administration of a disclosed AAV vector and/or a disclosed AAV particle and/or a disclosed pharmaceutical formulation can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.
  • CNS e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter
  • Administration can be continuous or intermittent.
  • a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered in combination with RNAi, a trans-splicing molecule, an antisense oligonucleotides, siRNA, shRNA, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more replacement enzymes, one or more immune modulators, and/or a gene editing system.
  • a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered via LNP administration.
  • a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed AAV vector can be concurrently and/or serially administered to a subject via multiple routes of administration.
  • administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can comprise IV administration.
  • a disclosed method can employ multiple routes of administration to the subject including retrograde ureteral infusion and/or arterial route.
  • a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.
  • a disclosed method of delivering a payload can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent.
  • a therapeutic agent can be any disclosed agent that effects a desired clinical outcome.
  • a disclosed therapeutic agent can be an enzyme or a recombinant enzyme.
  • a therapeutically effective amount of a disclosed replacement enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight.
  • a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.
  • the administering step can treat a subject in need thereof.
  • treating a subject can comprising administering one or more times to the subject one or more additional therapies.
  • a disclosed method of delivering a payload can further comprise monitoring the subject for adverse effects.
  • the method in the absence of adverse effects, can further comprise continuing to treat the subject.
  • the method in the presence of adverse effects, can further comprise modifying the treating step.
  • a disclosed method of delivering a payload can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway.
  • an agent can comprise an enzyme for ERT.
  • a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway.
  • a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.
  • a disclosed method of delivering a payload can further comprise administering one or more immune modulators.
  • a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof.
  • a disclosed immune modulator can be bortezomib or SVP-Rapamycin.
  • a disclosed immune modulator can be Tacrolimus.
  • a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose.
  • a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.
  • a disclosed method of delivering a payload can further comprise administering one or more immunosuppressive agents.
  • an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof.
  • a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time.
  • a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time.
  • a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.
  • a disclosed method of delivering a payload can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response.
  • a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD 19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof.
  • a disclosed method of delivering a payload can further comprise administering lipid nanoparticles (LNPs).
  • LNPs can be organ-targeted (such as, for example, one or more parts and/or structures of the CNS or PNS).
  • LNPs can be targeted to one or more parts or regions of the brain.
  • mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.
  • a disclosed method of delivering a payload can further comprise plasmapheresis and immunosuppression.
  • a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.
  • a disclosed method of delivering a payload can further comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed AAV particle or a disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.
  • a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed AAV particle or a disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a
  • a disclosed method of delivering a payload can comprise modifying one or more of the disclosed steps.
  • modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.
  • a disclosed method of delivering a payload can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.
  • a disclosed method of delivering a payload can further comprise concurrent administration of one or more of the following: one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.
  • a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.
  • a disclosed method of delivering a payload can further comprise generating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method of delivering a payload can further comprise generating a disclosed AAV particle or a disclosed AAV vector.
  • generating a disclosed AAV particle or a disclosed viral vector can comprise generating an AAV particle or a rAAV particle or an AAV vector or a recombinant AAV (such as those disclosed herein).
  • a disclosed method of delivering a payload can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.
  • relevant genes such as, for example, a missing, deficient, and/or mutant protein or enzyme
  • a payload can comprise one or more base-editing components and one or more sgRNA targeting the region to be edited.
  • a disclosed method of delivering a payload can further comprise administering an oligonucleotide therapeutic agent.
  • a disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, noncoding RNA (ncRNA), a trans-splicing molecule, a trans-splicing molecule, an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof.
  • a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi.
  • a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable.
  • a disclosed therapeutic agent can comprise one or more elements of a gene editing system.
  • a disclosed gene editing system can be a CRISPR gene editing system (e.g., a CRISPR-Cas of any class, type, or subtype), a prime editing system, a base editing system, a zinc-finger nuclease gene editing system, a TALN gene editing system, an ARCUS nuclease gene editing system, a meganuclease gene editing system, a recombinase gene editing system, a transposase gene editing system, an integrase gene editing system, or a homologous recombination gene editing system.
  • CRISPR gene editing system e.g., a CRISPR-Cas of any class, type, or subtype
  • Prime editing system e.g., a CRISPR-Cas of any class, type
  • a disclosed element of a gene editing system can a CRISPR-based endonuclease.
  • a disclosed CRISPR-based endonuclease can be derived from a CRISPR/Cas type I, type II, or type III system.
  • a disclosed endonuclease can be Cas9 or a Cas 12.
  • a disclosed Cas9 can be that of Staphylococcus aureus or Streptococcus pyogenes.
  • a disclosed Cas9 can be derived from Staphylococcus aureus or Streptococcus pyogenes.
  • a disclosed Cas9 can be that of or derived from a species other than S. aureus or S. Pyogenes.
  • a disclosed Cas9 can be any known Cas9.
  • a disclosed Casl2 can be any known Casl2.
  • a disclosed Cas9 can be any Cas9 analog.
  • Cas9 and Casl2 are well known to the art and the skilled person can identify and employ a Cas9 or a Cas 12 from one or more species without undue experimentation.
  • a disclosed element of a gene editing system can comprise a sgRNA. The art is familiar with sgRNAs and the skilled person can identify and employ a sgRNA without undue experimentation.
  • a disclosed sgRNA can be directed at any functional domain of a target sequence including, but not limited to those genes causing and/or related to a disclosed neurological disease or disorder, a disclosed neurodegenerative disorder, a disclosed muscular disorder, a disclosed neuromuscular disorder, and/or a disclosed neuro- oncological disorder.
  • a disclosed sgRNA can target a coding region of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, S0D1, SPG11, SQSIM1, TARDBP, TBK1, TBP, TDP-43, TRPC
  • a disclosed method of delivering a payload can further comprise generating and/or validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV capsid proteins, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.
  • a disclosed method of delivering a payload can further comprise reducing and/or minimizing vector-mediated immunotoxicity and/or transgene immunogenicity (e.g., the ability to induce specific immunity).
  • vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene.
  • vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.
  • a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method can further comprise measuring and/or determining a subject’s pre-treatment level of one or more clinical and/or metabolic indicators (such as, for example, the expression of some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1,
  • clinical and/or metabolic indicators
  • Disclosed herein is a method of treating a subject, the method comprising administering one or more times to a subject in need thereof a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector.
  • a method of treating a subject the method comprising administering one or more times to a subject in need thereof a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a disclosed AAV particle or a disclosed AAV vector.
  • a method of treating a subject the method comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle comprising a payload.
  • a method of treating a subject comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid.
  • a method of treating a subject comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid, wherein the contacting step allows for expression of the encoded payload, the encoded gene of interest, or the encoded transgene in the one or more cells.
  • a subject can be any age and can be male or female.
  • a subject can be treatment-naive.
  • a subject can have received treatment prior to the contacting step and/or administering step.
  • a subject in need thereof can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • a disclosed method can further comprise diagnosing the subject.
  • a disclosed method can comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art.
  • a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof.
  • a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.
  • a disclosed AAV vector can comprise a gene of interest and (i) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region IV (VR-IV), (ii) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region VIII (VR-VIII), or (iii) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region IV (VR-IV) and one or more substitutions in variable region VIII (VR-VIII).
  • a disclosed AAV vector can comprise a gene of interest and (i) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:01 with one or more substitutions in variable region IV (VR- IV), (ii) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:02 with one or more substitutions in variable region IV (VR-IV) and/or one or more substitutions in variable region VIII (VR-VIII), or (iii) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:03 with one or more substitutions in variable region IV (VR- IV)
  • a nucleic acid molecule comprising a
  • a disclosed AAV vector can comprise a gene of interest and (i) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10, or (ii) a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO: 10.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 585 - 590 relative to SEQ ID NO:01.
  • a disclosed AAV vector can comprise a gene of interest and (i) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 relative to SEQ ID NO:02, (ii) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 591 relative to SEQ ID NO:02, or (iii) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 455 - 461 and/or at positions 586 - 591 relative to SEQ ID NO:02.
  • a disclosed AAV vector can comprise a gene of interest and (i) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:03, (ii) a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 586 - 592 relative to SEQ ID NO:03, and (iii) a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 and/or at positions 586 - 592 relative to SEQ ID NO:03.
  • AAV particles and/or AAV vectors comprising disclosed AAV capsid proteins are discussed supra.
  • a disclosed encoded AAV capsid protein can be a variant of a parental wild-type capsid protein.
  • a disclosed parental wild-type capsid protein can be a capsid protein of AAV6, AAV8, or AAV9.
  • a disclosed parental wild-type capsid protein can be VP1 of AAV6, AAV8, or AAV9.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 585 - 590 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 142 - SEQ ID NO: 154, wherein the positions 585 - 590 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:82.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:70 - SEQ ID NO:82.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO: 11 - SEQ ID NO:26.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 11 - SEQ ID NO:26.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113, wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, comprising the amino acid sequence set forth in any one of SEQ ID NO:27 - SEQ ID NO:41.
  • a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:27 - SEQ ID NO:41.
  • a disclosed AAV vector can comprise a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, wherein positions 455 - 461 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:83 - SEQ ID NO:98, wherein the positions 455 - 461 of the AAV capsid protein is numbered with reference to SEQ ID NO:02, and wherein positions 586 - 591 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:99 - SEQ ID NO: 113 wherein the positions 586 - 591 of the AAV capsid protein is numbered with reference to SEQ ID NO:02.
  • a disclosed method can comprise expressing a disclosed payload, a disclosed transgene, or a disclosed heterologous nucleic acid in one or more target tissues.
  • a disclosed payload can comprise a nucleic acid that is encapsidated in the AAV particle.
  • a disclosed payload can encode a therapeutic RNA (e.g., a trans-splicing molecule, an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA) or a therapeutic protein.
  • a disclosed payload nucleic acid can encode a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA.
  • a disclosed payload can encode a messenger RNA (mRNA) can be encoded by a disclosed payload.
  • mRNA messenger RNA
  • a disclosed payload can encode a gene therapy product.
  • a gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect.
  • a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated.
  • a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.
  • a disclosed transgene or a heterologous nucleic acid can encode a therapeutic RNA or a therapeutic protein.
  • a disclosed therapeutic RNA can be a trans-splicing molecule, an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA.
  • a disclosed gene therapy product can be used to treat and/or prevent one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • a disclosed payload can encode one or more a base-editing components and/or one or more gRNA targeting the region to be edited.
  • a disclosed transgene or disclosed heterologous nucleic acid can encode a gene-editing molecule.
  • a disclosed gene-editing molecule can comprise a nuclease or a single guide RNA (sgRNA).
  • a disclosed transgene or a heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme.
  • a disclosed transgene or heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme.
  • a disclosed missing, deficient, and/or mutant protein or enzyme can be encoded by some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, SOD1, SPG11, SQSIM1, TARDBP, TBK
  • a disclosed gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect.
  • a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated.
  • a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.
  • a disclosed transgene can comprise a neurodegenerative disease (NDD)-relevant gene.
  • NDD neurodegenerative disease
  • NDDs can comprise Parkinson’s disease (PD), Dementia with Lewy Bodies (DLB), related synucleinopathies, Alzheimer’s disease (AD), Amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), or any combination thereof.
  • PD Parkinson’s disease
  • DLB Dementia with Lewy Bodies
  • AD Alzheimer’s disease
  • ALS Amyotrophic lateral sclerosis
  • HD Huntington’s disease
  • a disclosed method of treating can be used to improve and/or enhance gene transfer and/or expression in one or more regions and/or structures of the human brain when compared to a disclosed parental wild-type capsid protein.
  • gene transfer and/or expression can be improved in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at
  • a disclosed method of treating can be used to improve and/or enhance gene transfer and/or expression to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • gene transfer and/or expression can be improved and/or enhanced in one or more cells types in the CNS and/or the PNS (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at
  • a disclosed method of treating can be used to effect widespread transduction of cells in the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells of the human brain (e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed method of treating can be used to effect widespread transduction in one or more regions and/or structures of the human brain when compared to the wild-type capsid protein.
