Capsid polypeptide and method of use thereof

Abstract

This disclosure, in part, relates to dependent parvovirus capsid polypeptides that can be used to deliver payloads.

Description

Technical Field

[0001] 1. Cross - reference to related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 489,598, filed on March 10, 2023; U.S. Provisional Patent Application No. 63 / 495,614, filed on April 12, 2023; and U.S. Provisional Patent Application No. 63 / 535,725, filed on August 31, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0002] 2. Sequence listing This application contains a sequence listing that is electronically submitted in XML format, the entire contents of which are hereby incorporated by reference. The XML sequence listing created on February 29, 2024, is named DYN - 001WO_SL.xml and has a size of 525,148 bytes.

Background Art

[0003] 3. Background Dependoparvoviruses, such as adeno - associated dependoparvoviruses, such as adeno - associated virus (AAV), are of interest as vectors for delivering various payloads to cells, including human subjects.

Summary of the Invention

[0004] 4. Summary This disclosure relates, in part, to improved dependent parvovirus capsid polypeptides, e.g., VP1 capsid polypeptide, VP2 capsid polypeptide, and / or VP3 capsid polypeptide, methods for producing dependent parvoviruses comprising capsid polypeptides, compositions for use in the same, and viral particles produced thereby. In certain embodiments, this disclosure relates to viral particles comprising improved dependent parvovirus capsid polypeptides having increased central nervous system (CNS) biodistribution and / or transduction compared to viral particles that do not contain mutations in the improved dependent parvovirus capsid polypeptides. In certain embodiments, this disclosure relates to viral particles comprising improved dependent parvovirus capsid polypeptides having increased skeletal muscle biodistribution and / or transduction compared to viral particles that do not contain mutations in the improved dependent parvovirus capsid polypeptides.

[0005] Accordingly, this disclosure provides capsid polypeptides as described herein. In some embodiments, 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 with any one of the VP1 polypeptides from SEQ ID NOs. 12 to 73, or with its VP2 or VP3 portion. Typically, the capsid polypeptides of the present disclosure, compared to the capsid polypeptide of SEQ ID NO: 1, include mutations at positions corresponding to V596 (e.g., V596L) and / or N598 (e.g., N598S or N598T), and optionally further include mutations at one or more of the following positions compared to the capsid polypeptide of SEQ ID NO: 1: Q579 (e.g., Q579S, S579T, or Q579V), T593 (e.g., T593A, T593R, T593S, or T593V), W595 (e.g., W595A or W595Y), I601 (e.g., I601A or I601V), and Q592 (e.g., Q592A, Q592I, Q592N, or Q592S). In some embodiments, the capsid polypeptide contains mutations at V596, N598, and Q579 compared to the capsid polypeptide of SEQ ID NO: 1. In some embodiments, the capsid polypeptide contains mutations at V596, N598, and T593 compared to the capsid polypeptide of SEQ ID NO: 1. In some embodiments, the capsid polypeptide contains mutations at V596, N598, Q579, and T593 compared to the capsid polypeptide of SEQ ID NO: 1. In further embodiments, the capsid polypeptide contains mutations at W595, I601, Q592, or any combination of two or three of W595, I601, and Q592 compared to the capsid polypeptide of SEQ ID NO: 1, along with mutations at any of the aforementioned positional combinations. In some embodiments, the sequence identity percentage is calculated excluding any targeted peptide sequence insertion(s) in the capsid polypeptide sequence.In other embodiments, percentage sequence identity is calculated including any one or more targeted peptide sequence insertions within the capsid polypeptide sequence. Additional exemplary capsid polypeptides are disclosed in Section 5.2 and in numbered embodiments 1 to 531.

[0006] This disclosure further provides nucleic acids comprising nucleotide sequences encoding capsid polypeptides, e.g., those disclosed in Section 5.2 or any one of the numbered embodiments 1 to 531. In some embodiments, the nucleic acid molecule comprises one nucleotide sequence from SEQ ID NOs. 74 to SEQ ID NOs. 135, a fragment thereof (e.g., a fragment thereof encoding a VP2 polypeptide or a VP3 polypeptide), or any of the aforementioned variants having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to them. In some embodiments, the sequence identity percentage is calculated excluding any nucleotide sequences encoding a targeted peptide insertion(s). In some embodiments, the sequence identity percentage is calculated to include any nucleotide sequences encoding a targeted peptide insertion(s). In some embodiments, the nucleic acid is a vector, e.g., a plasmid. Exemplary nucleic acids are disclosed in Section 5.2 and in numbered embodiments 532–551.

