WO2026064428A2 - Polypeptides de capside et leurs procédés d'utilisation - Google Patents

Polypeptides de capside et leurs procédés d'utilisation

Info

Publication number
WO2026064428A2
WO2026064428A2 PCT/US2025/046835 US2025046835W WO2026064428A2 WO 2026064428 A2 WO2026064428 A2 WO 2026064428A2 US 2025046835 W US2025046835 W US 2025046835W WO 2026064428 A2 WO2026064428 A2 WO 2026064428A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
capsid polypeptide
amino acid
acid sequence
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/046835
Other languages
English (en)
Other versions
WO2026064428A3 (fr
Inventor
Samuel WOLOCK
Megan CRAMER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dyno Therapeutics Inc
Original Assignee
Dyno Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dyno Therapeutics Inc filed Critical Dyno Therapeutics Inc
Publication of WO2026064428A2 publication Critical patent/WO2026064428A2/fr
Publication of WO2026064428A3 publication Critical patent/WO2026064428A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C12N2810/405—Vectors comprising RGD peptide

Definitions

  • Dependoparvoviruses e.g., adeno-associated dependoparvoviruses, e.g., adeno-associated viruses (AAVs)
  • AAVs adeno-associated viruses
  • the present disclosure relates, in part, to improved dependoparvovirus capsid polypeptides, such as VP1 , VP2 and/or VP3 capsid polypeptides, methods of producing a dependoparvovirus comprising capsid polypeptides, compositions for use in the same, as well as viral particles produced by the same
  • the present disclosure relates to viral particles comprising the improved dependoparvovirus capsid polypeptides, with increased muscle biodistribution and/or transduction as compared to viral particles, e.g., without the mutations in the improved dependoparvovirus capsid polypeptides.
  • the disclosure provides capsid polypeptides comprising the sequence motif “RGD” and one or both of (i) an isoleucine at a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NO:7 and (ii) a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • the capsid polypeptides comprise an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NOV.
  • the “RGD” sequence motif is typically inserted into loop VIII (VR-VIII variable region) of the capsid polypeptide, e.g., immediately subsequent (C-terminal) to a position corresponding to Q585, S586, A587, Q588, A589, Q590, A591 , Q592, or T593 of the VP1 capsid polypeptide of SEQ ID NO:7
  • the disclosure provides capsid polypeptides comprising the amino acid sequence TRGDYAS (SEQ ID NO:16).
  • the amino acid sequence TRGDYAS (SEQ ID NO:16) is typically inserted into loop VIII (VR-VIII variable region) of the capsid polypeptide, e.g., immediately subsequent (C-terminal) to a position corresponding to Q585, S586, A587, Q588, A589, Q590, A591 , Q592, or T593 of the VP1 capsid polypeptide of SEQ ID NOV.
  • the amino acid sequence TRGDYAS (SEQ ID NO:16) is immediately subsequent to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • the capsid polypeptide further comprises an isoleucine at a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NO:7, a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NO:7, an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NO:7, or any combination of two or all three of the foregoing.
  • capsid polypeptides comprising the amino acid sequence RGDYSMT (SEQ ID NO:17)
  • the amino acid sequence RGDYSMT (SEQ ID NO:17) is typically inserted into loop VIII (VR-VIII variable region) of the capsid polypeptide, e.g., immediately subsequent (C-terminal) to a position corresponding to Q585, S586, A587, Q588, A589, Q590, A591 , Q592, or T593 of the VP1 capsid polypeptide of SEQ ID NOV.
  • the amino acid sequence RGDYSMT (SEQ ID NO:17) is immediately subsequent to a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NOV.
  • the capsid polypeptide further comprises a histidine at a position corresponding to T582 of the VP1 capsid polypeptide of the VP1 capsid polypeptide of SEQ ID NOV
  • the present disclosure provides a capsid polypeptide described herein.
  • the capsid polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VP1 polypeptide of SEQ ID NO: 12 or 14, or to a VP2 or VP3 portion thereof.
  • a capsid polypeptide of the disclosure comprises one or more mutation differences or a mutation set present in any one of V1 or V2.
  • the percentage sequence identity is calculated excluding any targeting peptide sequence insertion(s) in the capsid polypeptide sequence. In other embodiments, the percentage sequence identity is calculated including any targeting peptide sequence insertion(s) in the capsid polypeptide sequence
  • Exemplary VP1 amino acid sequences of the disclosure are set forth in SEQ ID NOS:12 or 14. Additional exemplary capsid polypeptides are disclosed in Section 6.2 and numbered embodiments 1 to 240
  • the present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding a capsid polypeptide as provided for herein, e.g., a capsid polypeptide disclosed in Section 6.2 or any one of numbered embodiments 1 to 240.
  • the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:13 or 15, a fragment thereof (e.g., a fragment thereof encoding a VP2 or VP3 polypeptide), or a variant of any of the foregoing having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.
  • the percentage sequence identity is calculated excluding any nucleotide sequence(s) encoding targeting peptide sequence insertion(s)
  • the percentage sequence identity is calculated including any nucleotide sequence(s) encoding targeting peptide sequence insertion(s).
  • the nucleic acid is a vector, e.g., a plasmid. Exemplary nucleic acids are disclosed in Section 6.2 and numbered embodiments 241 to 276.
  • the present disclosure further provides a dependoparvovirus particle comprising a capsid polypeptide, a capsid polypeptide disclosed in Section 6.2 or any one of numbered embodiments 1 to 240 and/or a nucleic acid described herein, e.g., a nucleic acid disclosed in Section 6.2 or any one of numbered embodiments 241 to 276 or a nucleic acid comprising a transgene as disclosed in Section 6.7.1.
  • the dependoparvovirus is an adeno-associated dependoparvovirus (AAV).
  • the AAV is AAV9, e.g., a variant AAV9.
  • Exemplary virus particles are disclosed in Section 6.4 and numbered embodiments 277 to 303 In some embodiments, the virus particles have one or more characteristics disclosed in Section 6.5 and numbered embodiments 290 to 295.
  • the disclosure is directed, in part, to a cell, cell-free system, or other translation system comprising a nucleic acid or vector described herein, e.g., comprising a sequence encoding a capsid polypeptide having one or more mutations described herein, for example a capsid polypeptide disclosed in Section 6.2 or any one of numbered embodiments 1 to 240.
  • the cell, cell-free system, or other translation system comprises a dependoparvovirus particle described herein, e.g., wherein the particle comprises a nucleic acid comprising a sequence encoding a capsid polypeptide, e.g., a capsid polypeptide disclosed in Section 6.2 or any one of numbered embodiments 1 to 240 and/or a nucleic acid described herein, e.g., a nucleic acid disclosed in Section 6.2 or any one of numbered embodiments 241 to 276 or a nucleic acid comprising a transgene as disclosed in Section 6.7.1
  • Exemplary cells, cell-free and other translation systems and their use to produce dependoparvovirus particles are disclosed in Section 6.6 and in numbered embodiments 556 to 567
  • the present disclosure further provides methods of using a dependoparvovirus disclosed herein, e.g., for delivering a payload to a cell or treating a disease or condition in a subject.
  • the methods typically comprise contacting the cell or administering to the subject a dependoparvovirus particle described herein in an amount effective to treat the disease or condition. Exemplary methods are disclosed in Section 6.7 and numbered embodiments 304 to 555.
  • the dependoparvovirus particles may be in the form of a composition, e.g., a pharmaceutical composition comprising the dependoparvovirus particles and a pharmaceutically acceptable carrier or excipient, for example as described in Section 6.7.2 and numbered embodiment 568.
  • capsid polypeptides [0015] Additional features, advantages and applications of the capsid polypeptides, nucleic acids, dependoparvovirus particles of the disclosure and methods of their production and use are more particularly described below.
  • FIGS. 1 A-1C Illustration of exemplary AAV serotype alignments. Amino acids that are present only in VP1 polypeptides are in normal text; amino acids that are present only in VP1 and VP2 polypeptides are in bold; amino acids that are present in VP1 , VP2 and VP3 polypeptides are underlined. Figure discloses SEQ ID NOS 3, 1 , 7, 5, and 9, respectively, in order of appearance. 6. DETAILED DESCRIPTION
  • the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 15 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Any disclosure herein of a value preceded by the term “about” or “approximately” is also a disclosure of the value per se. For example, disclosure of “about 10 pg/ml” is a disclosure of the value “10 pg/ml.”
  • the term “corresponds to” as used in reference to a position in a sequence can be used in reference to an entire capsid polypeptide or polynucleotide sequence, such as the full-length sequence of the capsid polypeptide that comprises a VP1 , VP2, and VP3 polypeptide, or a nucleic acid molecule encoding the same.
  • the term “corresponds to” can be used in reference to a region or domain of the capsid polypeptide.
  • a position that corresponds to a position in the VP1 section of the reference capsid polypeptide can correspond to the VP1 portion of the polypeptide of the variant capsid polypeptide.
  • the full-length polypeptide can be used or domains (regions) can be used to determine whether a position corresponds to a specific position.
  • the region is the VP1 polypeptide.
  • the region is the VP2 polypeptide.
  • the region is the VP3 polypeptide.
  • the variant polypeptide when the reference polypeptide is the wild-type sequence (e.g , full-length or region) of a certain serotype of AAV, the variant polypeptide can be of the same serotype with a mutation made at such corresponding position as compared to the reference sequence (e.g., full-length or region). In some embodiments, the variant capsid polypeptide is a different serotype as compared to the reference sequence
  • Dependoparvovirus capsid refers to an assembled viral capsid comprising dependoparvovirus polypeptides
  • a dependoparvovirus capsid is a functional dependoparvovirus capsid, e.g., is fully folded and/or assembled, is competent to infect a target cell, or remains stable (e.g., folded/assembled and/or competent to infect a target cell) for at least a threshold time.
  • Dependoparvovirus particle refers to an assembled viral capsid comprising dependoparvovirus polypeptides and a packaged nucleic acid, e.g., comprising a payload, one or more components of a dependoparvovirus genome (e.g., a whole dependoparvovirus genome), or both
  • a dependoparvovirus particle is a functional dependoparvovirus particle, e.g., comprises a desired payload, is fully folded and/or assembled, is competent to infect a target cell, or remains stable (e.g., folded/assembled and/or competent to infect a target cell) for at least a threshold time.
  • Dependoparvovirus X particle/capsid refers to a dependoparvovirus particle/capsid comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring dependoparvovirus X species or serotype.
  • a dependoparvovirus B particle refers to a dependoparvovirus particle comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring dependoparvovirus B sequence.
  • an AAVX particle/capsid refers to an AAV particle/capsid comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring AAV X serotype
  • an AAV9 particle refers to an AAV particle comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring AAV9 sequence
  • a dependoparvovirus X capsid is referred to as “Wild Type” or“wt” when such capsid comprises capsid polypeptides from a specified sequence identifier associated with such dependoparvovirus X capsid.
  • wild-type AAV9 capsid or wtAAV9 capsid are used interchangeably and refer to a capsid that comprises capsid polypeptides of SEQ ID NOV (e.g., a VP1 capsid of SEQ ID NOV and VP2 and VP3 portions thereof).
  • Edit Distance Sequences disclosed herein may be described in terms of “edit distance.” The minimum number of sequence edits, i.e. , additions, substitutions, or deletions of a single amino acid (for amino acid sequence) or a single nucleotide (for nucleotide sequences), which change one sequence into another sequence is the edit distance between the two sequences.
  • the term “edit distance” is often used interchangeably with the term “Levenshtein distance.”
  • Exogenous refers to a feature, sequence, or component present in a circumstance (e.g., in a nucleic acid, polypeptide, or cell) that does not naturally occur in said circumstance.
  • a nucleic acid sequence encoding a polypeptide can comprise an exogenous codon (e.g., codon encoding for an amino acid that does not naturally occur in that position, for example in a reference sequence), such as provided for herein
  • Use of the term exogenous in this fashion means that the codon in question at this position does not occur naturally, e.g., is not present in AAV9, e g., is not present in SEQ ID NO:7.
  • the codon replaces an endogenous codon.
  • the exogenous codon is inserted into the nucleic acid sequence, for example, relative to a reference sequence.
  • a sequence e.g., a codon
  • a sequence can be exogenous when provided in a particular sequence (e.g., that does not naturally comprise the codon at the site in question) but may not be exogenous in a second sequence (e g , that does naturally comprise that particular codon at the site in question).
  • a polypeptide component of a dependoparvovirus capsid e.g., Cap (e.g., VP1 , VP2, and/or VP3) or Rep
  • the term “functional” refers to a polypeptide which provides at least 50, 60, 70, 80, 90, or 100% of the activity of a naturally occurring version of that polypeptide component (e.g., when present in a host cell).
  • a functional VP1 polypeptide can stably fold and assemble into a dependoparvovirus capsid (e g., that is competent for packaging and/or secretion).
  • “functional” refers to a capsid or particle comprising one or more of the following production characteristics: comprises a desired payload, is fully folded and/or assembled, is competent to infect a target cell, or remains stable (e.g , folded/assembled and/or competent to infect a target cell) for at least a threshold time.
  • Mutation Difference means a single amino acid mutation (e.g., substitution, insertion or deletion) present in a subject polypeptide sequence, relative to a reference polypeptide sequence.
  • the reference polypeptide sequence is a polypeptide of any one of SEQ ID NOU , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, or a VP2 or VP3 portion thereof.
  • the reference polypeptide is any one of SEQ ID NO:7, 12, 14, or a VP2 or VP3 portion thereof.
  • the reference polypeptide is a polypeptide of SEQ ID NO:7.
  • the subject polypeptide is SEQ ID NO:12, 14, or a VP2 or VP3 portion thereof.
  • Mutation Set refers to the complete set of single amino acid mutations (substitutions, deletions and/or insertions) in a variant capsid polypeptide sequence (e.g., a polypeptide sequence of SEQ ID NO:12, 14, or a VP2 or VP3 portion thereof) relative to a reference sequence (e.g., a wild-type reference sequence).
  • the reference sequence is wild-type AAV9 VP1 capsid polypeptide (SEQ ID NO:7) or a VP2 or VP3 portion thereof.
  • part of the mutation set i.e.
  • a variant capsid polypeptide of the disclosure comprises a mutation set not consisting solely of a mutation set present in a capsid polypeptide of SEQ ID NO:12 or 14.
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to an individual nucleic acid monomer (e g , a nucleotide and/or nucleoside); in some embodiments, “nucleic acid’ refers to an oligonucleotide chain comprising individual nucleic acid monomers or a longer polynucleotide chain comprising many individual nucleic acid monomers.
  • a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA.
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
  • a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid is, comprises, or consists of one or more modified, synthetic, or non-naturally occurring nucleotides In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.
  • an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected).
  • the term “and/or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.
  • Percent Identity Sequences disclosed herein may be described in terms of “percent identity” (% identity). For calculating percent identity between two amino acid sequences or two nucleic acid sequences, the two sequences to be compared are aligned using the EMBOSS Needle Pairwise Sequence Alignment software tool based on the Needleman and Wunsch algorithm (Needleman & Wunsch, 1970, J. Mol.
  • Biol 48(3):443-53) (available at www.ebi.ac uk/Tools/psa/emboss_needle/) using the following parameters: Matrix: BLOSUM62 (for amino acid sequences) or DNAfull (for DNA sequences); Gap Open: 10; Gap Extend: 0.5; End Gap Penalty: false; End Gap Open: 10; and End Gap Extend: 0.5. Percent identity is determined by dividing the number of amino acid or nucleotide matches in the alignment by the length of the alignment and multiplying by 100. For example, if an alignment of two amino acid sequences has 95 matching amino acids and an alignment length of 100 amino acids, the two sequences have 95% identity.
  • percent identity when calculating percent identity of two capsid polypeptides, one or both of which contain(s) one or more targeting peptide insertions, percent identity can be determined without removing the targeting peptide insertion sequence(s) from the capsid polypeptide sequence(s) or, alternatively, percent identity can be determined after removing the targeting peptide insertion sequence(s) from the capsid polypeptide sequence(s).
  • a first capsid polypeptide has an identical sequence to a second capsid polypeptide, except that the first capsid polypeptide has a 7-mer targeting peptide insertion
  • the two capsid polypeptides have less than 100% sequence identity when percent identity is determined without removal of the targeting peptide insertion sequence from the first capsid polypeptide sequence, whereas the two capsid polypeptides have 100% sequence identity when the targeting peptide insertion sequence is removed from the first capsid polypeptide sequence prior to calculating percent identity.
  • references herein to percent identity of capsid polypeptides without mention of a targeting peptide refer to percent identity of the capsid polypeptides determined following removal of targeting polypeptide insertion sequence(s), if any, present in both capsid polypeptides, unless required otherwise by context.
  • References herein to percent identity calculated “taking targeting peptide insertions into account” means that the percent identity is calculated without removal of targeting polypeptide insertion sequence(s), if any, present in both capsid polypeptides.
  • References herein to percent identity calculated “without taking targeting peptide insertions into account” means that the percent identity is calculated following removal of targeting polypeptide insertion sequence(s), if any, present in both capsid polypeptides.
  • Polypeptide, peptide, and protein are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • Targeting Peptide refers to a peptide inserted into, or attached to, a capsid polypeptide to alter the tropism of the capsid polypeptide.
  • a targeting peptide can be inserted into an AAV capsid sequence for enhanced targeting to a desired cell-type, tissue, or organ, for example for enhanced targeting to muscle tissue.
  • a targeting peptide is typically 3 to 20 amino acids in length, for example, 3 to 12 amino acids, 5 to 12 amino acids, 5 to 10 amino acids, or 7 to 10 amino acids in length
  • Treating refers to treating a manifest disease or condition, for example, where the subject is already suffering from one or more symptoms of the disease or condition, or refers to treating a pre-manifest disease or condition, for example, where the subject is identified as having a disease or condition but is not yet exhibiting one or more symptoms of the disease or condition.
  • Pre-manifest conditions may be identified by, for example, genetic testing.
  • variant capsid polypeptide refers to a polypeptide that differs from a reference sequence (e.g, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11 , preferably SEQ ID NO:7), or sequence subunit thereof such as a VP2 or VP3 portion thereof).
  • the variant capsid polypeptide can, for example, comprise a mutation (e.g., substitution, deletion, or insertion).
  • the variant is about, or at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence.
  • any capsid polypeptide for example any capsid polypeptide disclosed herein, for example, a capsid polypeptide of SEQ ID NO: 12 or 1 , is a variant capsid polypeptide with respect to another capsid polypeptide having a different amino acid sequence, e g, another capsid polypeptide with a reference sequence as set forth above.
  • the term “variant capsid polypeptide” herein means, and is used interchangeably with, “capsid polypeptide,” and does not require any comparison to a specific reference sequence.
  • the reference sequence is a polypeptide comprising SEQ ID NO:7.
  • the reference sequence comprises or consists of a VP1 , VP2 or VP3 polypeptide, e g, of SEQ ID NO:7.
  • the term “variant” refers to a virus particle that includes a variant capsid polypeptide, e g, described herein.
  • the disclosure is directed, in part, to a variant capsid polypeptide, wherein the variant capsid polypeptide comprises a mutation (insertion, deletion, or substitution) as compared to the wild-type sequence
  • the wild-type sequence is SEQ ID NO:7.
  • the disclosure is directed, in part, to a variant capsid polypeptide comprising SEQ ID NO:7 with one or more mutations as compared to SEQ ID NO:7.