  • transduction can be increased and/or improved in cells in one or more regions and/or structures of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at
  • a disclosed method of treating can be used to increase and/or enhance tropism for one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • tropism for one or more cell types in the CNS and/or the PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof
  • a disclosed method of treating can be used to increase and/or enhance the level of viral genomes delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of viral genomes delivered to one or more cell types in the CNS and/or PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof
  • a disclosed method of treating can be used to increase and/or enhance the level of a payload delivered to one or more cell types in the human CNS and/or in the human PNS when compared to the wild-type capsid protein.
  • the level of a payload delivered to one or more cell types in the CNS and/or PNS e.g., excitatory neurons, astrocytes, oligodendrocytes, glial cells, inhibitory neurons, pericytes, smooth muscle cells, endothelial cells, microglia, macrophages, or any combination thereof
  • a disclosed method of treating can be used to increase penetrance through the BBB when compared to the wild-type capsid protein.
  • penetrance through the BBB can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed AAV capsid protein can be used to increase and/or widen distribution to one or more regions and/or parts of the human brain.
  • distribution to one or more regions and/or parts of the human brain can be increased and/or widened by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.
  • a disclosed method can be used to increase and/or enhance payload expression in one or more regions and/or parts of the human brain.
  • payload expression in one or more regions and/or parts of the human brain e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof) can be increased and/or enhanced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least
  • a disclosed method of treating can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level.
  • a disclosed AAV capsid protein can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.
  • a disclosed method of treating can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder.
  • a disclosed AAV capsid protein can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject.
  • a disclosed AAV capsid protein can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject.
  • a disclosed AAV capsid protein can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.
  • a disclosed method of treating can be used to improve and/or restore neurological function and/or neuromuscular function in the subject.
  • a disclosed method can be used to decrease the risk for a progressive neurological disease or disorder or the progress of a neurological disease or disorder in the subject and/or to decrease the risk for a progressive neuromuscular disease or disorder or the progress of a neuromuscular disease or disorder in the subject and/or to decrease the risk for a progressive neuro-oncologic disease or disorder or the progress of a neuro-oncologic disease or disorder in the subject.
  • a disclosed method can be used to reduce the risk of infection in one or more CNS or PNS cell types in the subject.
  • a disclosed method can be used to reduce the risk of developing inflammation in one or more regions and/or structures of the human brain and/or spinal cord.
  • inflammation can affect the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, the spinal cord, or any combination thereof.
  • a disclosed method can be used to repair diseased and/or dysfunctional cell types in the human CNS and/or in the human PNS.
  • a disclosed method can be used to treat a subject in need thereof.
  • a subject can be any age and can be male or female.
  • a subject can be treatment-naive.
  • a subject can have received treatment prior to the contacting step and/or administering step.
  • a subject in need thereof can have one or more neurological diseases or disorders, neurodegenerative disorders, muscular disorders, neuromuscular disorders, neuro-oncological disorders, or any combination thereof.
  • a disclosed method can be used in a method of delivering gene therapy to a subject in need thereof.
  • the contacting step allows for expression of the payload in the one or more target cells.
  • the contacting step allows for expression of the transgene or the heterologous nucleic acid in the one or more target cells.
  • a disclosed AAV particle can comprise a disclosed AAV capsid protein.
  • a disclosed AAV capsid protein can comprise any AAV capsid protein disclosed herein.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 10 vg/kg to about 2 x 10 14 vg/kg.
  • a disclosed AAV particle or disclosed vector can be administered at a dose of about 1 x 10 11 to about 8 x 10 13 vg/kg or about 1 x 10 12 to about 8 x 10 13 vg/kg or about 1 x 10 13 to about 6 x 10 13 vg/kg.
  • a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of at least about 1 x 10 10 , at least about 5 x 10 10 , at least about 1 x 10 11 , at least about 5 x 10 11 , at least about 1 x 10 12 , at least about 5 x 10 12 , at least about 1 x 10 13 , at least about 5 x 10 13 , or at least about 1 x 10 14 vg/kg.
  • a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of no more than about 1 x 10 10 , no more than about 5 x 10 10 , no more than about 1 x 10 11 , no more than about 5 x 10 11 , no more than about 1 x 10 12 , no more than about 5 x 10 12 , no more than about 1 x 10 13 , no more than about 5 x 10 13 , or no more than about 1 x 10 14 vg/kg.
  • a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of about 1 x 10 12 vg/kg.
  • a disclosed AAV particle or a disclosed vector can be administered at a dose of about 1 x 10 11 vg/kg.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total to about 1 x 10 17 vg per subject total.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total, about 1 x 10 13 vg per subject total, about 1 x 10 14 vg per subject total, about 1 x 10 15 vg per subject total, about 1 x 10 16 vg per subject total, or about 1 x 10 17 vg per subject total.
  • a disclosed AAV particle or a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range determined by a skilled person.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total to about 1 x 10 17 vg per subject total.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10 12 vg per subject total, about 1 x 10 13 vg per subject total, about 1 x 10 14 vg per subject total, about 1 x 10 15 vg per subject total, about 1 x 10 16 vg per subject total, or about 1 x 10 17 vg per subject total.
  • a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can be by delivered retrograde ureteral infusion and/or renal arterial administration and can comprise a range of about 1 x 10 12 vg per subject total to about 1 x 10 17 vg per subject total.
  • a disclosed method of treating, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme e.g., some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA
  • the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level.
  • restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme).
  • restoration can be a partial or incomplete restoration.
  • restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.
  • a disclosed method of treating, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.
  • a disclosed method of treating a subject can further comprise monitoring the subject’s metabolic and/or physiologic improvement following the administering step and/or following the administering steps.
  • a clinician can measure and/or determine the subject’s metabolic and/or physiologic status over time to identify one or more improvements and/or one or more dimini shments.