[0007] This disclosure further provides dependent parvovirus particles comprising a capsid polypeptide, a capsid polypeptide disclosed in any one of Section 5.2 or any one of the numbered embodiments 1 to 531, and / or nucleic acids described herein, e.g., nucleic acids disclosed in any one of Section 5.2 or any one of the numbered embodiments 532 to 551, or nucleic acids comprising a transgene / payload disclosed in Section 5.7. In some embodiments, the dependent parvovirus is adeno-associated dependent parvovirus (AAV). In some embodiments, the AAV is AAV9, e.g., variant AAV9. Exemplary virus particles are disclosed in Section 5.3 and the numbered embodiments 552 to 736. In some embodiments, the virus particles have one or more features disclosed in Section 5.4 and the numbered embodiments 570 to 736.

[0008] In some embodiments, the Disclosure covers cells, cell-free systems, or other translating systems, in part, a nucleic acid or vector described herein, for example, a capsid polypeptide having one or more mutations described herein, for example, a sequence encoding a capsid polypeptide disclosed in Section 5.2 or any one of the numbered embodiments 1 to 531. In some embodiments, the cells, cell-free systems, or other translating systems include dependent parvovirus particles described herein, for example, a particle containing a nucleic acid comprising a sequence encoding a capsid polypeptide, for example, a capsid polypeptide disclosed in Section 5.2 or any one of the numbered embodiments 1 to 531, and / or a nucleic acid described herein, for example, a nucleic acid disclosed in Section 5.2 or any one of the numbered embodiments 532 to 551, or a nucleic acid comprising a transgene disclosed in Section 5.7. Exemplary cellular, cell-free, and other translation systems, as well as their use for producing dependent parvovirus particles, are disclosed in Section 5.5, and in numbered embodiments 737, 738, 1807–1812, and 1816–1827.

[0009] This disclosure further provides methods for using the dependent parvovirus disclosed herein, for example, to deliver a payload to cells or to treat a disease or condition in a subject. The methods typically involve contacting cells or administering to a subject an amount of dependent parvovirus particles described herein that is effective in treating a disease or condition. Exemplary methods are disclosed in Section 5.6 and numbered embodiments 748-1323. The dependent parvovirus particles may also be in the form of a pharmaceutical composition, for example, comprising a composition, e.g., dependent parvovirus particles and a pharmaceutically acceptable carrier or excipient, as described, for example, in Section 5.8 and numbered embodiment 1813. This disclosure further provides compositions disclosed herein for use in treating a disease or condition in a subject and for use in the manufacture of pharmaceuticals for use in treating a disease or condition in a subject. Exemplary compositions for use are described in numbered embodiments 1814 and 1815.

[0010] Additional features, advantages, and applications of the capsid polypeptides, nucleic acids, and dependent parvovirus particles of this disclosure, as well as methods for their manufacture and use, are described more specifically below. [Brief explanation of the drawing]