  • the mutation can be, for example, an insertion, deletion, or substitution as compared to the wild-type sequence
  • the wild-type sequence is SEQ ID NO:7.
  • variant capsid polypeptides that increase muscle (cardiac and/or skeletal muscle) biodistribution and/or transduction when incorporated into a viral particle as compared to reference capsid polypeptides, e.g, wild-type capsid polypeptides.
  • the variant capsid polypeptides of the disclosure are engineered to include an amino acid sequence comprising the integrin-targeting sequence motif “RGD”, e.g, via a peptide insertion.
  • capsid polypeptides comprising the sequence motif “RGD” and one or both of (i) an isoleucine at a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NO:7 and (ii) a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • the capsid polypeptides comprise an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • the RGD-containing sequence motif is typically in a VR-VIII variable region and/or in a surface loop of the capsid polypeptide.
  • the RGD-containing sequence motif can be positioned (a) C- terminal to a position corresponding to Q585, S586, or A587 of the VP1 capsid polypeptide of SEQ ID NO:7, e g., immediately C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NO:7, and/or (b) N-terminal to a position corresponding to Q588, A589, or Q590 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • the RGD sequence motif comprises a subsequence Y or F amino acid to produce an RGDY (SEQ ID NO: 513) or RGDF (SEQ ID NO: 514) motif.
  • the RGD sequence motif comprises the amino acid sequence RGDX1X2X3X4, with Xi to X4 each being any amino acid.
  • RGDX1X2X3X4, Xi, X2, and X3 are each independently selected from L, G, V, and A and/or X4 is S, V, A, G, or L.
  • at least one of X2 and X3 is G.
  • the RGD sequence motif comprises the amino acid sequence TRGDYAS (SEQ ID NO:16). In some embodiments, the RGD sequence motif comprises the amino acid sequence RGDYSMT (SEQ ID NO:17). In some embodiments, the RGD sequence motif comprises the amino acid sequence RGDLGLS (SEQ ID NO:504). In some embodiments, the RGD sequence motif comprises the amino acid sequence RGDLSTP (SEQ ID NO:505). In some embodiments, the RGD sequence motif comprises the amino acid sequence SNSRGDYNSL (SEQ ID NO:506). In some embodiments, the RGD sequence motif comprises the amino acid sequence ENRRGDFNNT (SEQ ID NQ:507).
  • the RGD sequence motif comprises the amino acid sequence SRGDYNSL (SEQ ID NO:508). In some embodiments, the RGD sequence motif comprises the amino acid sequence RGDYNSL (SEQ ID NO:509). In some embodiments, the RGD sequence motif comprises the amino acid sequence RGDLST (SEQ ID NO:510). In some embodiments, the RGD sequence motif comprises the amino acid sequence RGDYVGL (SEQ ID NO:511 ). In some embodiments, the RGD sequence motif comprises the amino acid sequence RGDAVGV (SEQ ID NO:512). Additional RGD- containing sequence motifs are disclosed in WO 2020/206189A1, WO 2022/226374 A1 , WO 2022/053630 A1, and WO 2019/207132 A1 , which are incorporated by reference herein.
  • the disclosure provides a variant capsid polypeptide comprising the (a) the amino acid sequence X1X2RGDX3X4X5X6, where Xi is any amino acid or absent, each of X2-X5 is any amino acid, and XB is any amino acid or absent (b) one or both of (i) an isoleucine at a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NO:7 and (ii) a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • the capsid polypeptide comprise an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • At least a portion of the amino acid sequence X1X2RGDX3X4X5X6 is typically in a VR- VIII variable region and/or in a surface loop of the capsid polypeptide
  • the amino acid sequence X1X2RGDX3X4X5X6 can be positioned (a) C-terminal to a position corresponding to Q585, S586, or A587 of the VP1 capsid polypeptide of SEQ ID NOV, e.g, immediately C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NOV, and/or (b) N-terminal to a position corresponding to Q588, A589, or Q590 of the VP1 capsid polypeptide of SEQ ID NOV.
  • Xi is any amino acid or absent;
  • X2 is threonine,
  • X3 is tyrosine,
  • X4 is alanine,
  • X5 is serine,
  • Xs is any amino acid (optionally isoleucine) or absent, or (g) any combination of two or more (or optionally all) of (a) through (f).
  • variant capsid polypeptides comprising the amino acid sequence TRGDYAS (SEQ ID NO:16). At least a portion of SEQ ID NO:16 is typically in a VR- VIII variable region and/or in a surface loop of the capsid polypeptide
  • the amino acid sequence of SEQ ID NO:16 can be positioned (a) C-terminal to a position corresponding to Q585, S586, or A587 of the VP1 capsid polypeptide of SEQ ID NOV, e.g., immediately C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NOV, and/or (b) N-terminal to a position corresponding to Q588, A589, or Q590 of the VP1 capsid polypeptide of SEQ ID NOV, e.g., immediately N-terminal to a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ
  • the variant capsid polypeptide comprises a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NOV and/or an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NOV.
  • variant capsid polypeptides comprising the amino acid sequence TRGDYAS (SEQ ID NO:16)
  • the disclosure provides a variant capsid polypeptide comprising the amino acid sequence TRGDYASI (SEQ ID NO:18). At least a portion of SEQ ID NO:18 is typically in a VR-VIII variable region and/or in a surface loop of the capsid polypeptide.
  • the amino acid sequence of SEQ ID NO: 18 can be positioned (a) C-terminal to a position corresponding to Q585, S586, or A587 of the VP1 capsid polypeptide of SEQ ID NOV, e.g., immediately C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NOV, and/or (b) N- terminal to a position corresponding to A589, or Q590 of the VP1 capsid polypeptide of SEQ ID NOV, e.g., immediately N-terminal to a position corresponding to A589 of the VP1 capsid polypeptide of SEQ ID NOV.
  • the variant capsid polypeptide lacks an amino acid at a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NOV and/or comprises a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NOV and/or comprises an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NOV.
  • variant capsid polypeptides comprising the amino acid sequence TRGDYAS (SEQ ID NO:16)
  • the disclosure provides a variant capsid polypeptide comprising the amino acid sequence TRGDYASIAQAQTQ (SEQ ID NO:19), or an amino acid sequence having one, two, or three amino acid differences as compared to the amino acid sequence of SEQ ID NO:19
  • the amino acid of SEQ ID NO: 19 or the amino acid sequence having one, two or three amino acid differences as compared to the amount acid sequence of SEQ ID NO:19 can be positioned N-terminal to a position corresponding to Q597, V596 or W595 of the VP1 capsid polypeptide of SEQ ID NOV, for example immediately N-terminal to a position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NOV.
  • variant capsid polypeptides comprising the amino acid sequence TRGDYAS (SEQ ID NO:16)
  • the disclosure provides a variant capsid polypeptide comprising the amino acid sequence TRGDYASIAQAQTQWVQNQGA (SEQ ID NO:20), or an amino acid sequence having one, two, or three amino acid differences as compared to the amino acid sequence of SEQ ID NO:20.
  • variant capsid polypeptides comprising the amino acid sequence TRGDYAS (SEQ ID NO:16)
  • the disclosure provides a variant capsid polypeptide comprising the amino acid sequence SATRGDYASIAQAQTQWVQNQGAL (SEQ ID NO:21), or an amino acid sequence having one, two, or three amino acid differences as compared to the amino acid sequence of SEQ ID NO:21.
  • variant capsid polypeptides comprising the amino acid sequence RGDYSMT (SEQ ID NO:17) At least a portion of SEQ ID NO:17 is typically in a VR- VIII variable region and/or in a surface loop of the capsid polypeptide
  • the amino acid sequence of SEQ ID NO: 17 can be positioned (a) C-terminal to a position corresponding to Q585, S586, or A587 of the VP1 capsid polypeptide of SEQ ID NOV, e.g., immediately C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NOV, and/or (b) N-terminal to a position corresponding to Q588, A589, or Q590 of the VP1 capsid polypeptide of SEQ ID NOV, , e.g., immediately N-terminal to a position corresponding to Q588 of the VP1 capsid polypeptide
  • variant capsid polypeptides comprising the amino acid sequence RGDYSMT (SEQ ID NO: 17)
  • the disclosure provides a variant capsid polypeptide comprising the amino acid sequence HNHQSAQRGDYSMT (SEQ ID NO:22), or an amino acid sequence having one, two, or three amino acid differences as compared to the amino acid sequence of SEQ ID NO:21.
  • Variant capsid polypeptides of the disclosure comprising the sequence motif “RGD” (e.g., the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:17) typically comprise an amino acid sequence having at least 80%, at least 85%, 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%, or at least 99% sequence identity to a reference AAV serotype, e.g., as described herein, e.g., to SEQ ID NOV.
  • the percent sequence identity is calculated not taking into account any peptide insertions (e.g., not taking into account the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:17).
  • Exemplary variant capsid polypeptides comprising the amino acid sequence TRGDYAS (SEQ ID NO: 16) include the VP1 capsid polypeptide referred to herein as V1, corresponding to a capsid polypeptide of SEQ ID NO: 12, as well as VP2 and VP3 polypeptides thereof.
  • Exemplary variant capsid polypeptides comprising the amino acid sequence RGDYSMT (SEQ ID NO:17) include the VP1 capsid polypeptide referred to herein as V2, corresponding to a capsid polypeptide of SEQ ID NO: 14, as well as VP2 and VP3 polypeptides thereof.
  • a variant capsid polypeptide of the disclosure comprises the entire mutation set of V1 or the entire mutation set of V2, e.g., as compared to a VP1 capsid polypeptide of SEQ ID NOG
  • the variant capsid polypeptide is, but for the amino acid sequence TRGDYAS (SEQ ID NO:16) or the amino acid sequence RGDYSMT (SEQ ID NO:17), at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference AAV serotype described herein.
  • the variant capsid polypeptide is, but for the mutation differences or mutation set associated with V1 or V2, at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:1 (e.g., a VP1, VP2 or VP3 sequence of SEQ ID NO:1 ).
  • the variant capsid polypeptide is, but for the amino acid sequence TRGDYAS (SEQ ID NO:16) or the amino acid sequence RGDYSMT (SEQ ID NO:17), at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NOG (e g , a VP1 , VP2 or VP3 sequence of SEQ ID NOG).
  • the variant capsid polypeptide is, but for the amino acid sequence TRGDYAS (SEQ ID NO:16) or the amino acid sequence RGDYSMT (SEQ ID NO:17), at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NOG (e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NOG).
  • a capsid polypeptide of SEQ ID NOG e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NOG.
  • the variant capsid polypeptide is, but for the amino acid sequence TRGDYAS (SEQ ID NO:16) or the amino acid sequence RGDYSMT (SEQ ID NO:17), at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NOG (e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NOG)
  • a capsid polypeptide of SEQ ID NOG e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NOG
  • the variant capsid polypeptide is, but for the amino acid sequence TRGDYAS (SEQ ID NO:16) or the amino acid sequence RGDYSMT (SEQ ID NO:17), at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NOG (e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NOG).
  • a capsid polypeptide of SEQ ID NOG e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NOG.
  • the variant capsid polypeptide is, but for the amino acid sequence TRGDYAS (SEQ ID NO:16) or the amino acid sequence RGDYSMT (SEQ ID NO:17), at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:11 (e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:11).
  • a capsid polypeptide of SEQ ID NO:11 e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:11.
  • a variant capsid polypeptide that comprises a variant capsid polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a variant capsid polypeptide as provided herein, e g., a VP1 capsid polypeptide of SEQ ID NO:12 (and optionally comprising the amino acid sequence TRGDYAS (SEQ ID NO:16)) or SEQ ID NO:14 (and optionally comprising the amino acid sequence RGDYSMT (SEQ ID NO:17)), or a VP2 or VP3 portion thereof.
  • the variant capsid polypeptide comprises a VP1 , VP2, VP3, or any combination thereof, that each has about 1 to about 20 mutations as compared to a VP1 polypeptide of SEQ ID NO:12 or SEQ ID NO:14, and comprises one or more of the mutation differences or the entire mutation set of V1 or V2.
  • the variant capsid polypeptide comprises a VP1 , VP2, VP3, or any combination thereof, that each has about 1 to about 10 mutations as compared to a VP1 polypeptide of SEQ ID NO:12 or SEQ ID NO:14, and comprises one or more of the mutation differences or the entire mutation set of V1 or V2
  • the variant capsid polypeptide comprises a VP1 , VP2, VP3, or any combination thereof, that each has about 1 to about 5 mutations as compared to a VP1 polypeptide of SEQ ID NO:12 or SEQ IDNO:14, and comprises one or more of the mutation differences or the entire mutation set of V1 or V2.
  • capsid polypeptide sequences described herein are described in relation to a position and/or amino acid at a position within a reference sequence, e.g., SEQ ID NO:7.
  • the capsid polypeptides described herein are variant capsid polypeptides of the reference sequence, e.g., SEQ ID NO:7, e.g., include capsid polypeptides comprising at least 80%, at least 85%, 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%, or at least 99% identity to the reference capsid polypeptide sequence (e.g., reference capsid polypeptide VP1, VP2 and/or VP3 sequence), e g., SEQ ID NOY (or VP2 or VP3 sequence comprised therein) and include the amino acid sequence TRGDYAS (SEQ ID NO:
  • each amino acid position within a reference sequence corresponds to a position within the sequence of other reference capsid polypeptides such as capsid polypeptides derived from dependoparvoviruses with different serotypes.
  • sequence alignment tools known in the art.
  • a particularly preferred sequence alignment tool is EMBOSS Needle Pairwise Sequence Alignment software tool based on the Needleman and Wunsch algorithm (Needleman & Wunsch, 1970, J. Mol.
  • the variant capsid polypeptides of the invention include variants of reference capsid polypeptides that include one or more mutations described herein in such reference capsid polypeptides at positions corresponding to the position of the mutation described herein in relation to a different reference capsid polypeptide.
  • a mutation described as XnnnY relative to SEQ ID NO:7 (where X is the amino acid present at position nnn in SEQ ID NOV and Y is the amino acid mutation at that position, e.g., described herein)
  • the disclosure provides variant capsid polypeptides comprising at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% identity to a reference capsid polypeptide sequence (e.g., reference capsid polypeptide VP1 , VP2 and/or VP3 sequence) other than SEQ ID NO:7 (or VP2 or VP3 sequence comprised therein) and further comprising the disclosed mutation at a position corresponding to position nnn of SEQ ID NO:7 (e.g., comprising Y at the position in the position in
  • the variant is a variant of the AAV9 capsid polypeptide, which can be referred to as a “AAV9 variant capsid polypeptide” or “variant AAV9 capsid polypeptide ”
  • the disclosure provides variant capsid polypeptide sequences that are variants of a reference sequence other than SEQ ID NOV, e g., a reference sequence other than SEQ ID NOV as described herein, which include one or more mutation corresponding to the mutations described herein.
  • such variants include mutations corresponding to all of the mutations associated with SEQ ID NO:12 or SEQ ID NO:14.
  • variant capsid polypeptides described herein are optionally variants of reference capsids serotypes known in the art.
  • Non-limiting examples of such reference AAV serotypes include AAV1 , AAVrhI O, AAV-DJ, AAV-DJ8, AAV5, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B- 13, AAVTH1.1-32, AAVTH1.1- 35, AAVPHP.B2 (PHP B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B- DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHRB-GGT-T, AAVPHP.B-SGS, AAVPHP B-AQP, AAVPH
  • AAV SM 10-8 AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11 , AAVhu.53, AAV4- 8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.
  • the reference AAV capsid sequence comprises an AAV2 sequence In some embodiments, the reference AAV capsid sequence comprises an AAV5 sequence In some embodiments, the reference AAV capsid sequence comprises an AAV8 sequence In some embodiments, the reference AAV capsid sequence comprises an AAV9 sequence In some embodiments, the reference AAV capsid sequence comprises an AAVrh74 sequence. While not wishing to be bound by theory, it is understood that a reference AAV capsid sequence comprises a VP1 region. In certain embodiments, a reference AAV capsid sequence comprises a VP1 , VP2 and/or VP3 region, or any combination thereof A reference VP1 sequence may be considered synonymous with a reference AAV capsid sequence
  • SEQ ID NO: 1 wild-type AAV2
  • SEQ ID NO:2 An example nucleic acid sequence encoding SEQ ID NO:1 is SEQ ID NO:2
  • SEQ ID NO:3 is the reference sequence.
  • a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 193-724 of SEQ ID NO:3
  • the sequence found in both VP1 and VP2 is in bold (e.g , a VP2 capsid polypeptide includes, e.g., consists of, the sequence corresponding to amino acids 137-724 of SEQ ID NO:3)
  • the sequence that is not underlined or bold is found only in VP1 (e g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-724 of SEQ ID NO:3).
  • the wild-type reference sequence of SEQ ID NO:3 can be encoded by a reference nucleic acid molecule sequence of SEQ ID NO:4.
  • SEQ ID NO:6 An example nucleic acid sequence encoding SEQ ID NO:5 is SEQ ID NO:6
  • a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 203-736 of
  • VP2 capsid polypeptide includes, e.g., consists of, the sequence corresponding to amino acids 138-736 of SEQ ID NO:7) and the sequence that is not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-736 of SEQ ID NO:7).
  • SEQ ID NO:8 An example nucleic acid sequence encoding SEQ ID NO:7 is SEQ ID NO:8
  • SEQ ID NO:11 (alternate wild-type AAVrh74) is as follows: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPV NAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGL VESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGP SGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQI SNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT QNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTL
  • a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 204-738 of SEQ ID NO:9
  • the sequence found in both VP1 and VP2 is in bold
  • a VP2 capsid polypeptide includes, e.g , consists of, the sequence corresponding to amino acids 137-738 of SEQ ID NO:9
  • the sequence that is not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide includes, e g., consists of, amino acids corresponding to amino acids 1-738 of SEQ ID NO:9).
  • SEQ ID NO:9 An example nucleic acid sequence encoding SEQ ID NO:9 is SEQ ID NO:10.
  • the present disclosure refers to structural capsid proteins (including VP1 , VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (i.e. capsid) of a viral vector such as AAV.
  • VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Met1 ), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence
  • a first-methionine (Met1 ) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases.
  • This “Met/AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.).
  • Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins. Where the Met/AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid can be produced, some of which include a Met1/AA1 amino acid (Met+/AA+) and some of which lack a Met1/AA1 amino acid as a result of Met/AA-clipping (Met-/AA-).
  • Met/AA-clipping in capsid proteins see Jin, et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins.
  • references to capsid polypeptides is not limited to either clipped (Met-/AA-) or unclipped (Met+/AA+) and, in context, also refer to independent capsid polypeptides, viral capsids comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid polypeptides of the present disclosure.
  • a direct reference to a “capsid polypeptide” also comprises VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA+) as well as corresponding VP capsid polypeptide which lack the Met1/AA1 amino acid as a result of Met/AA-clipping (Met-/AA-).
  • a reference to a specific SEQ ID NO: which comprises one or more capsid polypeptides which include a Met1/AA1 amino acid should be understood to teach the VP capsid polypeptides which lack the Met1/AA1 amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Met1/AA1 ).
  • reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Met1” amino acid (Met+) encoded by the AUG/ATG start codon is also understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Met1” amino acid (Met-) of the 736 amino acid Met+ sequence
  • reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon can also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1-) of the 736 amino acid AA1+ sequence.
  • references to viral capsids formed from VP capsid proteins can incorporate VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA1+), corresponding VP capsid proteins which lack the Met1/AA1 amino acid as a result of Met/AA1 -clipping (Met-/AA1-), and combinations thereof (Met+/AA1+ and Met-/AA1-).
  • an AAV capsid serotype can include VP1 (Met+/AA1+), VP1 (Met-/AA1-), or a combination of VP1 (Met+/AA1+) and VP1 (Met- /AA1-).
  • An AAV capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met-/AA1-), or a combination of VP3 (Met+/AA1+) and VP3 (Met-/AA1-); and can also include similar optional combinations of VP2 (Met+/AA1) and VP2 (Met-/AA1-).
  • the reference AAV capsid sequence comprises an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the those described above
  • a variant capsid polypeptide of the disclosure is an isolated or purified polypeptide (e.g , isolated or purified from a cell, other biological component, or contaminant).