  • a clinician can use the subject’s metabolic and/or physiologic status and/or the trend of the subject’s metabolic and/or physiological status and/or trend to make a treatment decision and/or to modify an aspect of a disclosed method and/or to continue treating the subject and/or continue to administer a disclosed vector, a disclosed pharmaceutical formulation, a disclosed AAV vector, a disclosed AAV particle, a disclosed composition, a disclosed therapeutic agent, and/or a disclosed immune modulator, or any combination thereof.
  • metabolic and/or physiologic data can inform the clinician.
  • contacting a cell can comprising methods known to the art.
  • contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof .
  • administering can comprise a variety of different routes.
  • routes include, but are not limited to, the following routes: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration.
  • Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV).
  • HPV hepatic portal vein
  • Administration of a disclosed AAV vector and/or a disclosed AAV particle and/or a disclosed pharmaceutical formulation can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.
  • CNS e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter
  • Administration can be continuous or intermittent.
  • a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered in combination with RNAi, trans-splicing molecules, antisense oligonucleotides, siRNA, shRNA, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more replacement enzymes, one or more immune modulators, and/or a gene editing system.
  • a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered via LNP administration.
  • a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed AAV vector can be concurrently and/or serially administered to a subject via multiple routes of administration.
  • administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can comprise IV administration.
  • a disclosed method can employ multiple routes of administration to the subject including retrograde ureteral infusion and/or arterial route.
  • a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.
  • a therapeutic agent can be any disclosed agent that effects a desired clinical outcome.
  • a disclosed therapeutic agent can be an enzyme or a recombinant enzyme.
  • a therapeutically effective amount of a disclosed replacement enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight.
  • a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.
  • the administering step can treat a subject in need thereof.
  • treating a subject can comprising administering one or more times to the subject one or more additional therapies.
  • a disclosed method of treating a subject can further comprise monitoring the subject for adverse effects.
  • the method in the absence of adverse effects, can further comprise continuing to treat the subject.
  • the method in the presence of adverse effects, can further comprise modifying the treating step.
  • a disclosed method of treating can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway.
  • an agent can comprise an enzyme for enzyme replacement therapy.
  • a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway.
  • a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.
  • a disclosed method of treating can further comprise administering one or more immune modulators.
  • a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof.
  • a disclosed immune modulator can be bortezomib or SVP-Rapamycin.
  • a disclosed immune modulator can be Tacrolimus.
  • a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose.
  • a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight.
  • a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.
  • a disclosed method of treating can further comprise can further comprise administering one or more immunosuppressive agents.
  • an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), steroids, or a combination thereof.
  • a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time.
  • a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time.
  • a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.
  • a disclosed method of treating can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response.
  • a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof.
  • Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV).
  • a disclosed method can further comprise administering lipid nanoparticles (LNPs).
  • LNPs can be organ-targeted (such as, for example, one or more cells in the human CNS and/or PNS or one or more regions and/or structures of the human brain).
  • LNPs can be targeted to one or more parts or regions of the human brain (e.g., the occipital lobe, the parietal lobe, the frontal lobe, the temporal lobe, the cortex, the hippocampus, the striatum, the olfactory bulb, the thalamus, the midbrain, the hindbrain, the cerebellum, the hypothalamus, the lateral ventricle, the corpus callosum, the cerebral cortex, the central sulcus, the brain stem, the medulla, the pons, or any combination thereof).
  • mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.
  • a disclosed method of treating can further comprise plasmapheresis and immunosuppression.
  • a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.
  • a disclosed method can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed AAV particle or a disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.
  • a disclosed method of treating can further comprise modifying one or more of the disclosed steps.
  • modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered
  • a disclosed method can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.
  • a disclosed method of treating can further comprise concurrent administration of one or more of the following: one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.
  • a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.
  • a disclosed method of treating a subject can further comprise generating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method of treating can further comprise generating a disclosed AAV particle or a disclosed AAV vector.
  • generating a disclosed AAV particle or a disclosed viral vector can comprise generating an AAV particle or a rAAV particle or an AAV vector or a recombinant AAV (such as those disclosed herein).
  • a disclosed method of treating can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.
  • relevant genes such as, for example, a missing, deficient, and/or mutant protein or enzyme
  • a payload can comprise one or more base-editing components and one or more sgRNA targeting the region to be edited.
  • a disclosed method of treating can further comprise administering an oligonucleotide therapeutic agent.
  • a disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), a trans-splicing molecule, an antisense molecule, miRNA, a morpholino, a peptidenucleic acid (PNA), or an analog or conjugate thereof.
  • a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi.
  • a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable.
  • a disclosed therapeutic agent can comprise one or more elements of a gene editing system.
  • a disclosed gene editing system can be a CRISPR gene editing system (e.g., a CRISPR-Cas of any class, type, or subtype), a prime editing system, a base editing system, a zinc- finger nuclease gene editing system, a TALN gene editing system, an ARCUS nuclease gene editing system, a meganuclease gene editing system, a recombinase gene editing system, a transposase gene editing system, an integrase gene editing system, or a homologous recombination gene editing system.
  • CRISPR gene editing system e.g., a CRISPR-Cas of any class, type, or subtype
  • a prime editing system e.g., a CRISPR-Cas of any class, type, or subtype
  • a prime editing system e.g., a CRISPR-Ca
  • a disclosed element of a gene editing system can a CRISPR- based endonuclease.
  • a disclosed CRISPR-based endonuclease can be derived from a CRISPR/Cas type I, type II, or type III system.
  • a disclosed endonuclease can be Cas9 or a Cas 12.
  • a disclosed Cas9 can be that of Staphylococcus aureus or Streptococcus pyogenes.
  • a disclosed Cas9 can be derived from Staphylococcus aureus or Streptococcus pyogenes.
  • a disclosed Cas9 can be that of or derived from a species other than S. aureus or S. Pyogenes.
  • a disclosed Cas9 can be any known Cas9.
  • a disclosed Casl2 can be any known Casl2.