  • the variant capsid polypeptide is present in a dependoparvovirus particle, e g., described herein.
  • the variant capsid polypeptide is present in a cell, cell-free system, or translation system, e.g., described herein.
  • the variant capsid polypeptide is present in a dependoparvovirus B (e.g., AAV9) particle.
  • the capsid particle has increased muscle transduction as compared to a reference capsid polypeptide, e.g., a wild-type capsid polypeptide such as a VP1 capsid polypeptide of SEQ ID NO:7 or a VP2 or VP3 portion thereof 6.3.
  • the disclosure is further directed, in part, to a nucleic acid comprising a sequence encoding a variant capsid polypeptide as provided for herein, e.g., as described in Section 6.2.
  • the nucleic acid encodes a VP1 variant capsid polypeptide.
  • the nucleic acid encodes a VP2 variant capsid polypeptide.
  • the nucleic acid encodes a VP3 variant capsid polypeptide.
  • the nucleic acid encodes a VP1, VP2 and VP3 variant capsid polypeptide.
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a variant capsid polypeptide that comprises one or more of the mutation differences or the entire mutation set associated with the capsid polypeptide of SEQ ID NO:12 or 14.
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide comprising one or more mutation differences or the entire mutation set associated with the capsid polypeptide of SEQ ID NO:12 or 14, and has at least 80%, at least 85%, 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%, or at least 99% sequence identity to a reference AAV serotype, e.g., as described herein, e.g., to SEQ ID NO:7.
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which, but for the mutation differences or mutation set associated with the capsid polypeptide of SEQ ID NO: 12 or 14, is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference AAV serotype described herein
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which, but for the mutation differences associated with the capsid polypeptide of SEQ ID NO:12 or 14, is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:1 (e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:1).
  • a capsid polypeptide of SEQ ID NO:1 e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:1
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which, but for the mutation differences associated with the capsid polypeptide of SEQ ID NO:12 or 14, is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:3 (e g , a VP1 , VP2 or VP3 sequence of SEQ ID NO:3).
  • a capsid polypeptide of SEQ ID NO:3 e g , a VP1 , VP2 or VP3 sequence of SEQ ID NO:3
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which, but for the mutation differences associated with the capsid polypeptide of SEQ ID NO:12 or 14, is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:5 (e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:5).
  • a capsid polypeptide of SEQ ID NO:5 e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:5
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which, but for the mutation differences associated with the capsid polypeptide of SEQ ID NO:12 or 14, is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:7 (e.g. , a VP1 , VP2 or VP3 sequence of SEQ ID NO:7).
  • a capsid polypeptide of SEQ ID NO:7 e.g. , a VP1 , VP2 or VP3 sequence of SEQ ID NO:7.
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which, but for the mutation differences associated with the capsid polypeptide of SEQ ID NO:12 or 14, is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:9 (e g , a VP1 , VP2 or VP3 sequence of SEQ ID NO:9).
  • a capsid polypeptide of SEQ ID NO:9 e g , a VP1 , VP2 or VP3 sequence of SEQ ID NO:9
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which, but for the mutation differences associated with the capsid polypeptide of SEQ ID NO:12 or 14, is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide of SEQ ID NO:11 (e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:11 ).
  • a capsid polypeptide of SEQ ID NO:11 e.g., a VP1 , VP2 or VP3 sequence of SEQ ID NO:11 .
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide which is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a variant capsid polypeptide as provided herein, e.g., a VP1 capsid polypeptide of the capsid polypeptide of SEQ ID NO:12 or 14, or a VP2 or VP3 portion thereof.
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a variant capsid polypeptide as provided herein.
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide comprising a VP1 , VP2, VP3, or any combination thereof, that each has about 1 to about 20 mutations as compared to a VP1 polypeptide of the capsid polypeptide of SEQ ID NO: 12 or 14, and comprises one or more of the mutation differences or the entire mutation set of the capsid polypeptide of SEQ ID NO:12 or 14.
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide comprising a VP1 , VP2, VP3, or any combination thereof, that each has about 1 to about 10 mutations as compared to a VP1 polypeptide of the capsid polypeptide of SEQ ID NO: 12 or 14, and comprises one or more of the mutation differences or the entire mutation set of the capsid polypeptide of SEQ ID NO:12 or 14.
  • the nucleic acid comprises a nucleotide sequence that encodes a variant capsid polypeptide comprising a VP1 , VP2, VP3, or any combination thereof, that each has about 1 to about 5 mutations as compared to a VP1 polypeptide of the capsid polypeptide of SEQ ID NO: 12 or 14, and comprises one or more of the mutation differences or the entire mutation set of the capsid polypeptide of SEQ ID NO:12 or 14.
  • the nucleic acid comprises a nucleotide sequence of SEQ ID NO:13 (which encodes a capsid polypeptide of SEQ ID NO:12). In some embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:15 (which encodes a capsid polypeptide of SEQ ID NO:14).
  • a nucleic acid of the disclosure (e.g., encoding a variant capsid polypeptide as described in Section 6.2) comprises conventional control elements or sequences which are operably linked to the nucleic acid molecule in a manner which permits transcription, translation and/or expression in a cell transfected with the nucleic acid (e.g., a plasmid vector comprising said nucleic acid) or infected with a virus comprising said nucleic acid
  • “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the disclosure is also directed, in part, to a dependoparvovirus particle (e.g , a functional dependoparvovirus particle) comprising a nucleic acid or polypeptide described herein or produced by a method described herein.
  • a dependoparvovirus particle e.g , a functional dependoparvovirus particle
  • the viral particle comprising a variant capsid polypeptide exhibits increased muscle (cardiac and/or skeletal) transduction as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO:7)
  • a dependoparvovirus particle comprises an amino acid sequence that has at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequences provided for herein (e.g., a capsid polypeptide described in Section 6.2 such as the VP1 capsid polypeptide of SEQ ID NO:12 or 14 or a VP2 or VP3 portion thereof).
  • a capsid polypeptide described in Section 6.2 such as the VP1 capsid polypeptide of SEQ ID NO:12 or 14 or a VP2 or VP3 portion thereof.
  • the variant capsid polypeptide comprises an amino acid sequence that differs by no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids from the amino acid sequence of a variant capsid polypeptide provided for herein.
  • the additional alteration improves a production characteristic of a dependoparvovirus particle or method of making the same. In some embodiments, the additional alteration improves or alters another characteristic of a dependoparvovirus particle, e.g., tropism
  • AAV serotypes and to a degree, dependoparvovirus species, are significantly interrelated structurally and functionally.
  • AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins.
  • heteroduplex analysis reveals extensive cross-hybridization between serotypes along the length of the genome, further suggesting interrelatedness.
  • Dependoparvoviruses genomes also comprise selfannealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs).
  • AAV serotypes The genomic organization of naturally occurring dependoparvoviruses, e.g., AAV serotypes, is very similar.
  • the genome of AAV is a linear, single-stranded DNA molecule that is approximately 5,000 nucleotides (nt) in length or less.
  • Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural capsid (Cap) proteins.
  • Rep non-structural replication
  • Cap structural capsid
  • Three different viral particle (VP) proteins form the capsid.
  • the terminal approximately 145 nt of the genome are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed.
  • the Rep genes encode the Rep proteins: Rep78, Rep68, Rep52, and Rep40.
  • Rep78 and Rep68 are transcribed from the p5 promoter
  • Rep 52 and Rep40 are transcribed from the p19 promoter
  • the cap genes encode the VP proteins, VP1, VP2, and VP3
  • the cap genes are transcribed from the p40 promoter.
  • a dependoparvovirus particle of the disclosure comprises a nucleic acid comprising a variant capsid polypeptide provided for herein.
  • the particle comprises a polypeptide as provided for herein.
  • the dependoparvovirus particle of the disclosure is an AAV9 particle.
  • the AAV9 particle comprises a variant capsid polypeptide as provided for herein or a nucleic acid molecule encoding the same.
  • the dependoparvovirus particle comprises a variant capsid comprising a variant capsid polypeptide described herein. In some embodiments, the dependoparvovirus particle comprises variant capsid polypeptide described herein and a nucleic acid molecule.
  • the dependoparvovirus particle comprises variant capsid polypeptide described herein and a nucleic acid molecule comprising one or more inverted terminal repeat sequences (ITRs), for example, ITRs derived from an AAV9 dependoparvovirus or an AAV2 dependoparvovirus, one or more regulatory elements (for example, a promoter), and a payload (e.g., as described herein, e.g., a heterologous transgene)
  • ITRs inverted terminal repeat sequences
  • the nucleic acid molecule is single-stranded.
  • the nucleic acid molecule is double stranded, for example, self-complementary.
  • the disclosure is directed, in part, to nucleic acids, polypeptides, cells, cell free systems, translation systems, viral particles, and methods associated with using and making the same to produce viral particles that have increased distribution to tissues and cells of the muscle and/or muscle tissue transduction as compared to a viral particle comprising a reference sequence that does not otherwise comprise the mutations described herein (or mutations corresponding thereto), for example, as compared with a viral particle comprising a capsid polypeptide sequence of SEQ ID NO:7.
  • a use of a viral particle (e.g., a viral particle as described in Section 6.4) comprising the variant capsid polypeptides as described in Section 6.2 or any one of numbered embodiments 1 to 240 (e.g., V1 or V2) leads to increased muscle biodistribution of the viral particle and/or increased transduction of a transgene virus particle in the cells of the muscle, and, therefore, increased expression of the payload (transgene) in the muscle (e.g., increased cardiac muscle expression and/or increased skeletal muscle expression).
  • a viral particle e.g., a viral particle as described in Section 6.4
  • the variant capsid polypeptides as described in Section 6.2 or any one of numbered embodiments 1 to 240 e.g., V1 or V2
  • a viral particle e.g., a viral particle as described in Section 6.4
  • a viral particle comprising the variant capsid polypeptides as described in Section 6.2 or any one of numbered embodiments 1 to 240
  • peripheral tissues e.g., (i) liver, (ii) spleen, (Hi) dorsal root ganglia, (iv) brain / central nervous system, or any combination of two, three, or all four of (i), (ii), (Hi), and (iv).
  • biodistribution and transduction are measured as described herein, for example as described in Section 8.1 (e.g., Example 1) for example by relative quantification (e.g., via qPCR) of transgene mRNA in one or more samples isolated from the relevant tissue type, e.g., muscle.
  • biodistribution and/or transduction of a virus particle having a variant capsid polypeptide can be measured using virus particles having a transgene operably linked to a promoter that is active in a target cell or tissue type of interest.
  • the promoter is a ubiquitous promoter
  • the promoter is selective or specific to a target cell or tissue type (e.g , muscle), and, optionally, is less (or not) active in a cell or tissue type where transgene expression is not desired (e.g., liver).
  • a promoter that is selective for a first cell or tissue type over a second cell or tissue type is active in the first cell or tissue type and less active or silent in the second cell or tissue type.
  • the promoter is a muscle-specific promoter-specific or muscle-selective promoter.
  • biodistribution and/or transduction of a virus particle having a variant capsid polypeptide can be measured using virus particles having a transgene operably linked to a ubiquitous promoter or a muscle-specific promoter
  • the transgene is a transgene encoding a capsid polypeptide or any other suitable heterologous transgene, for example a nucleic acid sequence encoding a synthetic, mammalian or human therapeutic protein or nucleic acid (e g., mRNA or RNAi) or reporter gene such as, for example a nucleic acid encoding a GFP or mCherry reporter
  • the virus particle e.g., as described herein, e.g., comprising a variant capsid polypeptide described herein, exhibits increased transduction of myocytes (muscle cells) relative to a virus particle comprising a reference capsid polypeptide, e.g., a reference capsid polypeptide of SEQ ID NO:7.
  • a viral particle comprising the variant capsid polypeptide e.g., the variant capsid polypeptide described herein, exhibits improved properties, e.g., improved biodistribution, transduction and/or production.
  • improvement rates are presented as fold-improvement over the rates exhibited by a virus particle comprising capsid polypeptides of SEQ ID NO:7.
  • improvement means an increase, e.g., in the case of muscle biodistribution or muscle transduction.
  • improvement means a decrease, e.g., in the case of liver biodistribution or liver transduction
  • a virus particle having increased biodistribution or transduction in target cells or target tissue types, e.g., muscle, and/or decreased biodistribution or transduction in off-target cells or off-target tissue types, e.g., liver may have improved specificity for a target cell or target tissue type. This improvement may be beneficial in the use of the viral particle to deliver a therapeutic transgene to the target cell or target tissue type in a subject afflicted with a disease affecting the target cell or target tissue type, for example.
  • one or more improved properties is exhibited in a mammal, e g , a primate, e g , a human
  • the increased or decreased biodistribution and/or transduction is exhibited upon administration of the virus particle or pharmaceutical composition comprising the virus particle, e g., as described herein, by systemic administration, e.g , intravenous administration.
  • a method of making dependoparvovirus particle comprises providing a cell, cell-free system, or other translation system, comprising a nucleic acid described encoding a variant capsid polypeptide provided for herein, or a polypeptide provided for herein (e.g., a variant capsid polypeptide); and cultivating the cell, cell-free system, or other translation system under conditions suitable for the production of the dependoparvovirus particle, thereby making the dependoparvovirus particle.
  • nucleic acid or polypeptide described herein is produced by a method known to one of skill in the art.
  • the nucleic acids, polypeptides, and fragments thereof of the disclosure are produced by any suitable means, including recombinant production, chemical synthesis, or other synthetic means. Such production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention
  • a host cell comprising a nucleic acid of the disclosure (e.g., encoding a variant capsid polypeptide as described in Section 6.2).
  • a host cell of the disclosure e.g., a host cell useful to general AAV virus particles comprising a variant AAV capsid as described herein, generally comprises one or more nucleic acids comprising a coding sequence encoding a variant capsid polypeptide of the disclosure (e.g., as described in Section 6.2), together with a payload (e g , transgene) and one or more coding sequences encoding additional components useful for promoting packaging of the payload into a dependoparvovirus capsid.
  • a payload e g , transgene
  • Additional components include, for example, coding sequences for a rep protein and dependoparvovirus inverted terminal repeats (ITRs), as well as helper sequences which promote dependoparvovirus particle production and/or secretion.
  • helper sequences include E1a, E1b, E2a, E4, and VA.
  • helper sequences may be included endogenously within the host cell (e g., the host cell may be engineered to express such helper sequences, e g., integrated into the host cell genome) or may be provided exogenously (e.g., transduced on the same or a different nucleic acid as the variant capsid polypeptide, payload, rep, and/or ITRs).
  • the helper sequences include AdV5 helper sequences
  • An exemplary AdV5 genome is disclosed in the art as NCBI Reference Sequence AC_000008.1 (disclosed as SEQ ID NO:1 of PCT Patent Application Publication No WO/2022/079429 A1 ).
  • a host cell disclosed herein comprises a portion of an AdV5 genome encoding for one or more helper protein sequences (e.g., E1a, E1 b, E2a, E4) or RNA sequences (e.g., VA).
  • a host cell disclosed herein comprises a nucleic acid sequence encoding one or more AdV5 helper protein sequences. Certain exemplary AdV5 helper protein sequences are provided below
  • AdV5 E1A [0123] AdV5 E1A:
  • AdV5 E1B 19K [0124] AdV5 E1B 19K:
  • AdV5 E1B 55K [0125] AdV5 E1B 55K:
  • AdV5 E3 12.5K [0126] AdV5 E3 12.5K:
  • AdV5 E3 CR1-alpha0 [0127]
  • AdV5 E3 gp19K MNNSSNSTGYSNSGFSRIGVGVILCLVILFILILTLLCLRLAACCVHICIYCQLFKRWGRHPR (SEQ ID NO: 519)
  • AdV5 E3 CR1-beta0 [0129]
  • VLHPNNDGIHRLDGLKHMFFSLTV (SEQ ID NO: 521 )
  • AdV5 E3 RID-alpha [0130] AdV5 E3 RID-alpha:
  • HHPQYRDRTIAELLRIL SEQ ID NO: 522
  • AdV5 E3 RID-beta [0131] AdV5 E3 RID-beta:
  • AdV5 E4 ORF6/7 [0133] AdV5 E4 ORF6/7:
  • AdV5 E4 34K [0134] AdV5 E4 34K:
  • AdV5 E4 ORF4 [0135] AdV5 E4 ORF4:
  • AdV5 E4 ORF3 [0136] AdV5 E4 ORF3:
  • AdV5 E4 ORFB [0137]
  • AdV5 E4 ORF1 [0138]
  • the helper sequences include AdV2 helper sequences.
  • An exemplary AdV2 genome is disclosed in the art as NCBI Reference Sequence AC_000007.1 (disclosed as SEQ ID NO:2 of PCT Patent Application Publication No WQ/2022/079429 A1 ).
  • a host cell disclosed herein comprises a portion of an AdV2 genome encoding for one or more helper protein sequences (e.g., E1a, E1 b, E2a, E4) or RNAsequences (e.g., VA).
  • a host cell disclosed herein comprises a nucleic acid sequence encoding one or more AdV2 helper protein sequences. Certain exemplary AdV2 helper protein sequences are provided below
  • AdV2 E1A [0140] AdV2 E1A:
  • AdV2 E1B 19K [0141] AdV2 E1B 19K:
  • AdV2 E1B 55K [0142] AdV2 E1B 55K:
  • AdV2 E3 12.5K [0143] AdV2 E3 12.5K:
  • AdV2 E3 CR1-alphap0 [0144]
  • AdV2 E3 gp19K MSNSSNSTSLSNFSGIGVGVILTLVILFILILALLCLRVAACCTHVCTYCQLFKRWGQHPR (SEQ ID NO: 535) [0145] AdV2 E3 gp19K:
  • AdV2 E3 CR1-beta0 [0146]
  • AdV2 E3 RID alpha [0147] AdV2 E3 RID alpha:
  • HPQYRDRTIADLLRIL SEQ ID NO: 5378
  • AdV2 E3 RID beta [0148] AdV2 E3 RID beta:
  • AdV2 E3 14.7K [0149] AdV2 E3 14.7K:
  • AdV2 E4 ORF6/7 [0150] AdV2 E4 ORF6/7:
  • AdV2 E4 34K [0151] AdV2 E4 34K:
  • AdV2 E4 ORF4 [0152] AdV2 E4 ORF4:
  • AdV2 E4 ORF3 [0153] AdV2 E4 ORF3:
  • AdV2 E4 ORF2 [0154] AdV2 E4 ORF2:
  • AdV2 E4 ORF1 [0155]
  • Additional AAV helper sequences are recognized in the art and include, for example, those described in U.S. Patent Application Publication Nos. 2004/0248288 A1 and 2022/0259572A1, and in PCT Patent Application Publication Nos. WO/1997/017458 A1 , WO/2024/143429 A1, and WO/2020/208379 A1, each of which is incorporated herein by reference.
  • Expression control sequences include efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; appropriate transcription initiation, termination, promoter and enhancer sequences; sequences that stabilize cytoplasmic mRNA; sequences that enhance protein stability; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); and in some embodiments, sequences that enhance secretion of the encoded transgene product.
  • polyA polyadenylation
  • Expression control sequences including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and can be utilized with the compositions and methods disclosed herein.
  • the native promoter for the transgene is used. Without wishing to be bound by theory, the native promoter can mimic native expression of the transgene, or provide temporal, developmental, or tissue-specific expression, or expression in response to specific transcriptional stimuli.
  • the transgene is operably linked to other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences, e.g., to mimic the native expression
  • the transgene is operably linked to a tissue-specific promoter, e.g., a promoter active specifically in one or more muscle cell types.
  • a vector e.g., a plasmid, carrying a transgene includes a selectable marker or a reporter gene.
  • selectable reporters or marker genes can be used to signal the presence of the vector, e.g , plasmid, in bacterial cells.
  • Other components of the vector, e.g., plasmid include an origin of replication. Selection of these and other promoters and vector elements are conventional and many such sequences are available (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, and references cited therein).
  • an insect cell may be used in production of the compositions described herein or in the methods of making a dependoparvovirus particle described herein
  • an insect cell line used can be from Spodoptera frugiperda, such as Sf9, SF21, SF900+, drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g., Bombyxmori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines.
  • the insect cells are susceptible to baculovirus infection, including High Five, Sf9, Se301 , SelZD2109, SeUCRI , SP900+, Sf21 , BTI- TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5 and Ao38.
  • Use of insect cells for expression of heterologous proteins is well recognized in the art, as are methods of introducing nucleic acids, such as vectors, e g., insect-cell compatible vectors, into such cells and methods of maintaining such cells in culture. See, for example, O'Reilly et al., 1994, Baculovirus Expression Vectors, A Laboratory Manual. Oxford Univ.
  • insect host cell systems in combination with baculoviral systems (e.g., as described by Luckow et al., 1988, Bio/Technology 6:47) is used.
  • the expression system is a Trichoplusia ni, Tn 5B1-4 insect cells/ baculoviral system, which can be used for production of high levels of proteins, as described in U.S. Pat. No. 6,660,521, incorporated herein by reference in its entirety
  • Expansion, culture, transfection, infection, and storage of insect cells can be carried out in any cell culture media, cell transfection media or storage media known in the art.
  • Nonlimiting examples of media are Hyclone SFX Insect Cell Culture Media, Expression System ESF AF Insect Cell Culture Medium, Basal IPL-41 Insect Cell Culture Media, ThermoFisher Sf90011 media, ThermoFisher Sf900111 media, and ThermoFisher Grace's Insect Media.
  • Insect cell mixtures and/or media can also comprise appropriate formulation additives or elements, including but not limited to salts, acids, bases, buffers, and surfactants (such as Poloxatner 188/Pluronic F-68).
  • the methods of the disclosure can be carried out with a mammalian cell type which allows for replication of dependoparvovirus or production of biologic products, and which can be maintained in culture.
  • Host cells include cells derived from mammalian species including but not limited to, human, monkey, mouse, rat, rabbit, and hamster.
  • Host cells can be of any suitable cell type, including but not limited to cell lines, fibroblasts, hepatocytes, tumor cells, and transformed cells.
  • the mammalian cells used can be HEK293, HEK293T, HeLa, CHO, NS0, SP2/0, PER C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, MRC-5, WEH1, 3T3, 1.0T1/2, MDCK, COS 1 , COS 7, BSC 1 , BSC 40, BMT 10, W138, Saos, C2C12, HepG2, L cells, primary fibroblast, hepatocyte and myoblast cells derived from mammals, COS cells, C127, 3T3, CHO, HeLa cells, KB cells, BHK, and other mammalian cell lines as described in U.S. Pat. Nos. 6,156,303, 5,387,484, 5,741 ,683, 5,691 ,176, 6,428,988 and 5,688,676, 6,541 ,258, the contents of each of which are incorporated herein by reference.
  • the host cell comprises a nucleic acid encoding a variant capsid polypeptide disclosed herein, where the nucleic acid is integrated into the host cell genome.
  • host cells include adenovirus rep and cap genes integrated into the genome. T ranscription of the integrated rep and cap genes may be dependent upon introduction of certain helper virus sequences (e.g., adenovirus E4, E2a and/or VA RNA) into the cell by transduction or other suitable means.
  • helper virus sequences e.g., adenovirus E4, E2a and/or VA RNA
  • Example plasmid free host cells are described in U.S. Patent Application Publication No. 2022/0025396 A1 , and U.S. Patent No. US 5,658,785, incorporated herein by reference.
  • the host cells are trans-complementing packaging cell lines that provide functions deleted from a replication-defective helper virus, e.g., HEK293 cells or other Ea trans-complementing cells.
  • the packaging cell line 293-10-3 is used as described in U.S. Pat. No. 6,281 ,010, incorporated herein by reference.
  • mammalian host cells e g. 293T cells
  • an adherent state e.g., adhered/attached to a suitable surface of a cell culture flask, vial, tray, well, tube, etc.
  • mammalian host cells can be in a suspended state (e.g., suspended in a medium).
  • the nucleic acids of the disclosure are situated as a part of any genetic element (vector) which can be delivered to a host cell, e.g., naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in a non-viral delivery vehicle (e.g., a lipid-based carrier), virus, etc , which transfer the sequences carried thereon
  • a vector can be delivered into a host cell by any suitable method, including transfection, liposome delivery, electroporation, membrane fusion techniques, viral infection, high velocity DNA- coated pellets, and protoplast fusion.
  • a person of skill in the art possesses the knowledge and skill in nucleic acid manipulation to construct any embodiment of this invention and said skills include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • a vector of the disclosure comprises sequences encoding a dependoparvovirus variant capsid polypeptide as provided for herein or a fragment thereof.
  • a vector of the disclosure comprises sequences encoding a dependoparvovirus Rep protein or a fragment thereof.
  • Such a Rep coding region encodes at least for AAV Rep78, Rep68, Rep52, and Rep40, or functional homologs thereof.
  • the Rep coding region is not required to include all wild-type genes but may be altered (e.g., by insertion, deletion, or mutation of one or more nucleotides) so long as the rep genes present provide for sufficient replication functions when expressed in the recombinant cell.
  • the Rep coding region may be derived from any AAV serotype.
  • the Rep coding region is or comprises a rep gene encoding AAV2 Rep proteins, exemplary sequences of which are provided below.
  • VNVDLDDCIFEQ SEQ ID NO: 545)
  • the Rep coding region encodes for Rep sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to one or more AAV2 Rep proteins.
  • the Rep coding region comprises a nucleotide sequence encoding for a Rep78 protein having at least 80%, at least about 85%, at least about 90%, at least about 95%, at least 98%, at least 99%, or 100% sequence identity to AAV2 Rep78.
  • the Rep coding region comprises a nucleotide sequence encoding for a Rep68 protein having at least 80%, at least about 85%, at least about 90%, at least about 95%, at least 98%, at least 99%, or 100% sequence identity to AAV2 Rep68. In some embodiments, the Rep coding region comprises a nucleotide sequence encoding for a Rep52 protein having at least 80%, at least about 85%, at least about 90%, at least about 95%, at least 98%, at least 99%, or 100% sequence identity to AAV2 Rep52.
  • the Rep coding region comprises a nucleotide sequence encoding for a Rep40 protein having at least 80%, at least about 85%, at least about 90%, at least about 95%, at least 98%, at least 99%, or 100% sequence identity to AAV2 Rep40.
  • the Rep coding sequence encodes for wild type Rep proteins.
  • the Rep coding sequence may encode for one or more mutant Rep proteins having improved properties compared with wild type Rep proteins Examples of such mutant Rep proteins are described in U.S. Patent No. 11,060,070, incorporated herein by reference.
  • such vectors contain both dependoparvovirus cap and rep proteins.
  • the dependoparvovirus rep and dependoparvovirus cap sequences are both of the same dependoparvovirus species or serotype origin.
  • the present embodiments also provide vectors in which the rep sequences are from a dependoparvovirus species or serotype which differs from that which is providing the cap sequences (e.g., AAV2 rep sequences and AAV9 cap sequences).
  • the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell genome and a vector).
  • the rep sequences are fused in frame to cap sequences of a different dependoparvovirus species or serotype to form a chimeric dependoparvovirus vector.
  • a vector of the disclosure comprises one or more helper sequences.
  • helper sequences include E1a, E1b, E2a, E4, and VA.
  • Certain exemplary helper protein sequences are provided in Section 6.6.1. Additional AAV helper sequences are recognized in the art and include, for example, those described in U.S. Patent Application Publication Nos. 2004/0248288 A1 and 2022/0259572A1, and in PCT Patent Application Publication Nos. WO/1997/017458 A1 , WO/2024/143429 A1, and WO/2020/208379 A1 , each of which is incorporated herein by reference
  • the vectors of the disclosure further contain a payload, e.g , a minigene comprising a selected transgene, e.g., flanked by dependoparvovirus 5' ITR and dependoparvovirus 3' ITR.
  • the ITR is from the same serotype as the variant capsid polypeptide.
  • the ITR is of a different serotype than the variant capsid polypeptide.
  • the viral genome comprises two ITR sequence regions, wherein the ITRs are of the same serotype as one another In some embodiments, the viral genome comprises two ITR sequence regions, wherein the ITRs are of different serotypes.
  • Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid.
  • both ITRs of the viral genome of the AAV particle are AAV2 ITRs Independently, each ITR may be about 100 to about 150 nucleotides in length.
  • An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146- 150 nucleotides in length.
  • the ITRs are 140-142 nucleotides in length.
  • Nonlimiting examples of ITR lengths are 102, 105, 130, 140, 141 , 142, 145 nucleotides in length.
  • the vectors described herein are useful for a variety of purposes, but are particularly well suited for use in production of recombinant dependoparvovirus particles comprising dependoparvovirus sequences or a fragment thereof, and in some embodiments, a payload.
  • the disclosure provides a method of making a dependoparvovirus particle (e.g., a dependoparvovirus B particle, e.g., an AAV9 particle), or a portion thereof.
  • the method comprises culturing a host cell which contains a nucleic acid sequence encoding a dependoparvovirus variant capsid protein as provided for herein, or fragment thereof; a functional rep gene (e g., encoding Rep proteins as described herein); a payload, e g., a minigene comprising dependoparvovirus inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to promote packaging of the payload, e.g., minigene, into the dependoparvovirus capsid.
  • ITRs dependoparvovirus inverted terminal repeats
  • the components necessary to be cultured in the host cell to package a payload, e.g., minigene, in a dependoparvovirus capsid are provided to the host cell in trans.
  • any one or more of the required components e.g., payload (e.g., minigene), rep sequences, cap sequences, and/or helper functions
  • a host cell which has been engineered to stably comprise the required component(s) comprises it under the control of an inducible promoter.
  • the required components are under the control of a constitutive promoter
  • suitable inducible and constitutive promoters are provided herein, and further examples are known to those of skill in the art.
  • a selected host cell which has been engineered to stably comprise one or more components comprises a component under the control of a constitutive promoter and another component under the control of one or more inducible promoters
  • a host cell which has been engineered to stably comprise the required components is generated from HEK 293 cells (e.g., which comprise helper functions under the control of a constitutive promoter), which comprises the rep and/or cap proteins under the control of one or more inducible promoters.
  • the payload e.g., minigene
  • rep sequences e.g., cap sequences, and helper functions required for producing a dependoparvovirus particle of the disclosure
  • the payload e.g., minigene
  • rep sequences e.g., cap sequences, and helper functions required for producing a dependoparvovirus particle of the disclosure
  • the genetic element may be delivered by any suitable method, including those described herein. Methods used to construct genetic elements, vectors, and other nucleic acids of the disclosure are known to those with skill and include genetic engineering, recombinant engineering, and synthetic techniques.
  • dependoparvovirus ITRs are readily selected from among any dependoparvovirus species and serotypes, e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh74, or AAV9.
  • ITRs or other dependoparvovirus components may be readily isolated using techniques available to those of skill in the art from a dependoparvovirus species or serotype.
  • Dependoparvovirus species and serotypes may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA).
  • the dependoparvovirus sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank or PubMed.
  • proteins e.g., recombinant or heterologous proteins, e.g., dependoparvovirus polypeptides
  • methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors, into such cells and methods of maintaining such cells in culture See, for example, METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, N J (1995); O'Reilly et al , BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Vir.
  • a nucleic acid construct encoding dependoparvovirus polypeptides is an insect cellcompatible vector.
  • an “insect cell-compatible vector” as used herein refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell.
  • exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell compatible
  • the vector may integrate into the insect cell's genome or remain present extra-chromosomally.
  • the vector may be present permanently or transiently, e.g , as an episomal vector.
  • Vectors may be introduced by any means known in the art. Such means include but are not limited to chemical treatment of the cells, electroporation, or infection.
  • the vector is a baculovirus, a viral vector, or a plasmid.
  • Methods of dependoparvovirus capsid (e.g., AAV) production in insect cells include, for example, those described in U.S. Patent Application Publication No. 2024/0093231 A1 , U.S. Patent No. 11 ,306,291 , Joshi et al., 2024, Methods Mol Biol, 2829:203-214, and Marwidi et al , 2024, Mol Ther Methods Clin Dev , 32(2)101228, incorporated herein by reference.
  • a nucleic acid sequence encoding a dependoparvovirus polypeptide is operably linked to regulatory expression control sequences for expression in a specific cell type, such as Sf9 or HEK cells.
  • a specific cell type such as Sf9 or HEK cells.
  • Techniques known to one skilled in the art for expressing foreign genes in insect host cells or mammalian host cells can be used with the compositions and methods of the disclosure. Methods for molecular engineering and expression of polypeptides in insect cells are described, for example, in Summers and Smith. A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex. (1986); Luckow. 1991 .
  • providing a cell comprising a nucleic acid described herein comprises acquiring a cell comprising the nucleic acid
  • cultivating a host cell comprises providing the cell with suitable media and incubating the cell and media for a time suitable to achieve viral particle production.
  • a method of making a dependoparvovirus particle further comprises a purification step comprising isolating the dependoparvovirus particle from one or more other components (e.g., from a cell or media component).
  • production of the dependoparvovirus particle comprises one or more (e.g., all) of: expression of dependoparvovirus polypeptides, assembly of a dependoparvovirus capsid, expression (e.g., duplication) of a dependoparvovirus genome, and packaging of the dependoparvovirus genome into the dependoparvovirus capsid to produce a dependoparvovirus particle.
  • production of the dependoparvovirus particle further comprises secretion of the dependoparvovirus particle into the media.
  • the dependoparvovirus particle can be isolated from the collected media.
  • dependoparvovirus particles are isolated from host cells. For instance, adherent host cells can subsequently be collected by scraping and/or pelleting and suspended cells can be collected by pelleting and transferred into a receptacle
  • Collection steps can be repeated as necessary for full collection of produced cells. If necessary, host cell lysis can be achieved by consecutive freeze-thaw cycles (-80°C to 37°C), chemical lysis (such as adding detergent, e.g., triton), mechanical lysis, or by allowing the cell culture to degrade after reaching ⁇ 0% viability. Cellular debris can be removed by centrifugation and/or depth filtration.
  • the nucleic acid molecule encoding the variant capsid polypeptide is disposed in a dependoparvovirus genome. In some embodiments, and as described elsewhere herein, the nucleic acid molecule encoding the variant capsid polypeptide is packaged into a dependoparvovirus particle along with the dependoparvovirus genome as part of a method of making a dependoparvovirus particle described herein In other embodiments, the nucleic acid molecule encoding the variant capsid polypeptide is not packaged into a dependoparvovirus particle made by a method described herein.
  • a method of making a dependoparvovirus particle described herein produces a dependoparvovirus particle comprising a payload (e.g., a payload described herein) and the variant capsid polypeptide.
  • the payload comprises a second nucleic acid (e.g., in addition to the dependoparvovirus genome), and production of the dependoparvovirus particle comprises packaging the second nucleic acid into the dependoparvovirus particle.
  • a cell, cell-free system, or other translation system for use in a method of making a dependoparvovirus particle comprises the second nucleic acid.
  • the second nucleic acid comprises an exogenous sequence (e.g., exogenous to the dependoparvovirus, the cell, or to a target cell or subject who will be administered the dependoparvovirus particle).
  • the exogenous sequence encodes an exogenous polypeptide.
  • the exogenous sequence encodes a therapeutic product.
  • virus particles of the disclosure have a similar production efficiency to viral particles with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NOV).
  • production efficiency of viral particles of the disclosure is (a) at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, or at least 0.9-fold and/or (b) up to 1-fold, e.g., as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO:1), or the production efficiency is within any range bounded by a value in (a) and a value in (b) In some embodiments, production efficiency is at least 0 5-fold as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NOV).
  • Production efficiency can be evaluated by producing viral particles having a variant capsid with a genome encoding a unique barcode and a fluorescent reporter gene under the control of a ubiquitous (e.g., CBh) or a muscle-specific promoter via transient triple transfection of adherent HEK293T cells followed by iodixanol gradient purification.
  • a ubiquitous e.g., CBh
  • a muscle-specific promoter e.g., a muscle-specific promoter
  • compositions comprising a nucleic acid, polypeptide, or particles described herein.
  • disclosure is further directed, in part, to methods utilizing a composition, nucleic acid, polypeptide, or particles described herein.
  • nucleic acids, polypeptides, particles, and methods disclosed herein have a variety of utilities.
  • a vector comprising a nucleic acid described herein, e.g., a nucleic acid encoding a variant capsid polypeptide.
  • a vector comprises a plasmid.
  • the vector is an isolated vector, e.g., removed from a cell or other biological components.
  • the disclosure is directed, in part to a cell, cell-free system, or other translation system, comprising a nucleic acid or vector described herein, e.g., a nucleic acid or vector comprising a nucleic acid molecule encoding a variant capsid polypeptide.
  • the cell, cell-free system, or other translation system is capable of producing dependoparvovirus particles comprising the variant capsid polypeptides.
  • the cell, cell-free system, or other translation system comprises a nucleic acid comprising a dependoparvovirus genome or components of a dependoparvovirus genome sufficient to promote production of dependoparvovirus particles comprising the variant capsid polypeptides.
  • the cell, cell-free system, or other translation system further comprises one or more non-dependoparvovirus nucleic acid sequences that promote dependoparvovirus particle production and/or secretion. Said sequences are referred to herein as helper sequences.
  • a helper sequence comprises one or more genes from another virus, e.g., an adenovirus or herpes virus
  • the presence of a helper sequence is necessary for production and/or secretion of a dependoparvovirus particle.
  • a cell, cell- free system, or other translation system comprises a vector, e.g., plasmid, comprising one or more helper sequences
  • a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises sequences encoding one or more dependoparvovirus genes (e.g , a Cap gene, a Rep gene, or a complete dependoparvovirus genome) and a helper sequence, and wherein the second nucleic acid comprises a payload
  • a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises sequences encoding one or more dependoparvovirus genes (e.g , a Cap gene, a Rep gene, or a complete dependoparvovirus genome) and a payload
  • the second nucleic acid comprises a helper sequence
  • a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises sequences encoding one or more dependopar
  • a cell, cell-free system, or other translation system comprises a first nucleic acid, a second nucleic acid, and a third nucleic acid, wherein the first nucleic acid comprises sequences encoding one or more dependoparvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependoparvovirus genome), the second nucleic acid comprises a helper sequence, and the third nucleic acid comprises a payload.
  • dependoparvovirus genes e.g., a Cap gene, a Rep gene, or a complete dependoparvovirus genome
  • the second nucleic acid comprises a helper sequence
  • the third nucleic acid comprises a payload.
  • the first nucleic acid, second nucleic acid, and optionally third nucleic acid are situated in separate molecules, e.g., separate vectors or a vector and genomic DNA.
  • one, two, or all of the first nucleic acid, second nucleic acid, and optionally third nucleic acid are integrated (e.g., stably integrated) into the genome of a cell.
  • a cell of the disclosure is generated by transfecting a suitable cell with a nucleic acid described herein.