  • a disclosed Cas9 can be any Cas9 analog.
  • Cas9 and Casl2 are well known to the art and the skilled person can identify and employ a Cas9 or a Cast 2 from one or more species without undue experimentation.
  • a disclosed element of a gene editing system can comprise a sgRNA. The art is familiar with sgRNAs and the skilled person can identify and employ a sgRNA without undue experimentation.
  • a disclosed sgRNA can be directed at any functional domain of a target sequence including, but not limited to those genes causing and/or related to a disclosed neurological disease or disorder, a disclosed neurodegenerative disorder, a disclosed muscular disorder, a disclosed neuromuscular disorder, and/or a disclosed neuro-oncological disorder.
  • a disclosed sgRNA can target a coding region of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN1, SMN2, SNCA, S0D1, SPG11, SQSIM1, TARDBP, TBK1, TBP, TDP-43, TRPC
  • a disclosed method of treating can further comprise generating and/or validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV capsid proteins, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.
  • a disclosed method of treating can further comprise reducing and/or minimizing vector-mediated immunotoxicity and/or transgene immunogenicity (e.g., the ability to induce specific immunity).
  • vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene.
  • vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.
  • a disclosed method of treating can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.
  • a disclosed method of treating can further comprise measuring and/or determining a subject’s pre-treatment level of one or more clinical and/or metabolic indicators (such as, for example, the expression of some or all coding regions of AADC, ACTN4, APOE, APP, ASPA, ATXN1, ATXN2, ATXN3, ATXN7, BACE, CD2AP, CHMP2B, CLN2, COL4A3, COI.4A4, COL4A5, COL4A6, COQ2, DCTN1, FIG4, FN1, FUS, GAN, GBA1, GLB1, GRN, HNRNPA1, HIT, IDS, LAMB2, EMX1B, LRRK2, MAPT, MATR3, NAGLU, NPC1, NPHS1, NPHS2, OPTN, PARK7, PDSS2, PFN1, PLCE1, PRPH, PSEN1, PSEN2, PTEN, SCARB2, SETX, SGSH, SIGMAR1, SMARCAL1, SMN
  • a disclosed method the expression of a disclosed payload can be restored and/or returned to a wild-type, normal, or control expression level.
  • a disclosed method can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.
  • a disclosed method can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi -systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof.
  • restoring one or more aspects of cellular homeostasis can comprise
  • the human CNS is comprised of a diverse array of cell types and subtypes implicated in the underlying causes of many neurodegenerative disorders. CNS disorders do not arise from neurons alone and mutations in non-neuronal cell types, such as astrocytes, oligodendrocytes, and microglia, can be causative for major disease indications. Additionally, treatment of select CNS disorders, including Parkinson’s and AADC deficiency may benefit from less systemic dosing, and more direct administration to target areas.
  • Efficient gene therapy vectors can provide numerous clinical benefits, including lowering total dose, increasing specificity of expression, mitigating expression in off-target tissue, and minimizing therapeutic side effects such as immune activation.
  • Natural AAV serotypes transduce the nonhuman primate and human CNS, though localized delivery of available natural serotypes can result in incomplete coverage of the CNS, or restricted cell-type access.
  • Administration of high doses of AAV via both systemic and direct routes can lead to dorsal root ganglia toxicity.
  • CNS-tropic vectors have been created with intended use via systemic delivery. But, systemic delivery of gene therapies via AAV has limitations including as high vector dose and off target expression in non-target organs due to circulating AAV following administration.
  • AAV capsids that gain greater therapeutic access to the CNS.
  • Diverse capsids have been designed on mouse, pig, non-human primate, and in vitro cultured neuron systems hoping that AAV tropism will exhibit clinical translatability to the human CNS.
  • translatability from one species to another can be hampered by species-to-species variability in expression of target binding moieties. Therefore, only a small number of AAV vectors have been tested directly on human brain tissue or assayed for cell-type specificity.
  • AAV capsids derived from three natural parental serotypes, AAV6, AAV8, and AAV9 were evolved in human brain tissue.
  • the expression profiles of AAV8, AAV9, and their derived novel capsids were characterized in human tissue with cell-type specificity. Glia are implicated in the onset of neurodegenerative disorders, and glial dysregulation is indicated to be partially causative for some CNS disorders compared to the role of neurons.
  • the Examples provided below identify AAV9 capsids harboring a RGD motif in VR4 to highly specifically target human astroglia. From directed evolution of AAV8, two novel capsids, AAV.hb21 and AAV.hb25, were created.
  • AAV8-derived novel capsids improved transduction in human neurons and astrocytes compared to their parental serotype. From in vivo experiments in mice, AAV8-derived novel capsids were - from other organs compared to wildtype AAV8 when delivered systemically, indicating that the novel transduction properties of AAV8-derived capsids are CNS specific.
  • mice All animal procedures were conducted in accordance with IACUC safety standards and regulations. Mice were housed in a temperature and humidity-controlled environment, with a 12-hour light/dark cycle. Mice were provided enrichment, water, and standard chow. The mouse strains used in this study were maintained with the assistance of the Division of Laboratory Animal Resources at Duke University School of Medicine.
  • a sterile bent syringe needle was used to scoop the entire slice into a 1.5 mL centrifuge tube containing 500 pL of PBS.
  • the PBS was aspirated from the tube, and samples were frozen in a Dry Ice/200 proof EtOH slurry, and then placed at -80 °C for storage.
  • slices were fixed in 4% PFA for 24 hr. and stored in PBS/azide until further processed.
  • Genomic DNA Isolation Tissues were thawed from the -80 °C. A pipette was used to remove the slice from the tube onto a sterile petri dish. The slice was minced using a clean, new razor blade. Half of the slice was put into one tube, and the second half put into a second tube (each piece ⁇ 25 mg of tissue). Genomic DNA extraction was carried out using the Thermo Fisher Purelink Genomic DNA kit. A 200 pL mix of RnaseA/Genomic Lysis buffer was used to remove any remaining bits from the petri dish, then pipetted into the slice tube, suspending the slice in media. Some of the liquid was used to wash the original tube in case any DNA remained.