  • a method of making a dependoparvovirus particle comprising a variant capsid polypeptide as provided for herein or improving a method of making a dependoparvovirus particle comprises providing a cell described herein.
  • providing a cell comprises transfecting a suitable cell with one or more nucleic acids described herein.
  • the cell is a human cell.
  • the cell is an immortalized cell or a cell from a cell line known in the art.
  • the cell is an HEK293 cell.
  • the cell is an HEK293T cell.
  • the disclosure is directed, in part, to a method of delivering a payload to a cell, e.g., a cell in a subject or in a sample.
  • a method of delivering a payload to a cell comprises contacting the cell with a dependoparvovirus particle comprising a variant capsid polypeptide (e.g., described herein) comprising the payload.
  • the disclosure also includes a dependoparvovirus particle comprising a variant capsid polypeptide (e.g., described herein) comprising a payload described herein for use in the methods of delivering a payload described herein.
  • the dependoparvovirus particle is a dependoparvovirus particle described herein and comprises a payload described herein.
  • the cell is a myocyte (muscle cell).
  • skeletal muscles include the biceps, the triceps, the quadriceps, the tibialis interior, the gastrocnemius muscle, and diaphragm.
  • the method is conducted ex vivo.
  • the cell is a cell in an ex vivo sample that has been obtained from a subject.
  • the payload comprises a transgene.
  • the transgene is a nucleic acid sequence heterologous to the vector sequences flanking the transgene which encodes a polypeptide, RNA (e g , a miRNA or siRNA) or other product of interest
  • RNA e g , a miRNA or siRNA
  • the nucleic acid of the transgene is operatively linked to a regulatory component in a manner sufficient to promote transgene transcription, translation, and/or expression in a host cell.
  • a transgene is any polypeptide- or RNA-encoding sequence and the transgene selected will depend upon the use envisioned
  • a transgene comprises a reporter sequence, which upon expression produces a detectable signal.
  • reporter sequences include, without limitation, DNA sequences encoding colorimetric reporters (e.g., P-lactamase, p- galactosidase (LacZ), alkaline phosphatase), cell division reporters (e.g., thymidine kinase), fluorescent or luminescence reporters (e.g., green fluorescent protein (GFP) or luciferase), resistance conveying sequences (e.g., chloramphenicol acetyltransferase (CAT)), or membrane bound proteins including to which high affinity antibodies directed thereto exist or can be produced by conventional means, e.g., comprising an antigen tag, e.g., hemagglutinin or Myc.
  • colorimetric reporters e.g., P-lactamase, p- galactosidase (LacZ), alkaline phosphatase
  • cell division reporters e.g., thymidine kinase
  • a reporter sequence operably linked with regulatory elements which drive their expression provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry
  • the transgene encodes a product which is useful in biology and medicine, such as RNA, proteins, peptides, enzymes, dominant negative mutants.
  • the RNA comprises a tRNA, ribosomal RNA, dsRNA, catalytic RNAs, small hairpin RNA, siRNA, trans-splicing RNA, and antisense RNAs.
  • the RNA inhibits or abolishes expression of a targeted nucleic acid sequence in a treated subject (e.g., a human or animal subject).
  • the transgene is used to correct or ameliorate gene deficiencies.
  • gene deficiencies include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed
  • the transgene encodes a therapeutic protein or polypeptide which is expressed in a host cell.
  • a dependoparvovirus particle comprises or delivers multiple transgenes, e.g., to correct or ameliorate a gene defect caused by a multi-subunit protein.
  • a different transgene (e.g., each situated/delivered in a different dependoparvovirus particle, or in a single dependoparvovirus particle) is used to encode each subunit of a protein, or to encode different peptides or proteins, e.g., when the size of the DNA encoding the protein subunit is large, e.g., for immunoglobulin, platelet-derived growth factor, or dystrophin protein.
  • different subunits of a protein are encoded by the same transgene, e.g, a single transgene encoding each of the subunits with the DNA for each subunit separated by an internal ribozyme entry site (IRES).
  • IRS internal ribozyme entry site
  • the DNA is separated by sequences encoding a 2A peptide, which self-cleaves in a post-translational event. See, e.g., Donnelly et al, J. Gen. Virol., 78(Pt 1 ): 13-21 (January 1997); Furler, et al, Gene Then, 8(11 ):864-873 (June 2001 ); Klump et al., Gene Then 8( 10): 811 -817 ( May 2001 ) (incorporated herein by reference in their entirety).
  • virus particles comprising a genome are provided, wherein the genome includes a nucleic acid expression construct.
  • the nucleic acid expression construct can include a heterologous transgene and one or more regulatory elements.
  • the regulatory elements include a promotor, e g, a promoter that is active in a target cell or tissue type of interest.
  • the promoter is a ubiquitous promoter.
  • the promoter is selective or specific to a target cell or tissue type (e.g., muscle (either broad muscle expression or skeletal muscle)), and, optionally, is less (or not) active in a cell or tissue type where transgene expression is not desired (e.g., liver).
  • a promoter that is selective for a first cell or tissue type over a second cell or tissue type is active in the first cell or tissue type and less active or silent in the second cell or tissue type.
  • the promoter is a muscle-specific promoter-specific or muscle-selective promoter
  • the promoter is a ubiquitous or constitutive promoter active in a mammalian cell, for example a human cell, for example, in a human cell type of interest.
  • the cell type is a muscle cell such as, for example, a cardiac myocyte, skeletal muscle myocyte, or smooth muscle myocyte
  • ubiquitous promoters include, but are not limited, to a CAG promoter (hybrid from a cytomegalovirus early enhancer element, a chicken-beta actin promoter, e.g., the first exon and the first intron of the chicken beta actin gene, and the splice acceptor of the rabbit beta globin gene), chicken-beta actin promoter, CBA promoter, OBh promoter, CB6 promoter, CMV promoter, human EF1-alpha promoter, PGK promoter, ubiquitin C (UBC) promoter and fragments thereof.
  • CAG promoter hybrid from a cytomegalovirus early enhancer element, a chicken-
  • the promoter is a tissue-specific promoter, for example, a promoter specific in muscle tissue or muscle cells, e.g., a desmin, MCK, TNNT2, or smooth muscle 22 (SM22) promoter. Further exemplary muscle specific promoters are described below.
  • the promoter is a CBh promoter.
  • An exemplary CBh promoter sequence is set forth as SEQ ID NO:500.
  • the CBh promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:500.
  • the promoter is a EF1a promoter, for example a human EF1a promoter.
  • An exemplary human EF1a promoter sequence is set forth as SEQ ID NQ:501.
  • the human EF1a promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:501.
  • the promoter is a tissue-specific promoter.
  • tissue-specific promoters include muscle-specific promoters.
  • the promoter is a promoter specific in muscle tissue or muscle cells, e.g., a desmin, MCK, TNNT2, or smooth muscle 22 (SM22) promoter. Further exemplary muscle specific promoters are described below.
  • the nucleic acid expression construct comprises an intron
  • the intron is disposed between the promoter and the heterologous transgene.
  • the intron is disposed 5’ to the heterologous transgene on the expression construct, for example immediately 5’ to the heterologous transgene or 100 nucleotides or less 5’ to the heterologous transgene.
  • the intron is a chimeric intron derived from human b-globin and Ig heavy chain (also known as b- globin splice donor/immunoglobulin heavy chain splice acceptor intron, or b-globin/IgG chimeric intron; Reed, R., et al. Genes and Development, 1989, incorporated herein by reference in its entirety)
  • the intron is a VH4 intron or a SV40 intron
  • virus particles comprising a payload, wherein the payload includes a nucleic acid that includes a heterologous transgene are provided.
  • the heterologous transgene encodes an RNA interference agent, for example a siRNA, shRNA or other interfering nucleic acid.
  • the payload includes a heterologous transgene that encodes a therapeutic polypeptide.
  • the heterologous transgene is a human gene or fragment thereof.
  • the therapeutic polypeptide is a human protein.
  • the heterologous transgene of the virus particle encodes a molecule useful in treating a disease, and the virus particle is administered to a patient in need thereof to treat said disease
  • the payload comprises a molecule that is effective in treating muscle-related disease. Exemplary, nonlimiting muscle-related diseases are described below.
  • the virus particle comprises a heterologous transgene encoding a genome editing system.
  • a CRISPR genome editing system e.g., one or more components of a CRISPR genome editing system such as, for example, a guide RNA molecule and/or a RNA-guided nuclease such as a Cas enzyme such as Cas9, Cpf1 and the like
  • a zinc finger nuclease genome editing system e.g., human, genomic target sequence.
  • the virus particle includes a heterologous transgene encoding a targetable transcription regulator.
  • a CRISPR-based trascription regulator for example, one or more components of a CRISPR-based transcription regulator, for example, a guide RNA molecule and/or a enzymatically-inactive RNA-guided nuclease/transcription factor (“TF”) fusion protein such as a dCas9-TF fusion, dCpf1-TF fusion and the like
  • TF enzymatically-inactive RNA-guided nuclease/transcription factor
  • zinc finger transcription factor fusion protein such as a dCas9-TF fusion, dCpf1-TF fusion and the like
  • TALEN transcription regulator for example, a TALEN transcription regulator, a meganuclease transcription regulator.
  • components of a therapeutic molecule or system are delivered by more than one unique virus particle (e.g., a population that includes more than one unique virus particles)
  • the therapeutic molecule or components of a therapeutic molecule or system are delivered by a single unique virus particle (e.g , a population that includes a single unique virus particle).
  • the transgene encodes any biologically active product or other product, e.g , a product desirable for study. Suitable transgenes may be readily selected by persons of skill in the art, such as those, but not limited to, those described herein.
  • a method of delivering a payload to a subject comprises administering to the subject a dependoparvovirus particle comprising a variant polypeptide (e g , described herein) comprising the payload, e g , in a quantity and for a time sufficient to deliver the payload.
  • the dependoparvovirus particle is a dependoparvovirus particle described herein and comprises a payload described herein.
  • the particle delivers the payload to muscle tissue (e.g., cardiac muscle or skeletal muscle).
  • muscle tissue e.g., cardiac muscle or skeletal muscle.
  • the delivery to muscle is increased as compared to a particle without the variant capsid polypeptide or as compared to a wild-type capsid polypeptide, e.g., a particle with capsid polypeptides of SEQ ID NO:7.
  • virus particles comprising a genome are provided, wherein the genome includes a nucleic acid expression construct including a heterologous transgene and one or more regulatory elements, where the one or more regulatory elements include a muscle specific promoter.
  • muscle specific promoters that can be used to drive expression of a transgene in muscle include but are not limited to Desmin (DES), CAMK, Mb, myosin (e.g., myo-3), dystrophin, muscle creatine kinase (MCK), MHCK7, CK6, CK7, CK8, CK8e, dMCK, tMCK, MH, SPc-5-12 (also known as 05-12), alpha skeletal actin (ASKA), SP-301, E-syn, myosin light chain (MLC), myosin heavy chain (MHO), four and a half LIM domains protein 1 (FHL1), alpha 2 actinin (ACTN2), filamin-C (FLNC),
  • Promoters capable of driving expression in muscle are further described in Skopenkova et al., 2021 , Acta Naturae 13(1 ):47-58, Piekarowicz et al., 2019, Mol Ther Methods Clin Dev. 15:157-169, Wang, 2008 Gene Ther. 15:1489-1499, Coulon ef al., 2007, JBC 282(45):33192-33200, WO 2021/127655, and WO 2023/006890, the contents of each of which are incorporated herein by reference in their entireties.
  • the payload comprises a molecule that is effective in treating a muscle disease, such as, for example, a protein or an antisense oligonucleotide such as an RNA interference nucleotide (e.g., shRNA, siRNA or miRNA).
  • a protein such as, for example, a protein or an antisense oligonucleotide such as an RNA interference nucleotide (e.g., shRNA, siRNA or miRNA).
  • an RNA interference nucleotide e.g., shRNA, siRNA or miRNA
  • the disclosure provides a virus particle comprising (a) a capsid polypeptide described herein, for example, a capsid polypeptide described in Section 6.2 and (b) an engineered viral genome comprising (i) a nucleotide sequence encoding a molecule for the treatment of Duchenne muscular dystrophy (DMD), for example human Dystrophin (DMD) or a fragment or variant thereof (e.g., a variant thereof having at least 90% or at least 95% sequence identity to human DMD), and (ii) a promoter operably linked to said nucleotide sequence.
  • DMD Duchenne muscular dystrophy
  • DMD DMD
  • a fragment or variant thereof e.g., a variant thereof having at least 90% or at least 95% sequence identity to human DMD
  • a promoter operably linked to said nucleotide sequence e.g., a variant thereof having at least 90% or at least 95% sequence identity to human DMD
  • the viral genome can further comprise one or more (e.g., two, three, four, or all five) of (a) a pair of dependoparvovirus ITRs, (b) an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and (e) a polyA sequence.
  • the viral genome comprises ITRs flanking the nucleotide sequence and a polyA sequence operably linked to the nucleotide sequence.
  • the viral genome lacks rep and cap sequences, which are in trans by the host cell in which the virus particle is produced.
  • the viral genome is self- complementary.
  • the disclosure provides a virus particle comprising (a) a capsid polypeptide described herein, for example, a capsid polypeptide described in Section 6.2 and (b) an engineered viral genome comprising (i) a nucleotide sequence encoding a molecule for the treatment of X-linked myotubular myopathy, for example human myotubularin (MTM1) or a fragment or variant thereof (e.g., a variant thereof having at least 90% or at least 95% sequence identity to human MTM1), and (ii) a promoter operably linked to said nucleotide sequence.
  • MTM1 human myotubularin
  • An exemplary accession number for human MTM1 is set forth in Table M1.
  • the viral genome can further comprise one or more (e.g., two, three, four, or all five) of (a) a pair of dependoparvovirus ITRs, (b) an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and (e) a polyA sequence.
  • the viral genome comprises ITRs flanking the nucleotide sequence and a polyA sequence operably linked to the nucleotide sequence
  • the viral genome lacks rep and cap sequences, which are in trans by the host cell in which the virus particle is produced.
  • the viral genome is self- complementary.
  • the disclosure provides a virus particle comprising (a) a capsid polypeptide described herein, for example, a capsid polypeptide described in Section 6.2 and (b) an engineered viral genome comprising (i) a nucleotide sequence encoding a molecule for the treatment of Myotonic Dystrophy Type 1 (DM1 ), for example human myotonin-protein kinase (DMPK) or a fragment or variant thereof (e.g., a variant thereof having at least 90% or at least 95% sequence identity to human DMPK, and (ii) a promoter operably linked to said nucleotide sequence
  • DM1 Myotonic Dystrophy Type 1
  • DMPK myotonin-protein kinase
  • a promoter operably linked to said nucleotide sequence An exemplary accession number for human DMPK is set forth in Table M1.
  • the viral genome can further comprise one or more (e.g., two, three, four, or all five) of (a) a pair of dependoparvovirus ITRs, (b) an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and (e) a polyA sequence.
  • the viral genome comprises ITRs flanking the nucleotide sequence and a polyA sequence operably linked to the nucleotide sequence.
  • the viral genome lacks rep and cap sequences, which are in trans by the host cell in which the virus particle is produced.
  • the viral genome is self-complementary.
  • the disclosure provides a virus particle comprising (a) a capsid polypeptide described herein, for example, a capsid polypeptide described in Section 6.2 and (b) an engineered viral genome comprising (i) a nucleotide sequence encoding a molecule for the treatment of Limbgirdle muscular dystrophy (LGMD), for example human Gamma-sarcoglycan, Alpha-sarcoglycan, Beta-sarcoglycan, or Delta-sarcoglycan or a fragment or variant thereof (e g , a variant thereof having at least 90% or at least 95% sequence identity to human Gamma-sarcoglycan, Alpha-sarcoglycan, Beta-sarcoglycan, or Delta-sarcoglycan), and (II) a promoter operably linked to said nucleotide sequence Exemplary accession numbers for human Gamma-sarcoglycan, Alpha-sarcoglycan, Beta- s
  • the viral genome can further comprise one or more (e.g., two, three, four, or all five) of (a) a pair of dependoparvovirus ITRs, (b) an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and (e) a polyA sequence.
  • the viral genome comprises ITRs flanking the nucleotide sequence and a polyA sequence operably linked to the nucleotide sequence.
  • the viral genome lacks rep and cap sequences, which are in trans by the host cell in which the virus particle is produced.
  • the viral genome is self-complementary.
  • the disclosure provides a virus particle comprising (a) a capsid polypeptide described herein, for example, a capsid polypeptide described in Section 6.2 and (b) an engineered viral genome comprising (i) a nucleotide sequence encoding a molecule for the treatment of Pompe disease, for example human acid alpha-glucosidase (GAA) or a fragment (e.g., exon 2) or variant thereof (e.g., a variant thereof having at least 90% or at least 95% sequence identity to human GAA or a portion thereof), and (ii) a promoter operably linked to said nucleotide sequence.
  • GAA human acid alpha-glucosidase
  • exon 2 a fragment
  • variant thereof e.g., a variant thereof having at least 90% or at least 95% sequence identity to human GAA or a portion thereof
  • a promoter operably linked to said nucleotide sequence is set forth in Table M1 .
  • the viral genome can further comprise one or more (e.g., two, three, four, or all five) of (a) a pair of dependoparvovirus ITRs, (b) an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and (e) a polyA sequence.
  • the viral genome comprises ITRs flanking the nucleotide sequence and a polyA sequence operably linked to the nucleotide sequence.
  • the viral genome lacks rep and cap sequences, which are in trans by the host cell in which the virus particle is produced.
  • the viral genome is self-complementary.
  • the disclosure provides a virus particle comprising (a) a capsid polypeptide described herein, for example, a capsid polypeptide described in Section 6.2 and (b) an engineered viral genome comprising (i) a nucleotide sequence encoding a molecule for the treatment of Facioscapulohumeral muscular dystrophy (FSHD), for example an antisense oligonucleotide targeting human DUX4, and (ii) a promoter operably linked to said nucleotide sequence.
  • FSHD Facioscapulohumeral muscular dystrophy
  • the viral genome can further comprise one or more (e.g., two, three, four, or all five) of (a) a pair of dependoparvovirus ITRs, (b) an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and (e) a polyA sequence
  • the viral genome comprises ITRs flanking the nucleotide sequence and a polyA sequence operably linked to the nucleotide sequence.
  • the viral genome lacks rep and cap sequences, which are in trans by the host cell in which the virus particle is produced.
  • the viral genome is self-complementary
  • the disclosure provides a virus particle comprising (a) a capsid polypeptide described herein, for example, a capsid polypeptide described in Section 6.2 and (b) an engineered viral genome comprising (i) a nucleotide sequence encoding a molecule for the treatment of Facioscapulohumeral muscular dystrophy (FSHD), for example human SMCHD1 or a fragment or variant thereof (e g , a variant thereof having at least 90% or at least 95% sequence identity to human SMCHD1 or a portion thereof), and (ii) a promoter operably linked to said nucleotide sequence.
  • FSHD Facioscapulohumeral muscular dystrophy
  • An exemplary accession number for human SMCHD1 is set forth in Table M1.
  • the viral genome can further comprise one or more (e.g., two, three, four, or all five) of (a) a pair of dependoparvovirus ITRs, (b) an intron, (c) an enhancer or repressor sequence, (d) a stuffer sequence, and (e) a polyA sequence
  • the viral genome comprises ITRs flanking the nucleotide sequence and a polyA sequence operably linked to the nucleotide sequence.
  • the viral genome lacks rep and cap sequences, which are in trans by the host cell in which the virus particle is produced
  • the viral genome is self-complementary
  • Exemplary muscle tissue related diseases include but are not limited to Acid Maltase Deficiency (AMD), Amyotrophic Lateral Sclerosis (ALS), Andersen-Tawil Syndrome, Barth syndrome (TAZ), Becker Muscular Dystrophy (BMD), Becker Myotonia Congenita, Bethlem Myopathy, Bulbospinal Muscular Atrophy (Spinal- Bulbar Muscular Atrophy), Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency (CPT Deficiency), Central Core Disease (CCD), Centronuclear Myopathy, Charcot-Marie-Tooth Disease (CMT), Congenital Muscular Dystrophy (CMD), Congenital Myasthenic Syndromes (CMS), Congenital Myotonic Dystrophy, Cori Disease (Debrancher Enzyme Deficiency), Danon disease, Debrancher Enzyme Deficiency, Dejerine-Sottas Disease (DSD), Dermatomyo
  • ASD Acid Maltas
  • Payloads suitable for treating muscle-related disease include the following disease (suitable payload) combinations: Barth syndrome (TAZ), Primary merosin deficiency (LAMA2), Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Danon disease (LAMP2), Limb girdle muscular dystrophy (Subtypes and affected genes: LGMD1A (TTID), LGMD1B (LMNA), LGMD1C (CAV3), LGMD1D (DNAJB6), LGMD1 E (DES), LGMD1 F (TNP03), LGMD1G (HNRPDL), LGMD1H, LGMD2A (CAPN3), LGMD2B (DYSF), LGMD2C (SGCG), LGMD2D (SGCA), LGMD2E (SGCB), LGMD2F (SGCD), LGMD2G (TCAP), LGMD2H (TRIM32), LGMD2I (FKRP), LGMD2J (TTN), LGMD2K (PO), Barth syndrome (TAZ), Primary
  • the payload is selected from: TAZ, LAMA2, DMD, LAMP2, CAPN3, DYSF, SGCA, SGCB, FKRP, PABPN1 , MTM1, and GAA.
  • the payload is human dystrophin.
  • the payload is an antisense oligonucleotide effective in treating a muscle disease, for example by modulating expression of a target gene.
  • target genes of such antisense oligonucleotides for treatment of muscle disease include, for example, MSTN, INHBA, ACVR1 B, MLCK1.
  • Non-limiting, example antisense oligonucleotides that can be delivered via the viral particles of the disclosure (along with associated target genes and muscle tissue related diseases that can be treated) and are provided in Table M2. Where such antisense oligonucleotides are indicated by reference to a patent or patent application publication, such patent and patent applications are incorporated herein by reference in their entirety for their disclosed antisense oligonucleotide structures and sequences.
  • a method of treating a disease or condition in a subject comprises administering to the subject a dependoparvovirus particle comprising a variant polypeptide described herein, e.g., comprising a payload described herein.
  • the dependoparvovirus particle, which comprises a variant polypeptide, comprising a payload described herein is administered in an amount and/or time effective to treat the disease or condition.
  • the payload is a therapeutic product
  • the payload is a nucleic acid, e.g., encoding an exogenous polypeptide.
  • the disclosure is also directed to a dependoparvovirus particle comprising a variant polypeptide described herein, e.g., comprising a payload described herein, for use in the methods of treatment described herein.
  • the disclosure is also directed to the use of a dependoparvovirus particle comprising a variant polypeptide described herein, e.g., comprising a payload described herein for the manufacture of a medicament for the treatment of a disease or condition as described herein.
  • the dependoparvovirus particles comprising a variant polypeptide described herein or produced by the methods described herein can be used to express one or more therapeutic proteins to treat various diseases or disorders
  • the disease or disorder is a cancer, e.g., a cancer such as carcinoma, sarcoma, leukemia, lymphoma; or an autoimmune disease, e.g., multiple sclerosis.
  • Non-limiting examples of carcinomas include esophageal carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma; hepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma (various tissues); bladder carcinoma, including transitional cell carcinoma; lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma; sweat gland carcinoma; sebaceous gland carcinoma; thyroid carcinoma; pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; uterine carcinoma; testicular carcinoma; osteogenic carcinoma; ductal carcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor; cervical carcinoma; epithelial carcinoma; and nasopharyngeal carcinoma.
  • Non-limiting examples of sarcomas include fibrosarcoma, myxosarcoma, liposarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
  • Non-limiting examples of solid tumors include ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
  • Non-limiting examples of leukemias include chronic myeloproliferative syndromes; T-cell CLL prolymphocytic leukemia, acute myelogenous leukemias; chronic lymphocytic leukemias, including B-cell CLL, hairy cell leukemia; and acute lymphoblastic leukemias
  • lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt's lymphoma; and Hodgkin's lymphoma.
  • the disease or disorder is a genetic disorder.
  • the genetic disorder is sickle cell anemia, Glycogen storage diseases (GSD, e.g., GSD types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV), cystic fibrosis, lysosomal acid lipase (LAL) deficiency 1, Tay-Sachs disease, Phenylketonuria, Mucopolysaccharidoses, Galactosemia, muscular dystrophy (e.g., Duchenne muscular dystrophy), hemophilia such as hemophilia A (classic hemophilia) or hemophilia B (Christmas Disease), Wilson's disease, Fabry Disease, Gaucher Disease hereditary angioedema (HAE), and alpha 1 antitrypsin deficiency.
  • GSD Glycogen storage diseases
  • cystic fibrosis cystic fibrosis
  • LAL lysosomal acid lipas
  • the disease or condition is a disease of muscle.
  • exemplary diseases of the muscle include Acid Maltase Deficiency (AMD), Amyotrophic Lateral Sclerosis (ALS), Andersen-Tawil Syndrome, Becker Muscular Dystrophy (BMD), Becker Myotonia Congenita, Bethlem Myopathy, Bulbospinal Muscular Atrophy (Spinal- Bulbar Muscular Atrophy), Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency (CPT Deficiency), Central Core Disease (CCD), Centronuclear Myopathy, Charcot-Marie- Tooth Disease (CMT), Congenital Muscular Dystrophy (CMD), Congenital Myasthenic Syndromes (CMS), Congenital Myotonic Dystrophy, Cori Disease (Debrancher Enzyme Deficiency), Debrancher Enzyme Deficiency, Dejerine-Sottas Disease (DSD), Dermatomyositis (DM).
  • ASD
  • a dependoparvovirus particle comprising a variant polypeptide and comprising a payload (e.g. , a transgene as described in Section 6.7.1) to a subject induces expression of the payload (e.g., transgene) in a subject.
  • the expression is induced in muscle tissue.
  • the production is similar in the muscle tissue as compared to a similar particle with the wild-type capsid protein.
  • the production is increased in the muscle tissue as compared to a similar particle with the wild-type capsid protein.
  • the amount of a payload, e.g., transgene, e.g., heterologous protein, e.g., therapeutic polypeptide, expressed in a subject can vary.
  • the payload e.g., protein or RNA product of a transgene
  • the payload can be expressed in the serum of the subject in the amount of at least about 9 g/ml, at least about 10 pg/ml, at least about 50 g/ml, at least about 100 pg/ml, at least about 200 pg/ml, at least about 300 pg/ml, at least about 400 g/ml, at least about 500 pg/ml, at least about 600 pg/ml, at least about 700 pg/ml, at least about 800 g/ml, at least about 900 pg/ml, or at least about 1000 pg/ml
  • the payload e.g., the payload, e.g., the payload,
  • a viral particle comprising a capsid polypeptide as described herein is prepared as a pharmaceutical composition.
  • pharmaceutical composition refers to a composition comprising at least one active ingredient (e.g., the viral particle) and optionally, one or more pharmaceutically acceptable carriers or excipients.
  • Relative amounts of the active ingredient, pharmaceutically acceptable carrier or excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary Differences in the constitution of a pharmaceutical composition may depend upon the identity, size, and/or condition of the subject being treated, the route by which the composition is to be administered, and/or any other factor.
  • the composition may comprise between 0.0001 % and 99% (w/w) of the active ingredient.
  • the composition may comprise between 0.0001% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, or at least 80% (w/w) active ingredient.
  • Non limiting examples of carriers and/or excipients include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, or combination thereof
  • a capsid polypeptide comprising the amino acid sequence TRGDYAS (SEQ ID NO:16).
  • capsid polypeptide of embodiment 1 wherein at least a portion of the amino acid sequence of SEQ ID NO:16 is in a VR-VIII variable region of the capsid polypeptide
  • capsid polypeptide of any one of embodiments 1 to 5 wherein the amino acid sequence of SEQ ID NO: 16 is C-terminal to a position corresponding to S586 of the VP1 capsid polypeptide of SEQ ID NOV.
  • the capsid polypeptide of any one of embodiments 1 to 6 wherein the amino acid sequence of SEQ ID NO: 16 is C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • capsid polypeptide of any one of embodiments 1 to 11 wherein the amino acid sequence of SEQ ID NO:16 is immediately N-terminal to the position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • capsid polypeptide of any one of embodiments 1 to 16 which comprises an amino acid sequence having one, two, or three amino acid differences as compared to the amino acid sequence of TRGDYASIAQAQTQ (SEQ ID NO:19).
  • capsid polypeptide of any one of embodiments 1 to 16 which comprises the amino acid sequence of SEQ ID NO:19 19.
  • the capsid polypeptide of embodiment 21 which comprises the amino acid sequence of SEQ ID NO:21.
  • a capsid polypeptide comprising the amino acid sequence TRGDYASI (SEQ ID NO:18).
  • capsid polypeptide of any one of embodiments 23 to 31 wherein the amino acid sequence of SEQ ID NO:18 is N-terminal to a position corresponding to A589 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • capsid polypeptide of any one of embodiments 23 to 32 which lacks an amino acid at a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • capsid polypeptide of any one of embodiments 23 to 33 which comprises a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • capsid polypeptide of any one of embodiments 23 to 34 which comprises an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • a capsid polypeptide comprising an amino acid sequence which:
  • (a) comprises the amino acid sequence of SEQ ID NO:16;
  • (b) has no more than three, two, or one amino acid differences as compared to the amino acid sequence TRGDYASIAQAQTQ (SEQ ID NO:19).
  • capsid polypeptide of any one of embodiments 36 to 51 which comprises an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NOV.
  • a capsid polypeptide comprising:
  • the capsid polypeptide of embodiment 53 which comprises an isoleucine at a position corresponding to Q588 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • the capsid polypeptide of embodiment 53 or 54 which comprises a glutamine at a position corresponding to G594 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • capsid polypeptide of any one of embodiments 53 to 55 which comprises an alanine at a position corresponding to 1601 of the VP1 capsid polypeptide of SEQ ID NO:7.
  • capsid polypeptide of any one of embodiments 53 to 56 wherein at least a portion of the amino acid sequence X1X2RGDX3X4X5X6 is in a VR-VIII variable region of the capsid polypeptide.
  • capsid polypeptide of any one of embodiments 53 to 61 wherein the amino acid sequence X1X2RGDX3X4X5X6 is C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NOV.
  • a capsid polypeptide comprising the amino acid sequence RGDYSMT (SEQ ID NO:17).
  • capsid polypeptide of any one of embodiments 77 to 81 wherein the amino acid sequence of SEQ ID NO:17 is C-terminal to a position corresponding to A587 of the VP1 capsid polypeptide of SEQ ID NOV.
  • capsid polypeptide of any one of embodiments 77 to 88 which comprises a histidine at a position corresponding to T582 of the VP1 capsid polypeptide of SEQ ID NOV.
  • capsid polypeptide of any one of embodiments 77 to 89 which comprises the amino acid sequence HNHQSAQRGDYSMT (SEQ ID NO:22).
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof 92.
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof 103
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 1 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:3 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:5 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof 136
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:7 or the VP2 or VP3 portion thereof 147
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:9 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO:11 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 12 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 70% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 75% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • 191 The capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 80% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 85% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 90% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 91% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 92% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 93% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 94% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 95% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 96% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 97% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 98% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • the capsid polypeptide of any one of embodiments 1 to 90 which comprises an amino acid sequence having at least 99% sequence identity to a VP1 capsid polypeptide of SEQ ID NO: 14 or the VP2 or VP3 portion thereof.
  • a capsid polypeptide which is optionally a capsid polypeptide according to any one of embodiments 1 to 202, which comprises the amino acid sequence of a VP1 capsid polypeptide of SEQ ID NO:12 or the VP2 or VP3 portion thereof.
  • a capsid polypeptide which is optionally a capsid polypeptide according to any one of embodiments 1 to 202, which comprises the amino acid sequence of a VP1 capsid polypeptide of SEQ ID NO:14 or the VP2 or VP3 portion thereof.
  • a capsid polypeptide which:
  • (a) comprises the mutations in the mutation set present in the amino acid sequence of SEQ ID NO:12 as compared to the amino acid sequence of SEQ ID NO:7;
  • (b) comprises an amino acid sequence with an edit distance of 15 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 14 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 13 or less to the amino acid sequence of SEQ ID NO: 12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 12 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 11 or less to the amino acid sequence of SEQ ID NO: 12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 10 or less to the amino acid sequence of SEQ ID NO: 12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 9 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 8 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 7 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 6 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 5 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 4 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 3 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 2 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof)
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 1 or less to the amino acid sequence of SEQ ID NO:12 (or to the VP2 or VP3 portion thereof).
  • (a) comprises the mutations in the mutation set present in the amino acid sequence of SEQ ID NO:14 as compared to the amino acid sequence of SEQ ID NO:7; and (b) comprises an amino acid sequence with an edit distance of 15 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 14 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 13 or less to the amino acid sequence of SEQ ID NO: 14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 12 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 11 or less to the amino acid sequence of SEQ ID NO: 14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 10 or less to the amino acid sequence of SEQ ID NO: 14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 9 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 8 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof)
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 7 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 6 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 5 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 4 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 3 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 2 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • the capsid polypeptide of embodiment 205 comprising an amino acid sequence with an edit distance of 1 or less to the amino acid sequence of SEQ ID NO:14 (or to the VP2 or VP3 portion thereof).
  • capsid polypeptide of any one of embodiments 1 to 235 whose sequence has an edit distance of (a) 12 or lower, (b) 11 or lower, or (c) 10 or lower to:
  • capsid polypeptide of any one of embodiments 1 to 235 whose sequence has an edit distance of (a) 12 or lower, (b) 11 or lower, or (c) 10 or lower to:
  • capsid polypeptide of any one of embodiments 1 to 237 which is a VP1 capsid polypeptide.
  • capsid polypeptide of any one of embodiments 1 to 237 which is a VP2 capsid polypeptide.
  • 240 The capsid polypeptide of any one of embodiments 1 to 237, which is a VP3 capsid polypeptide.
  • nucleic acid molecule of embodiment 241 wherein the nucleic acid molecule comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO:13 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of embodiment 241 wherein the nucleic acid molecule comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:13 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of embodiment 241, wherein the nucleic acid molecule comprises a nucleotide sequence having at least 91% sequence identity to the nucleotide sequence of SEQ ID NO:13 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of embodiment 241, wherein the nucleic acid molecule comprises a nucleotide sequence having at least 92% sequence identity to the nucleotide sequence of SEQ ID NO:13 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of embodiment 241, wherein the nucleic acid molecule comprises a nucleotide sequence having at least 93% sequence identity to the nucleotide sequence of SEQ ID NO:13 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of any one of embodiments 241 to 255 wherein the nucleic acid molecule comprises a fragment of the nucleotide sequence of SEQ ID N0:13 that encodes a VP3 capsid polypeptide.
  • nucleic acid molecule of embodiment 241 wherein the nucleic acid molecule comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO:15 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of embodiment 241, wherein the nucleic acid molecule comprises a nucleotide sequence having at least 93% sequence identity to the nucleotide sequence of SEQ ID NO:15 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of embodiment 241 wherein the nucleic acid molecule comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:15 or a fragment thereof (e.g., a VP1-encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of embodiment 241, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:15 or a fragment thereof (e.g., a VP1 -encoding, a VP2-encoding or a VP3-encoding fragment thereof).
  • nucleic acid molecule of any one of embodiments 241 to 275 wherein the nucleic acid molecule is double-stranded or single-stranded, and wherein the nucleic acid molecule is linear or circular, e.g., wherein the nucleic acid molecule is a plasmid 277 A virus particle (e.g., adeno-associated virus (“AAV”) particle) comprising a capsid polypeptide of any one of embodiments 1 to 240 or comprising a capsid polypeptide encoded by the nucleic acid molecule of any one of embodiments 241 to 276.
  • a virus particle e.g., adeno-associated virus (“AAV”) particle
  • a virus particle e.g., adeno-associated virus (“AAV”) particle
  • AAV adeno-associated virus
  • the improvement comprising including in the capsid polypeptide the amino acid sequence TRGDYAS (SEQ ID NO:16), optionally wherein the capsid polypeptide is the capsid polypeptide of any one of embodiments 1 to 76.
  • a virus particle e.g., adeno-associated virus (“AAV”) particle
  • AAV adeno-associated virus
  • the improvement comprising including in the capsid polypeptide the amino acid sequence TRGDYAS (SEQ ID NO:16), optionally wherein the capsid polypeptide is the capsid polypeptide of any one of embodiments 1 to 76.
  • a virus particle e.g., adeno-associated virus (“AAV”) particle
  • AAV adeno-associated virus
  • the improvement comprising including in the capsid polypeptide the amino acid sequence RGDYSMT (SEQ ID NO:17), optionally wherein the capsid polypeptide is the capsid polypeptide of any one of embodiments 77 to 90.
  • a virus particle e.g., adeno-associated virus (“AAV”) particle
  • AAV adeno-associated virus
  • the improvement comprising including in the capsid polypeptide the amino acid sequence RGDYSMT (SEQ ID NO:17), optionally wherein the capsid polypeptide is the capsid polypeptide of any one of embodiments 77 to 90.
  • virus particle of embodiment 277, 278, or 280 comprising a nucleic acid comprising a payload (e.g., a heterologous transgene) and one or more regulatory elements
  • a payload e.g., a heterologous transgene
  • CBh promoter 288 The virus particle of embodiment 287, wherein the CBh promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:500.
  • virus particle of any one of embodiments 277 to 289 wherein said virus particle exhibits increased cardiac muscle biodistribution, e.g., as measured in a mammal, e.g., in NHP, e.g., as described herein, relative to wild-type AAV9 (e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8), optionally wherein the increased cardiac muscle biodistribution is measured using a method described in Section 8.1.
  • wild-type AAV9 e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8
  • virus particle of any one of embodiments 277 to 290 wherein said virus particle exhibits increased skeletal muscle biodistribution, e.g., as measured in a mammal, e.g., in NHP, e.g., as described herein, relative to wild-type AAV9 (e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8), optionally wherein the increased skeletal muscle biodistribution is measured using a method described in Section 8.1.
  • wild-type AAV9 e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8
  • virus particle of any one of embodiments 277 to 291 wherein said virus particle exhibits increased cardiac muscle transduction, e.g., as measured in a mammal, e.g., in NHP, e.g., as described herein, relative to wild-type AAV9 (e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8), optionally wherein the increased cardiac muscle transduction is measured using a method described in Section 8.1.