  • the suspended slice was vortexed and incubated at 55 °C.
  • the slice was vortexed every 20 minutes until it was completely digested, about 1.5 hours, then the standard kit directions were followed.
  • the concentration of genomic DNA from a 20 pg human cortical slice was typically 20 ng/pL, for a total of 300 ng.
  • NEB DH10B electrocompetent cells were transformed with 6 pL -8 pL of the library in a 1 mm cuvette, then recovered in 2 mL SOC for 1 hr. Libraries were plated on large-format bacterial plates, then incubated at 32 °C for 15 hours. Colonies were collected, and library plasmid purified using the Zymo Maxiprep kit. [0411] Viral Library Cycling. New AAV libraries for each serotype and variable region were generated by transfecting AAV library plasmid with a helper plasmid in producer HEK293T either in adherent (cycle 1) or suspension (cycle 2,3) cultures.
  • Plasmid copies of the ITR-AAV genomes were limited to 5,000 copies per cell to limit cross-packaging to ⁇ 5%.
  • Virus was purified from collected media (AAV8, AAV9), or media and cell lysate (AAV6), then purified on an iodixanol gradient (Gonzalez TJ, et al. (2023) Nat Protoc. 18(11):3413-3459).
  • Viruses suspended in formulation buffer were sterile filtered and concentrated using Thermo Fisher protein concentrators, then stored at 4 °C for short term use or -80 °C for long-term storage.
  • U87 Cell Transduction U87 cells were split into 150k cells per well of a 24-well plate. 24 hr. later U87 cells had doubled, and were transduced in duplicate by adding virus into the media at 10k MOI (3e9 per well). Cells and virus were incubated for 48 hr., then either fixed (for imaging) or dissociated with trypsin (for flow), washed, and resuspended in PBS with 10% FBS. Cells were sorted for live/dead (DAPI) and GFP or tomato on a FACS Canto flow sorter. Statistics were calculated in FlowJo and plotted in R, Excel, or Prism.
  • Sequence abundance and enrichment was determined using custom Perl scripts (Gonzalez TJ, et al. (2023) Nat Protoc. 18(11) :3413-3459) and then plotted in R or python.
  • Novel AAV Capsid Variant Cloning Top-enriched novel AAV capsid constructs were generated by subcloning human-codon optimized nucleotides encoding the enriched, mutagenized 6-mers or 7-mers into a recombinant ITR-plasmid, replacing the AAV8 or AAV9 wild-type sequence at either VR4 or VR8, respectively, via site-directed mutagenesis.
  • AAV was generated via triple-transfection in HEK293T cells. After 6 days, media was harvested and AAV precipitated in 12% PEG overnight at 4 °C. AAV-containing PEG pellets (3500 prm, 45 minutes) were resuspended in DPBS containing 0.0001% F-68 and 1 mm MgCl. Preparations were treated with DNAse for 1 hr. at 45 °C. Sodium Chloride was added to a final concentration of 300 mM, then preparations were loaded onto an iodixanol gradient, and purified on the gradient in an ultracentrifuge (30,000 g, 18 hrs.).
  • Blocking buffer was removed, then slices were incubated in primary antibody (Table xx) diluted in blocking buffer for 48 hrs. - 72 hrs. at 4 °C. Slices were then further incubated in primary for 3 hrs. at room temperature. Slices were washed in PBS/0.5% Triton 3x for 15 min. each, then incubated with secondary antibody and DAPI in blocking buffer overnight at 4 °C. Slices were then further incubated at room temperature for 3 hrs. Slices were washed in PBS/0.5% Triton 3x for 15 min. each, then Tissue cleared in TDE. Briefly, tissues were immersed step-wise in 30%/60%/75% TDE in PBS for at least 20 minutes each step in the gradient, then were mounted in 75% TDE and stored at 4 °C.
  • images were rotated in ImageJ and cropped to either 500 gm (mice) or 1000 gm (human) cortical windows. Images were analyzed and counted using ImageJ, Imaris, and Excel software, and data was graphed in python or Prism.
  • each capsid variant plasmid was triple-transfected with a helper plasmid, and a TR-plasmid encoding a CBh-promoter driving NLS-eGFP with a unique 10-nucleotide barcode in the 3’UTR.
  • the media from capsid preparations was pooled and purified on an iodixanol gradient. Virus was sterile-filtered, then either stored at -80 °C for later use or PCR amplified and sent to sequencing.
  • mice Barcoded AAV Capsid Pools in Human Slice and Mouse IV Experiments.
  • barcoded AAV Capsid pools were administered intravenously to 4 month old male C57BL/6J mice at lel2 vector genomes in 100 pL volume. After 2 weeks, mice were sacrificed and tissues were dissected into RNAlater at 4 °C (for high throughput sequencing) or fixed in 4% PFA (for immunofluorescence).
  • capsid barcodes from human cortical tissue, 300 pm human cortical slices were transduced with 8 pL lel l AAV capsid barcode pools applied dropwise at Day 0.
  • RNA and gDNA were isolated and purified using Trizol/Qiagen RNeasy Plus columns/back extraction buffer, and cDNA was generated using the High-Capacity RNA-to-cDNA kit from Invitrogen.
  • Barcodes were PCR amplified and abundance was assessed via high throughput sequencing on an Illumina NovaSeq6000 S-Prime chip or NextSeqlOOO with 150-base paired-end reads. Data was analyzed with custom python scripts, Microsoft excel, and graphed in python and prism.
  • mice [0420] Mouse Intravenous and Intracerebroventricular AAV Administration.
  • the barcoded capsid pools were administered via tail-vein injection at lel2 total viral genomes into 4-month old C57BL/6J mice.
  • lelO total vector genomes was administered to in-house bred P0-P1 male and female C57BL/6J mice. Briefly, pups were anesthetized on ice and injected with a 26-gauge needle attached to a Microinjection Syringe Pump (World Precision Instruments) on a chilled stereotactic apparatus (KOPF Instruments). Injection coordinates used were relative to lambda: 0.8 mm sagittal, 1.5 mm rostral, and a depth of 1.5 mm.