  • wild-type AAV9 e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8
  • wild-type AAV9 e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8
  • wild-type AAV9 e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8
  • wild-type AAV9 e.g., a virus particle comprising capsid polypeptides of SEQ ID NO:7 or encoded by SEQ ID NO:8
  • An adeno-associated virus particle comprising (a) a capsid polypeptide of any one of embodiments 1 to 240 or comprising a capsid polypeptide encoded by the nucleic acid molecule of any one of embodiments 241 to 276 and (b) a nucleic acid comprising a payload (e.g., a heterologous transgene) and one or more regulatory elements.
  • a payload e.g., a heterologous transgene
  • virus particle of any one of embodiments 277 to 302 (suitable) for use in gene therapy.
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • a method of treating a disease or condition in a subject comprising administering to the subject in an amount effective to treat the disease or condition:
  • composition e.g., a pharmaceutical composition, comprising the virus particle of (a) and, optionally, a pharmaceutically acceptable carrier.
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • ALS Amyotrophic Lateral Sclerosis
  • the method of any one of embodiments 309 to 312, wherein the disease or condition isease (CCD) The method of any one of embodiments 309 to 312, wherein the disease or condition Myopathy.
  • CMT disease or condition-Tooth Disease
  • CMT disease or conditionscular Dystrophy
  • CMS disease or conditionasthenic Syndromes
  • CMS disease or conditionotonic Dystrophy
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionnzyme Deficiency The method of any one of embodiments 309 to 312, wherein the disease or conditions Disease (DSD).
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionumeral Muscular Dystrophy (FSH to FSHD) The method of any one of embodiments 309 to 312, wherein the disease or condition) Distal Myopathy.
  • the method of any one of embodiments 309 to 312, wherein the disease or conditione (Debrancher Enzyme Deficiency).
  • the method of any one of embodiments 309 to 312, wherein the disease or condition Type 11 The method of any one of embodiments 309 to 312, wherein the disease or condition Type 2.
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionyopathy The method of any one of embodiments 309 to 312, wherein the disease or conditionent Congenital Muscular Dystrophy.
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionase (Spinal-Bulbar Muscular Atrophy)
  • the method of any one of embodiments 309 to 312, wherein the disease or condition rogenase Deficiency The method of any one of embodiments 309 to 312, wherein the disease or conditionn Myasthenic Syndrome (LEMS).
  • the method of any one of embodiments 309 to 312, wherein the disease or condition isease Amyotrophic Lateral Sclerosis).
  • the method of any one of embodiments 309 to 312, wherein the disease or condition se Phosphtoylase Deficiency).
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionient Congenital Muscular Dystrophy The method of any one of embodiments 309 to 312, wherein the disease or condition ases of Muscle.
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionathy The method of any one of embodiments 309 to 312, wherein the disease or condition Myopathy
  • the method of any one of embodiments 309 to 312, wherein the disease or conditione Disease The method of any one of embodiments 309 to 312, wherein the disease or conditionrain Disease.
  • MTM to MM disease or condition opathy
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionease (Nemaline Myopathy).
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionse Myotonic Muscular Dystrophy).
  • the method of any one of embodiments 309 to 312, wherein the disease or condition (Phosphofructokinase Deficiency).
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionase Myotonia Congenita
  • the method of any one of embodiments 309 to 312, wherein the disease or condition ital Muscular Dystrophy.
  • the method of any one of embodiments 309 to 312, wherein the disease or conditionrg Syndrome Congenital Muscular Dystrophy.
  • the method of any one of embodiments 309 to 312, wherein the disease or conditional Myopathy The method of any one of embodiments 309 to 312, wherein the disease or condition ann Disease (Spinal Muscular Atrophy). 408 The method of any one of embodiments 309 to 312, wherein the disease or condition is ZASP-Related Myopathy.
  • 410 The method of any one of embodiments 309 to 312, wherein the disease or condition is Amyotrophic lateral sclerosis (ALS) (Lou Gehrig’s disease) and/to wherein the heterologous nucleic acid sequence encodes Superoxide dismutase-1 (SOD1 ) (e.g., a polypeptide represented by UniProt Accession number P00441 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto).
  • SOD1 Superoxide dismutase-1
  • 440 The method of any one of embodiments 309 to 312, wherein the disease or condition is Congenital Myasthenic Syndrome and/to wherein the heterologous nucleic acid sequence encodes COLQ (e.g., a polypeptide represented by UniProt Accession number Q9Y215 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto).
  • COLQ e.g., a polypeptide represented by UniProt Accession number Q9Y215 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • GFPT1 e.g., a polypeptide represented by UniProt Accession number Q06210 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • DOK7 e.g., a polypeptide represented by UniProt Accession number Q18PE1 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • CHAT e.g., a polypeptide represented by UniProt Accession number P28329 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • ACTA1 e.g., a polypeptide represented by UniProt Accession number P68133 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • STAC3 e.g., a polypeptide represented by UniProt Accession number Q96MF2 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • TPM3 e.g., a polypeptide represented by UniProt Accession number P06753 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • DMPK e.g., a polypeptide represented by UniProt Accession number Q09013 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • EGR2 e.g., a polypeptide represented by UniProt Accession number P11161 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • TIA1 e.g., a polypeptide represented by UniProt Accession number P31483 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • DMD Dystrophin
  • SYNE2 e.g., a polypeptide represented by UniProt Accession number Q8WXH0 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • TMEM43 e.g., a polypeptide represented by UniProt Accession number Q9BTV4 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • LMNA e.g., a polypeptide represented by UniProt Accession number P02545 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • SMCHD1 e.g., a polypeptide represented by UniProt Accession number A6NHR9 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • 480 The method of any one of embodiments 309 to 312, wherein the disease or condition is Glycogenosis Type 11 (Glycogen sttoage disease XI) and/to wherein the heterologous nucleic acid sequence encodes LDHA (e.g., a polypeptide represented by UniProt Accession number P00338 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto).
  • LDHA e.g., a polypeptide represented by UniProt Accession number P00338 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • Glycogenosis Type 5 Glycogen sttoage disease V to McArdle Disease or Myophosphtoylase Deficiency
  • PYGM e.g., a polypeptide represented by UniProt Accession number P11217 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • PFKM e g., a polypeptide represented by UniProt Accession number P08237 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • AR Androgen receptto
  • SMN1 a polypeptide represented by UniProt Accession number Q16637 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • CACNB2 e.g., a polypeptide represented by UniProt Accession number Q08289 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • POMGNT1 e.g., a polypeptide represented by UniProt Accession number Q8WZA1 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • 501 The method of any one of embodiments 309 to 312, wherein the disease or condition is Mitochondrial Myopathy and/to wherein the heterologous nucleic acid sequence encodes CHCHD10 (e.g., a polypeptide represented by UniProt Accession number Q8WYQ3 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto).
  • CHCHD10 e.g., a polypeptide represented by UniProt Accession number Q8WYQ3 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • DYSF e.g., a polypeptide represented by UniProt Accession number 075923 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • AMPD1 e.g., a polypeptide represented by UniProt Accession number P23109 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • DES e.g., a polypeptide represented by UniProt Accession number P17661 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • ZASP Myofibrillar Myopathy 4
  • FLNC e.g., a polypeptide represented by UniProt Accession number Q14315 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • KY e.g., a polypeptide represented by UniProt Accession number Q8NBH2 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • DMPK Myotonin-protein kinase
  • TPM3 e.g., a polypeptide represented by UniProt Accession number P06753 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • TNNT1 a polypeptide represented by UniProt Accession number P13805 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • ACTA1 e.g., a polypeptide represented by UniProt Accession number P68133 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • TPM2 e.g., a polypeptide represented by UniProt Accession number P07951 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • LMOD3 e.g., a polypeptide represented by UniProt Accession number Q0VAK6 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • PABPN1 e.g., a polypeptide represented by UniProt Accession number Q86U42 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • SMN Survival motto neuron protein
  • SMN1 a polypeptide represented by UniProt Accession number Q16637 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto.
  • MTM1 Myotubularin
  • SMN1 a polypeptide represented by UniProt Accession number Q16637 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto
  • LMNA human lamin A
  • SMA Spinal Muscular Atrophy
  • heterologous nucleic acid sequence encodes an antisense oligonucleotide targeting human Survival Housing Neuron (SMN) (e.g , a polypeptide represented by UniProt Accession number Q16637 to a polypeptide comprising an amino acid sequence having at least 95% sequence identity thereto).
  • STN Survival Housing Neuron
  • GAA human acid alpha-glucosidase
  • DMPK Dystrophia Myotonica-Protein Kinase
  • MSTN human Myostatin
  • a cell, cell-free system, or other translation system comprising the capsid polypeptide of any one of embodiments 1 to 240, the nucleic acid molecule of any one of embodiments 241 to 276, or virus particle of any one of embodiments 277 to 303.
  • a method of making a virus comprising:
  • the second nucleic acid comprises a payload, e.g., a heterologous nucleic acid sequence encoding a therapeutic product, e.g., as described herein.
  • nucleic acid molecule of any one of embodiments 241 to 276 mediates the production of a virus particle at a level similar, or at least 10% greater than the production level mediated by a nucleic acid comprising SEQ ID NO:8 in an otherwise similar production system.
  • a virus e.g., a dependoparvovirus particle such as an adeno- associated virus (AAV) particle
  • a method of making a virus that comprises providing a cell, cell-free system, or other translation system comprising a nucleic acid molecule encoding a capsid polypeptide; and cultivating the cell, cell-free system, or other translation system under conditions suitable for the production of the virus particle, the improvement comprising utilizing a nucleic acid molecule encoding a capsid polypeptide comprising the amino acid sequence TRGDYAS (SEQ ID NO:16), optionally wherein the capsid polypeptide is the capsid polypeptide of any one of embodiments 1 to 76.
  • a dependoparvovirus particle such as an adeno- associated virus (AAV) particle
  • a virus e.g., a dependoparvovirus particle such as an adeno- associated virus (AAV) particle
  • a method of making a virus that comprises providing a cell, cell-free system, or other translation system comprising a nucleic acid molecule encoding a capsid polypeptide; and cultivating the cell, cell-free system, or other translation system under conditions suitable for the production of the virus particle, the improvement comprising utilizing a nucleic acid molecule encoding a capsid polypeptide comprising the amino acid sequence RGDYSMT (SEQ ID NO:17), optionally wherein the capsid polypeptide is the capsid polypeptide of any one of embodiments 77 to 90.
  • a virus e.g., a dependoparvovirus particle such as an adeno- associated virus (AAV) particle
  • AAV adeno- associated virus
  • nucleic acid molecule comprises a payload, e.g., a heterologous nucleic acid sequence encoding a therapeutic product, e.g., as described herein.
  • nucleic acid molecule encoding the capsid polypeptide mediates the production of a virus particle which does not include said nucleic acid molecule encoding the capsid polypeptide or fragment thereof.
  • nucleic acid molecule encoding the capsid polypeptide mediates the production of a virus particle at a level at least 40%, at least 50%, or at least 60% of the production level mediated by a nucleic acid molecule comprising SEQ ID NO:8 in an otherwise similar production system.
  • a composition e.g., a pharmaceutical composition, comprising a virus particle of any one of embodiments 277 to 303, or a virus particle produced by the method of any one of embodiments 557 to 567, and a pharmaceutically acceptable carrier or excipient.
  • a host cell comprising the nucleic acid of any one of embodiments 241 to 276.
  • a nucleic acid comprising a payload, e.g., a heterologous nucleic acid sequence encoding a therapeutic product, e.g., as described herein.
  • the host cell of any one of embodiments 571 to 574 which is a mammalian host cell.
  • the host cell of embodiment 575 which is a human host cell.
  • the host cell of embodiment 579 which is configured to package a virus (a) according to any one of embodiments 277 to 303 and/or (b) useful for performing a method according to any one of embodiments 306 to 555.
  • a library of AAV variant genomes encoding each variant’s capsid and a unique capsid variant barcode identifier was cloned into one ITR plasmid backbone as described previously (Ogden et al. Science, Nov. 19, 2019, 366,(6469):1139-1143; doi: 10.1126/science.aaw2900, hereby incorporated by reference in its entirety), with expression of the capsid gene under the control of a ubiquitous CBh promoter.
  • Each capsid polypeptide variant was included in combination with between 1-500 unique genomic identifiers (“barcodes”) to enable measurement of biological replicates for each virus comprising a unique capsid polypeptide.
  • Each unique barcode is used in combination with a random genomic identifier (“ID tag”) to aid in quantification and to remove polypeptide sequences that contain unintended mutations.
  • ID tag a random genomic identifier
  • Cardiac muscle was dissected to isolate subregions including ventricles, papillary muscle, and atria.
  • total DNA was extracted from tissue samples using MagMAX DNA Multi-Sample Ultra 2.0 Kit (Thermofisher). Samples were first homogenized in the supplied lysis buffer, then incubated at 65°C with Proteinase K before the lysates were added to a plate for automated extraction using the KingFisher Apex instrument (Thermofisher).
  • samples were prepared for next-generation sequencing by amplifying the transgene barcode regions with primers compatible with Illumina NGS platform and sequenced with NextSeq 2000 (Illumina).
  • the barcode tags were extracted from reads with the expected amplicon structure, and the abundance (number of reads, number of UMIs, or number of unique combinations of barcode and ID tags) of each barcode was recorded. Analyses were restricted to the set of barcodes that were present in the input plasmid sample, as measured by a separate sequencing assay that targeted the variant regions of the input plasmid sample
  • Virus packaging was calculated by normalizing aggregated production replicates to input plasmid abundance. Biodistribution and transduction of tissue were calculated by normalizing aggregated biodistribution or transduction samples to input virus abundance. The output was reported as fold change relative to the WT AAV9.
  • Both AAV capsid variants V1 and V2 showed an increase in biodistribution to skeletal and cardiac muscle tissues and a decrease in biodistribution to the liver relative to biodistribution associated with wildtype AAV9 in these tissues (Table 4)
  • AAV capsid variants V1 and V2 also displayed increased transduction in skeletal and cardiac muscle tissues, relative to transduction associated with wildtype AAV9 (Table 5)
  • the designed capsid polypeptides were cloned into plasmids to create a library of plasmids encoding the capsid variants
  • a library of AAV variant genomes encoding each variant’s capsid and a unique capsid variant barcode identifier was cloned into one ITR plasmid backbone as described previously (Ogden et al. Science, Nov. 19, 2019, 366,(6469):1139-1143; doi: 10.1126/science.aaw2900, hereby incorporated by reference in its entirety), with expression of the capsid gene under the control of a ubiquitous CBh promoter.
  • Each capsid polypeptide variant was included in combination with between 1-500 unique genomic identifiers (“barcodes”) to enable measurement of biological replicates for each virus comprising a unique capsid polypeptide.
  • barcodes unique genomic identifiers
  • Each unique barcode is used in combination with a random genomic identifier (“ID tag”) to aid in quantification and to remove polypeptide sequences that contain unintended mutations.
  • a library of AAV capsid variants, each comprising a genome encoding that variant’s capsid polypeptides was produced via transient triple transfection of adherent HEK293T Transfections were completed at a 1 :1:1/150 ratio of Helper, Rep, and ITR plasmid expressing Cap, which has been optimized with these plasmids to limit cross packaging.
  • Cells were harvested, lysed, and purified by a sequence of steps: (1 ) clarification via depth and sterilizing filtration, (2) ultrafiltration and diafiltration via tangential flow filtration (TFF), (3) iodixanol gradient purification, and (4) ultrafiltration and diafiltration via TFF.
  • the produced virus was tested for suitable sterility and endotoxin, and titer performed by ddPCR
  • mice were monitored for signs of inflammation and were treated with weekly IM injections of steroids (methylprednisolone, 40 mg) according to the animal facility’s SOPs and recommendations from the veterinarian Serum samples were collected 4 hours, 2 days, 5 days, and weekly after the injections. The animals were sacrificed 4 weeks after the injections and tissues were collected for biodistribution and transduction analyses. The tissues collected are shown in Table 3. Peripheral tissue samples were collected into RNA/ater® (Sigma-Aldrich) and incubated overnight at 4°C, after which the RNA/afer® was drained and samples were frozen at -80°C. The entire brain was sliced into 4mm coronal slabs using a chilled species-specific brain matrix.
  • steroids methylprednisolone, 40 mg
  • Each slab was bisected into left and right hemispheres along the sagittal midline.
  • the slabs from the right hemisphere were immersed in RNAIater®, drained and frozen.
  • the left hemisphere slabs were sub-dissected for sub regions of the brain, placed into cryo tubes and immediately frozen on dry-ice
  • Cardiac muscle was dissected to isolate basal and apex subregions.
  • total DNA was extracted from tissue samples using MagMAX DNA MultiSample Ultra 2.0 Kit (Thermofisher).
  • RNA samples were first homogenized in the supplied lysis buffer, then incubated at 65°C with Proteinase K before the lysates were added to a plate for automated extraction using the KingFisher Apex instrument (Thermofisher) RNAwas extracted using the MagMAXTM mirVanaTM Total RNA Isolation Kit (Thermofisher) according to the manufacturer's recommendations, and was further treated with DNase I- XT (New England Biolabs) to remove any vector DNA contamination in the RNA sample. Reverse transcription was done with Protoscript II Reverse Transcriptase (New England Biolabs) utilizing primers that were specific to the vector transgene and included unique molecular identifiers (UMIs). Control reactions lacking the reverse transcriptase enzyme (-RT control) were also prepared. Finally, samples were prepared for nextgeneration sequencing by amplifying the transgene barcode regions with primers compatible with Illumina NGS platform and sequenced with NextSeq 2000 (Illumina).
  • the barcode tags were extracted from reads with the expected amplicon structure, and the abundance (number of reads, number of UMIs, or number of unique combinations of barcode and ID tags) of each barcode was recorded. Analyses were restricted to the set of barcodes that were present in the input plasmid sample, as measured by a separate sequencing assay that targeted the variant regions of the input plasmid sample
  • Virus packaging was calculated by normalizing aggregated production replicates to input plasmid abundance. Biodistribution and transduction of tissue were calculated by normalizing aggregated biodistribution or transduction samples to input virus abundance. The output was reported as fold change relative to the WT AAV9.
  • variants V1, V3, and V4 (a) target liver less than WT AAV9, (b) target heart (e g., primate heart) more than WT AAV9, (c) target muscle (e.g., primate muscle) more than WT AAV9, and/or (d) target spleen (e.g., primate spleen) less than WT AAV9, with respect to biodistribution and/or transduction.
  • target liver less than WT AAV9
  • target heart e g., primate heart
  • target muscle e.g., primate muscle
  • target spleen e.g., primate spleen