  • AAV was administered in 2 pL at a controlled rate of 0.066 pL/s, and mice were recovered on a heating pad before being placed back in their cage. Brains were harvested 2 weeks post-injection and placed in cold 4% PFA for 24 hr., then stored in PBS/azide until processed for immunofluorescence.
  • AAV.hb# or “hb#” are synonymous and are used interchangeably when referring to disclosed compositions (e.g., capsid proteins, AAV particles, AAV vectors, etc.) and to disclosed methods of use.
  • compositions e.g., capsid proteins, AAV particles, AAV vectors, etc.
  • AAV.hb21 and “hb21” are synonymous
  • AAV.hb 14 and “hbl4” are synonymous
  • AAV.hb42 and “hb42” are synonymous.
  • EXAMPLE 1 GENERATION OF A MODEL FOR AAV DIRECTED EVOLUTION IN HUMAN TISSUE
  • AAV mutant capsid libraries were developed from three natural serotypes - AAV6, AAV8, and AAV9. For each serotype, two capsid variable regions were chosen for randomized mutagenesis, with the exception being AAV6, for which only one variable region (Variable region VIII) was chosen. Separate AAV libraries for each capsid and variable region were produced and sequenced for high diversity.
  • AAV libraries were applied to slices in biological duplicates. Unbound AAV capsids were washed from the tissue after 3 hr. of incubation, and AAV variants that transduced the tissue were captured 16 hr. or 24 hr. following transduction. Selected AAV genomes that transduced human cortical slices were captured and used to generate a second-round AAV capsid library. In this way, each AAV capsid library was cycled in duplicate across three rounds of directed evolution.
  • Enriched amino acid sequences of the AAV6 VR8 library converged on a general consensus motif “QxDGxx”.
  • QxDGxx general consensus motif
  • Directed evolution libraries can display enrichment of amino acid sequences that have become enriched due to factors other than ability to transduce target tissue effectively, including false-positive results from cross-packaging.
  • the approach to directed evolution did not initially assess ability of a given capsid to potentiate gene expression following transduction.
  • highly enriched capsid variants were individually cloned and then each capsid was coupled with a recombinant genome expressing a unique barcode in the 3’UTR.
  • Separate AAV preps were generated for each capsid/barcode pair, then pooled according to their parental serotype prior to application to human slices. Human slice cultures were generated as described above.
  • 1.5el 1 viral genomes (vg) were applied to slices. Slices were maintained for 8 days, then RNA and DNA were extracted from the tissue. Input and output values for genomes and RNA expression were captured via high throughput sequencing.
  • AAV9-derived capsids exhibited a high degree of correlation between capsid fold-change rank of viral transduction and RNA expression compared to AAV9.
  • AAV human brain capsid 32 AAV.hb32
  • AAV.hb38 originated from mutations in AAV9 VR4.
  • Almost all the amino acid sequences enriched in the AAV9 VR4 directed evolution contained a strong “RGD” motif, including AAV.hb32 (TRGDLGG) and AAV.hb38 (NRGDLGG), and were nearly invariant to the larger motif (xRGDLxG), suggesting that “LxG” contributes to high expression.
  • AAV.hb40 contained a mutation in the RGD motif, with the 7-mer being TLGDLGG, and differing from hb32 by a single amino acid.
  • AAV.hb40 showed greatly attenuated expression relative to AAV9 and AAV.hb32, indicating that loss of the RGD motif abolished the increased expression of AAV.hb32.
  • the inclusion of this capsid sequence as an enriched motif was due likely to sequencing error or possible capsid cross-packaging.
  • AAV8-derived capsids exhibited the highest RNA fold-enrichment among AAV8 VR8 mutations.
  • Several novel capsids showed consistently high expression across human cortex tissue cultures from different patients, including AAV.hb21 (TQRQTQ) and AAV.hb25 (NAGLSR).
  • AAV.hb21 comprised 64% of the total library reads following three rounds of directed evolution, and though AAV.hb21 is closely related to other capsid sequences, AAV.hb21 transduction resulted in some of the highest relative gene expression among the barcoded capsid pool.
  • There was a much lower correlation between RNA fold-enrichment and genome copy enrichment among AAV8-derived capsids indicating that cell entry ability was uncoupled from recombinant expression.
  • AAV.hb21 transduced significantly more cells than AAV8 across cortical slices and across patient samples.
  • AAV.hb21 exhibited broad expression encompassing cell types in both the white matter and grey matter.
  • markers for human neurons NeN
  • human astrocytes GFAP
  • AAV.hb21 co-labeled significantly more neurons and astrocytes than AAV8 in human cortical slices.
  • distribution of transduced neurons was localized to regions in the slice corresponding to the grey matter and specifically cortical excitatory pyramidal neuron layers. Labeling of astrocytes was more broadly distributed between the white matter and grey matter.
  • AAV.hb21 transduction resulted in higher numbers of GFP+ cells compared to AAV8 in as-yet unidentified cell types.
  • AAV.hb21 Given its propensity to transduce human neurons and astrocytes, whether AAV.hb21 exhibited species back-compatibility in certain cell types was examined. To assess this, hb21 packaging single-stranded CBA-tdTomato was delivered to the mouse CNS in vivo via intracerebroventricular (ICV) injection. Its expression was then compared to wild-type (parental) AAV8. lelO total vg was injected in 2 pL into the left lateral ventricle of P0-P1 pups, then tomato expression was analyzed 14 days later. AAV.hb21 transduced the mouse CNS, including the cortex and hippocampus at high levels.
  • ICV intracerebroventricular
  • AAV.hb21 Within the cortex, neurons spatially and morphologically resembling lower cortical plate pyramidal neuron layers (Layer 4/5) were highly transduced. AAV.hb21 also transduced cells morphologically resembling astroglia. AAV.hb21 delivery resulted in significantly higher tomato+ expression (mean fluorescence intensity, MFI) in the mouse cortex, and resulted in a higher number of tomato+ cells when compared to ICV delivery of CBA-tomato with wild-type AAV8. Thus, AAV.hb21 was effective at transducing the CNS in vivo following direct injection, and exhibited increased levels of expression of single-stranded recombinant AAV genomes. Several other AAV serotypes also exhibited improved transduction in mice following ICV dosing compared to their parental serotype.
  • AAV8 delivery resulted in no amplification from the brain.
  • AAV8-derived capsids largely exhibited no increase in transduction across organs compared to AAV8 following IV delivery (FIG. 7, plotted as log2 fold-change). Instead, many capsids, including AAV.hb21 and AAV.hb25, exhibited lower expression in multiple organs compared to AAV8. This indicates that AAV.hb21, AAV.hb25, and other capsids exhibited biased tropism toward the CNS, meaning that the differences in transduction properties between these capsids and their parent AAV8 serotype could be CNS specific, human specific, or both. Given that amplification of genomes or RNA barcodes from the AAV8 capsid pool from brain tissue following IV injection was not achieved, AAV8-derived capsids likely did not gain the ability to effectively cross the BBB.
  • AAV9-derived capsids displayed improved transduction within mouse heart and muscle tissue.
  • RNA expression from these capsids was significantly lower in the liver compared to AAV9.
  • RGD- based AAV capsids with BBB crossing abilities at the given dose were not observed in the mouse cohort.
  • This data supported the observation of strong AAV transduction of muscle and heart parenchyma with RGD peptide insertions into the VR8 position of AAV9. Together, these data show that the RGD peptide flexibly displayed on the AAV9 exterior surface resulted in high transduction of the brain, heart, and muscle, with transduction in the brain highly biased towards glial tropism.
  • FIG. 8A - FIG. 8D show digital resectioning of transduced human cortical explants.
  • FIG. 8A AAV8, hb21, hb25
  • FIG. 8B AAV9, hb32
  • FIG. 8C AAV8, hb21, hb25
  • FIG. 8D AAV9, hb32
  • MFI mean fluorescence intensity
  • capsid selection directly on human brain tissue evolved capsids (i) with improved transduction properties following direct CNS administration, (ii) that overcome the species-to-species barrier to delivery, and (iii) express gene therapies with high efficacy.
  • AAV capsid tropism directly on human brain explants. We characterize this system and demonstrate AAV-mediated expression persists for at least 2 weeks, enabling a multitude of basic research directions. We identify capsids with high tropism across multiple human CNS cell types.
  • AAV8-derived capsids AAV.hb21 and AAV.hb25 exhibit novel amino acid sequences with expression levels matching or exceeding AAV9.
  • Capsid AAV.hb32 contains a “RGD” motif within the VR4 7-mer which is a sequence known to bind integrins, and has been characterized in two previous capsid evolutions of AAV9 yet at a different capsid position of VR8.
  • AAV.hb21 exhibits improved tropism for both neurons and for GFAP+ astrocytes, in addition to transducing more cells in toto in the human CNS.
  • AAV.hb32 demonstrates that an integrin binding motif in VR4 confers high glial specificity within the human CNS in culture at the concentrations tested, packaging a recombinant transgene expressing eGFP using a ubiquitous promoter, and results in a high level of gene expression. It has not been demonstrated before that integrin binding and glial tropism in the context of the human CNS is linked to an RGD motif displayed within the VR4 surface of AAV9. Further, our evolution and the resulting capsid mutants discovered to transduce human CNS tissue is unique. Not to mention that this method of evolution - nucleotide and subsequently amino acid substitution - is markedly distinct from peptide insert approaches. Peptide insert approaches typically are localized to insertions of amino acids within the VR8 region of AAV serotypes, most notably AAV9.

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Abstract

Des vecteurs de virus adéno-associé recombinant (rAAV) sont des plateformes éminentes d'administration de gènes, et plusieurs thérapies médiées par rAAV ont récemment été approuvées pour une utilisation clinique. Sont divulguées dans la présente invention des compositions comprenant des protéines de capside d'AAV présentant un tropisme amélioré et une efficacité de transduction améliorée pour une ou plusieurs cellules dans le SNC humain, et des méthodes d'utilisation de particules AAV et des vecteurs AAV comprenant ces protéines de capside d'AAV pour administrer efficacement un gène d'intérêt ou un transgène à des cellules ou des tissus cibles et pour traiter un sujet dont l'état le nécessite.
PCT/US2024/022244 2023-03-31 2024-03-29 Compositions comprenant des aav tropiques du cerveau et leurs méthodes d'utilisation Pending WO2024206831A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019006418A2 (fr) * 2017-06-30 2019-01-03 Intima Bioscience, Inc. Vecteurs viraux adéno-associés destinés à la thérapie génique
WO2021226267A2 (fr) * 2020-05-05 2021-11-11 Duke University Compositions de virus adéno-associés compatibles entre espèces et leurs méthodes d'utilisation
US20220251145A1 (en) * 2017-09-20 2022-08-11 4D Molecular Therapeutics Inc. Adeno-associated virus variant capsids and methods of use thereof
WO2022232575A1 (fr) * 2021-04-30 2022-11-03 Duke University Compositions comprenant des protéines de capside chimériques de virus adéno-associé et leurs méthodes d'utilisation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019006418A2 (fr) * 2017-06-30 2019-01-03 Intima Bioscience, Inc. Vecteurs viraux adéno-associés destinés à la thérapie génique
US20220251145A1 (en) * 2017-09-20 2022-08-11 4D Molecular Therapeutics Inc. Adeno-associated virus variant capsids and methods of use thereof
WO2021226267A2 (fr) * 2020-05-05 2021-11-11 Duke University Compositions de virus adéno-associés compatibles entre espèces et leurs méthodes d'utilisation
WO2022232575A1 (fr) * 2021-04-30 2022-11-03 Duke University Compositions comprenant des protéines de capside chimériques de virus adéno-associé et leurs méthodes d'utilisation

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