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La divulgation concerne en partie des polypeptides de capside de dépendoparvovirus qui peuvent être utilisés pour délivrer des charges utiles.
PCT/US2025/046835 2024-09-18 2025-09-17 Polypeptides de capside et leurs procédés d'utilisation Pending WO2026064428A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463696218P 2024-09-18 2024-09-18
US63/696,218 2024-09-18

Publications (2)

Publication Number Publication Date
WO2026064428A2 true WO2026064428A2 (fr) 2026-03-26
WO2026064428A3 WO2026064428A3 (fr) 2026-04-23

Family

ID=97522327

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/046835 Pending WO2026064428A2 (fr) 2024-09-18 2025-09-17 Polypeptides de capside et leurs procédés d'utilisation

Country Status (1)

Country Link
WO (1) WO2026064428A2 (fr)

Citations (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4745051A (en) 1983-05-27 1988-05-17 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US5387484A (en) 1992-07-07 1995-02-07 International Business Machines Corporation Two-sided mask for patterning of materials with electromagnetic radiation
US5478745A (en) 1992-12-04 1995-12-26 University Of Pittsburgh Recombinant viral vector system
WO1997017458A1 (fr) 1995-11-09 1997-05-15 Avigen, Inc. Fonctions accessoires servant a produire des virions de vaa recombines
US5658785A (en) 1994-06-06 1997-08-19 Children's Hospital, Inc. Adeno-associated virus materials and methods
US5688676A (en) 1995-06-07 1997-11-18 Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US5691176A (en) 1990-10-30 1997-11-25 Applied Immune Sciences, Inc. Recombinant adeno-associated virus vector packaging cells and methods for use
US5741683A (en) 1995-06-07 1998-04-21 The Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
WO1999011764A2 (fr) 1997-09-05 1999-03-11 Targeted Genetics Corporation Procedes de generation de preparations de vecteurs de aav recombinants dont le titre est eleve et qui sont exemptes de virus assistant
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
US6281010B1 (en) 1995-06-05 2001-08-28 The Trustees Of The University Of Pennsylvania Adenovirus gene therapy vehicle and cell line
US6428988B1 (en) 1996-09-06 2002-08-06 The Trustees Of The University Of Pennsylvania Methods using cre-lox for production of recombinant adeno-associated viruses
US6541258B2 (en) 1996-12-18 2003-04-01 Targeted Genetics Corporation AAV split-packaging genes and cell lines comprising such genes for use in the production of recombinant AAV vectors
US20030148506A1 (en) 2001-11-09 2003-08-07 The Government Of The United States Of America, Department Of Health And Human Services Production of adeno-associated virus in insect cells
WO2003074714A1 (fr) 2002-03-05 2003-09-12 Stichting Voor De Technische Wetenschappen Systeme d'expression de baculovirus
US6660521B2 (en) 1998-12-04 2003-12-09 Genvec, Inc. Use of trans-activation and CIS-activation to increase the persistence of a transgene in an at least E4-deficient adenovirus
US20040029106A1 (en) 2000-06-01 2004-02-12 Samulski Richard Jude Duplexed parvovirus vectors
US20040248288A1 (en) 2000-01-05 2004-12-09 Miller Arthur Dusty Compositions and methods for efficient aav vector production
US8298818B2 (en) 2006-04-28 2012-10-30 University Of Florida Research Foundation, Inc. Self-complementary adeno-associated virus having a truncated CMV-chicken β-actin promoter
US9150882B2 (en) 2006-01-31 2015-10-06 The Board Of Trustees Of The Leland Stanford Junior University Self-complementary parvoviral vectors, and methods for making and using the same
WO2019207132A1 (fr) 2018-04-27 2019-10-31 Universität Heidelberg Polypeptides de capside d'aav modifiés pour le traitement de maladies musculaires
WO2020206189A1 (fr) 2019-04-04 2020-10-08 Regenxbio Inc. Virus adéno-associés recombinants et leurs utilisations
WO2020208379A1 (fr) 2019-04-12 2020-10-15 Freeline Therapeutics Limited Système plasmidique
WO2021113767A1 (fr) 2019-12-04 2021-06-10 Sangamo Therapeutics, Inc. Nouvelles compositions et procédés de production d'aav recombinants
WO2021127655A1 (fr) 2019-12-20 2021-06-24 Research Institute At Nationwide Children's Hospital Thérapie génique optimisée pour cibler un muscle dans des maladies musculaires
US11060070B2 (en) 2010-03-11 2021-07-13 Uniqure Ip B.V. Mutated rep encoding sequences for use in AAV production
WO2021156609A1 (fr) 2020-02-04 2021-08-12 Oxford Genetics Limited Procédé de fabrication de vecteurs viraux adénoassociés
US20220025396A1 (en) 2018-05-07 2022-01-27 Spark Therapeutics, Inc. Plasmid free aav vector producing cell lines
US20220064671A1 (en) 2019-01-18 2022-03-03 Voyager Therapeutics, Inc. Methods and systems for producing aav particles
WO2022053630A1 (fr) 2020-09-10 2022-03-17 Genethon Capside de aav modifiée par peptide
US11306291B2 (en) 2007-07-26 2022-04-19 Uniqure Ip B.V. AAV vectors produced by insect cells comprising REP52 and REP78 coding sequences with differential codon biases
WO2022079429A1 (fr) 2020-10-13 2022-04-21 Freeline Therapeutics Limited Système plasmidique
US20220175887A1 (en) 2016-08-19 2022-06-09 University Of Florida Research Foundation, Incorporated Methods and compositions for treating conditions using recombinant self-complementary adeno-associated virus
US20220259572A1 (en) 2021-02-11 2022-08-18 Lonza Houston, Inc. Adeno-associated virus (aav) production
WO2022226374A1 (fr) 2021-04-23 2022-10-27 Locanabio, Inc. Capsides d'aav modifiées ciblant des tissus et leurs méthodes d'utilisation
WO2023006890A1 (fr) 2021-07-28 2023-02-02 Vrije Universiteit Brussel Amélioration de l'efficacité d'une thérapie génique ciblant les muscles
US20230139985A1 (en) 2021-10-15 2023-05-04 Research Institute At Nationwide Children's Hospital Self-Complementary Adeno-Associated Virus Vector and its Use in Treatment of Muscular Dystrophy
WO2023143063A1 (fr) 2022-01-25 2023-08-03 广州派真生物技术有限公司 Procédé pour améliorer la production de virus adéno-associés par un système à baculovirus, et application
WO2023178220A1 (fr) 2022-03-16 2023-09-21 Regenxbio Inc. Compositions et procédés pour la production d'aav recombiné
WO2023239627A2 (fr) 2022-06-08 2023-12-14 Regenxbio Inc. Méthodes de production d'aav recombinant
US20240093231A1 (en) 2017-07-20 2024-03-21 Uniqure Ip B.V. Aav capsid production in insect cells
WO2024143429A1 (fr) 2022-12-27 2024-07-04 株式会社シンプロジェン Regulation du gene auxiliaire

Patent Citations (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4745051A (en) 1983-05-27 1988-05-17 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US5691176A (en) 1990-10-30 1997-11-25 Applied Immune Sciences, Inc. Recombinant adeno-associated virus vector packaging cells and methods for use
US5387484A (en) 1992-07-07 1995-02-07 International Business Machines Corporation Two-sided mask for patterning of materials with electromagnetic radiation
US5478745A (en) 1992-12-04 1995-12-26 University Of Pittsburgh Recombinant viral vector system
US5658785A (en) 1994-06-06 1997-08-19 Children's Hospital, Inc. Adeno-associated virus materials and methods
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
US6281010B1 (en) 1995-06-05 2001-08-28 The Trustees Of The University Of Pennsylvania Adenovirus gene therapy vehicle and cell line
US5688676A (en) 1995-06-07 1997-11-18 Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US5741683A (en) 1995-06-07 1998-04-21 The Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
WO1997017458A1 (fr) 1995-11-09 1997-05-15 Avigen, Inc. Fonctions accessoires servant a produire des virions de vaa recombines
US6428988B1 (en) 1996-09-06 2002-08-06 The Trustees Of The University Of Pennsylvania Methods using cre-lox for production of recombinant adeno-associated viruses
US6541258B2 (en) 1996-12-18 2003-04-01 Targeted Genetics Corporation AAV split-packaging genes and cell lines comprising such genes for use in the production of recombinant AAV vectors
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
WO1999011764A2 (fr) 1997-09-05 1999-03-11 Targeted Genetics Corporation Procedes de generation de preparations de vecteurs de aav recombinants dont le titre est eleve et qui sont exemptes de virus assistant
US6660521B2 (en) 1998-12-04 2003-12-09 Genvec, Inc. Use of trans-activation and CIS-activation to increase the persistence of a transgene in an at least E4-deficient adenovirus
US20040248288A1 (en) 2000-01-05 2004-12-09 Miller Arthur Dusty Compositions and methods for efficient aav vector production
US7465583B2 (en) 2000-06-01 2008-12-16 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
US20040029106A1 (en) 2000-06-01 2004-02-12 Samulski Richard Jude Duplexed parvovirus vectors
US20030148506A1 (en) 2001-11-09 2003-08-07 The Government Of The United States Of America, Department Of Health And Human Services Production of adeno-associated virus in insect cells
WO2003074714A1 (fr) 2002-03-05 2003-09-12 Stichting Voor De Technische Wetenschappen Systeme d'expression de baculovirus
US9150882B2 (en) 2006-01-31 2015-10-06 The Board Of Trustees Of The Leland Stanford Junior University Self-complementary parvoviral vectors, and methods for making and using the same
US8298818B2 (en) 2006-04-28 2012-10-30 University Of Florida Research Foundation, Inc. Self-complementary adeno-associated virus having a truncated CMV-chicken β-actin promoter
US11306291B2 (en) 2007-07-26 2022-04-19 Uniqure Ip B.V. AAV vectors produced by insect cells comprising REP52 and REP78 coding sequences with differential codon biases
US11060070B2 (en) 2010-03-11 2021-07-13 Uniqure Ip B.V. Mutated rep encoding sequences for use in AAV production
US20220175887A1 (en) 2016-08-19 2022-06-09 University Of Florida Research Foundation, Incorporated Methods and compositions for treating conditions using recombinant self-complementary adeno-associated virus
US20240093231A1 (en) 2017-07-20 2024-03-21 Uniqure Ip B.V. Aav capsid production in insect cells
WO2019207132A1 (fr) 2018-04-27 2019-10-31 Universität Heidelberg Polypeptides de capside d'aav modifiés pour le traitement de maladies musculaires
US20220025396A1 (en) 2018-05-07 2022-01-27 Spark Therapeutics, Inc. Plasmid free aav vector producing cell lines
US20220064671A1 (en) 2019-01-18 2022-03-03 Voyager Therapeutics, Inc. Methods and systems for producing aav particles
WO2020206189A1 (fr) 2019-04-04 2020-10-08 Regenxbio Inc. Virus adéno-associés recombinants et leurs utilisations
WO2020208379A1 (fr) 2019-04-12 2020-10-15 Freeline Therapeutics Limited Système plasmidique
WO2021113767A1 (fr) 2019-12-04 2021-06-10 Sangamo Therapeutics, Inc. Nouvelles compositions et procédés de production d'aav recombinants
WO2021127655A1 (fr) 2019-12-20 2021-06-24 Research Institute At Nationwide Children's Hospital Thérapie génique optimisée pour cibler un muscle dans des maladies musculaires
WO2021156609A1 (fr) 2020-02-04 2021-08-12 Oxford Genetics Limited Procédé de fabrication de vecteurs viraux adénoassociés
WO2022053630A1 (fr) 2020-09-10 2022-03-17 Genethon Capside de aav modifiée par peptide
WO2022079429A1 (fr) 2020-10-13 2022-04-21 Freeline Therapeutics Limited Système plasmidique
US20220259572A1 (en) 2021-02-11 2022-08-18 Lonza Houston, Inc. Adeno-associated virus (aav) production
WO2022226374A1 (fr) 2021-04-23 2022-10-27 Locanabio, Inc. Capsides d'aav modifiées ciblant des tissus et leurs méthodes d'utilisation
WO2023006890A1 (fr) 2021-07-28 2023-02-02 Vrije Universiteit Brussel Amélioration de l'efficacité d'une thérapie génique ciblant les muscles
US20230139985A1 (en) 2021-10-15 2023-05-04 Research Institute At Nationwide Children's Hospital Self-Complementary Adeno-Associated Virus Vector and its Use in Treatment of Muscular Dystrophy
WO2023143063A1 (fr) 2022-01-25 2023-08-03 广州派真生物技术有限公司 Procédé pour améliorer la production de virus adéno-associés par un système à baculovirus, et application
WO2023178220A1 (fr) 2022-03-16 2023-09-21 Regenxbio Inc. Compositions et procédés pour la production d'aav recombiné
WO2023239627A2 (fr) 2022-06-08 2023-12-14 Regenxbio Inc. Méthodes de production d'aav recombinant
WO2024143429A1 (fr) 2022-12-27 2024-07-04 株式会社シンプロジェン Regulation du gene auxiliaire

Non-Patent Citations (32)

* Cited by examiner, † Cited by third party
Title
"UniProt", Database accession no. Q9UBX2
BERNS: "Virology", 1990, RAVEN PRESS, pages: 1743 - 1764
CARTER, HANDBOOK OF PARVOVIRUSES, vol. 1, 1989, pages 169 - 228
COULON ET AL., JBC, vol. 282, no. 45, 2007, pages 33192 - 33200
DONNELLY ET AL., J. GEN. VIROL, vol. 78, January 1997 (1997-01-01), pages 13 - 21
FURLER ET AL., GENE THER, vol. 8, no. 11, June 2001 (2001-06-01), pages 864 - 873
HWANG ET AL.: "N- Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals", SCIENCE, vol. 327, no. 5968, 19 February 2010 (2010-02-19), pages 973 - 977, XP055369420, DOI: 10.1126/science.1183147
JIN ET AL.: "Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins", HUM GENE THER METHODS, no. 5, 28 October 2017 (2017-10-28), pages 255 - 267
JOSHI ET AL., METHODS MOL BIOL, vol. 2829, 2024, pages 203 - 214
K. FISHER ET AL., J. VIROL, vol. 70, 1993, pages 520 - 532
KAJIGAYA ET AL., PNAS, vol. 88, 1991, pages 4646 - 50
KAJIGAYA ET AL., PROC. NAT'I. ACAD. SCI. USA, vol. 88, 1991, pages 4646 - 50
KIRNBAUER ET AL., VIR, vol. 219, no. 9, 1996, pages 37 - 44
KLUMP ET AL., GENE THER, vol. 8, no. 10, May 2001 (2001-05-01), pages 811 - 817
LUCKOW ET AL., BIO/ TECHNOLOGY, vol. 6, 1988, pages 165 - 174
MARWIDI ET AL., MOL THER METHODS CLIN DEV, vol. 32, no. 2, 2024, pages 101228
MCCARTY, MOL. THER, vol. 16, no. 10, 2008, pages 1648 - 1656
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, no. 3, 1970, pages 443 - 53
OGDEN ET AL., SCIENCE, vol. 366, no. 6469, 19 November 2019 (2019-11-19), pages 1139 - 1143
O'REILLY ET AL.: "BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL", 1994, COLD SPRING HARBOR PRESS
O'REILLY, D. RL. K. MILLERV. A. LUCKOW, BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, 1992
PIEKAROWICZ ET AL., MOL THER METHODS CLIN DEV, vol. 15, 2019, pages 157 - 169
PROKOP ET AL., CLONING AND EXPRESSION OF HETEROLOGOUS GENES IN INSECT CELLS WITH BACULOVIRUS VECTORS' RECOMBINANT DNA TECHNOLOGY AND APPLICATIONS, 1986, pages 97 - 152
ROSE, COMPREHENSIVE VIROLOGY, vol. 3, 1974, pages 1 - 61
RUFFING ET AL., J. VIR, vol. 66, 1992, pages 6922 - 30
SATNULSKI ET AL., J. VIR, vol. 63, 1989, pages 3822 - 8
SKOPENKOVA ET AL., ACTA NATURAE, vol. 13, no. 1, 2021, pages 47 - 58
SUMMERSSMITH, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CULTURE PROCEDURES, 1986
W. H. FREEMANRICHARDSON, C. D: "Baculovirus Expression Protocols, Methods in Molecular Biology", vol. 39, 1995, HUMANA PRESS
WANG, GENE THER, vol. 15, 2008, pages 1489 - 1499
WILMOTT ET AL., HUM. GENE. THER. METHODS, vol. 30, no. 6, 2019, pages 206 - 213
ZHAO ET AL., VIR, vol. 272, 2000, pages 382 - 93

Also Published As

Publication number Publication date
WO2026064428A3 (fr) 2026-04-23

Similar Documents

Publication Publication Date Title
US11840704B2 (en) Adeno-associated virus variant capsids and methods of use thereof
AU2017248840B2 (en) Methods of enhancing biological potency of baculovirus system-produced recombinant adeno-associated virus
TW202106700A (zh) Aav 工程化
WO2012158757A1 (fr) Plasmides pro-viraux pour la production de virus adéno-associé recombinant
EP4200316B1 (fr) Procédé de fabrication d'une bibliothèque de vaa recombinant
KR20250169640A (ko) 캡시드 폴리펩타이드 및 이의 사용 방법
WO2026064428A2 (fr) Polypeptides de capside et leurs procédés d'utilisation
WO2026064444A1 (fr) Polypeptides de capside et leurs procédés d'utilisation
US20240271160A1 (en) Production of adeno-associated virus vector in insect cells
US20240294578A1 (en) Capsid variants and methods of using the same
US20240277869A1 (en) Capsid variants and methods of using the same
US20250163469A1 (en) Adeno-associated virus variant capsids and methods of use thereof
CN120865359B (zh) 一种aav衣壳蛋白突变体及其应用
EA050229B1 (ru) Изготовление и применение векторов на основе рекомбинантного aav
WO2025024227A2 (fr) Variants d'aav pour une administration cardiaque
CN117836421A (zh) 在昆虫细胞中制备腺相关病毒载体
WO2026064442A2 (fr) Polypeptides de capside et leurs procédés d'utilisation
HK40036161A (zh) 腺相关病毒变异衣壳和其使用方法