[0011] 5. Brief description of the drawing [Figure 1A] An example of an AAV serotype alignment. Amino acids present only in the VP1 polypeptide are in regular letters, amino acids present only in the VP1 and VP2 polypeptides are in bold, and amino acids present in the VP1, VP2, and VP3 polypeptides are underlined. [Figure 1B]An example of an AAV serotype alignment. Amino acids present only in the VP1 polypeptide are in regular letters, amino acids present only in the VP1 and VP2 polypeptides are in bold, and amino acids present in the VP1, VP2, and VP3 polypeptides are underlined. [Figure 1C] An example of an AAV serotype alignment. Amino acids present only in the VP1 polypeptide are in regular letters, amino acids present only in the VP1 and VP2 polypeptides are in bold, and amino acids present in the VP1, VP2, and VP3 polypeptides are underlined. [Figure 2A] Genome maps of plasmids used in Example 2. A) Wild-type AAV9 rep cap plasmid. B) VAR-B1 rep cap plasmid. C) VAR-B1 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-eGFP. D) Wild-type AAV9 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-mCherry. [Figure 2B] Genome maps of plasmids used in Example 2. A) Wild-type AAV9 rep cap plasmid. B) VAR-B1 rep cap plasmid. C) VAR-B1 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-eGFP. D) Wild-type AAV9 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-mCherry. [Figure 2C]Genome maps of plasmids used in Example 2. A) Wild-type AAV9 rep cap plasmid. B) VAR-B1 rep cap plasmid. C) VAR-B1 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-eGFP. D) Wild-type AAV9 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-mCherry. [Figure 2D] Genome maps of plasmids used in Example 2. A) Wild-type AAV9 rep cap plasmid. B) VAR-B1 rep cap plasmid. C) VAR-B1 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-eGFP. D) Wild-type AAV9 capsid: Packaging plasmid related to a heterogeneous nucleic acid sequence packaged in an ITR-containing plasmid encoding NLS-mCherry. [Figure 3A] Relative transduction (A) and in vivo distribution (B) of VAR-B1 (compared to wild-type AAV9) from the 2-capsid NHP experiment described in Example 2. [Figure 3B] Relative transduction (A) and in vivo distribution (B) of VAR-B1 (compared to wild-type AAV9) from the 2-capsid NHP experiment described in Example 2. [Figure 4A]A) Representative images of VAR-B1 and wild-type AAV9 immunofluorescence in the hippocampus and cerebellum. In the hippocampus, the number of cells expressing VAR-B1 GFP is approximately 25 times greater than the number of cells expressing AAV9 mCherry. This is also observed in the CA3 layer, where VAR-B1 GFP can be seen in CA3 pyramidal neurons that co-stain with the neuronal marker NeuN. In the cerebellum, VAR-B1 GFP is expressed in more cells than AAV9 mCherry, with the highest expression occurring in the Purkinje cell layer. B) Representative images of VAR-B1 and wild-type AAV9 immunofluorescence in the cervical spinal cord, frontal cortex, and caudate nucleus. In the spinal cord, the number of cells expressing VAR-B1 GFP is 9.4 times greater than that expressing AAV9 mCherry. Unlike AAV9 mCherry, VAR-B1 expressed GFP is detected in some neurons (co-staining with NeuN). In the prefrontal cortex and caudate nucleus, the total number of cells expressing VAR-B1 GFP increased compared to AAV9 mCherry (15.9-fold and 4.6-fold increases), and this was specifically increased up to 25-fold and 37-fold for neurons (co-staining with NeuN). [Figure 4B]A) Representative images of VAR-B1 and wild-type AAV9 immunofluorescence in the hippocampus and cerebellum. In the hippocampus, the number of cells expressing VAR-B1 GFP is approximately 25 times greater than the number of cells expressing AAV9 mCherry. This is also observed in the CA3 layer, where VAR-B1 GFP can be seen in CA3 pyramidal neurons that co-stain with the neuronal marker NeuN. In the cerebellum, VAR-B1 GFP is expressed in more cells than AAV9 mCherry, with the highest expression occurring in the Purkinje cell layer. B) Representative images of VAR-B1 and wild-type AAV9 immunofluorescence in the cervical spinal cord, frontal cortex, and caudate nucleus. In the spinal cord, the number of cells expressing VAR-B1 GFP is 9.4 times greater than that expressing AAV9 mCherry. Unlike AAV9 mCherry, VAR-B1 expressed GFP is detected in some neurons (co-staining with NeuN). In the prefrontal cortex and caudate nucleus, the total number of cells expressing VAR-B1 GFP increased compared to AAV9 mCherry (15.9-fold and 4.6-fold increases), and this was specifically increased up to 25-fold and 37-fold for neurons (co-staining with NeuN). [Figure 5] Representative images from in vitro cell transduction experiments demonstrating transduction of VAR-B1 and WT AAV9 in both primary human neurons and Sh-sy5y cell lines. Primary human neurons were treated with both VAR-B1 and WT AAV9 at 50K MOI, and Sh-sy5y cells were treated with virus at 100K MOI. Images were acquired from fixed samples using an EVOS M5000 with a 20× objective lens. [Figure 6] Neuronal transduction rates for VAR-1, VAR-2, VAR-3, VAR-18, VAR-54, and VAR-B1. Transduction rates were normalized to the cell and capsid abundances in the test material (TA) for the aggregated cortical and basal ganglia regions. [Modes for carrying out the invention]

[0012] 5.1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the implementation or testing of this disclosure. In case of any conflict, including definitions, this specification shall prevail. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, and protein and nucleic acid chemistry, as well as hybridization, described herein, are well known and commonly used in the art. Enzyme reactions and purification techniques shall be carried out in accordance with the manufacturer's specifications, either as commonly practiced in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. Throughout this specification and its embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” should be understood to mean that they include the elements or groups of elements described, but not that they exclude any other elements or groups of elements. All publications and other references mentioned herein are incorporated in their entirety by reference. While several documents are cited herein, this citation does not imply that any of these documents constitute part of the widely known general knowledge in the art.

[0013] a, an, the: As used herein, the singular forms a, an, and the referents include multiple objects unless otherwise clearly indicated by the context.

[0014] About, approximately: As used herein, the terms "about" and "approximately" are generally intended to mean an acceptable degree of error for the measured quantity in view of the nature or precision of the measurement. Exemplary degrees of error are within 15 percent (%) of a given value or range of values, typically within 10%, more typically within 5%. Any disclosure in this specification of a value preceded by the term "about" or "approximately" is also a disclosure of the value itself. For example, a disclosure of "about 10 μg / ml" is a disclosure of the value "10 μg / ml".

[0015] CNS: As used herein, "CNS" means one or more regions of the central nervous system. In embodiments, the CNS includes one or more of the brain and spinal cord.

[0016] Corresponding to: As used herein, the term "corresponding to" can be used in reference to a position within a sequence, such as an amino acid or nucleic acid sequence, to refer to an entire capsid polypeptide or polynucleotide sequence, such as the full-length sequence of a capsid polypeptide including VP1 polypeptide, VP2 polypeptide, and VP3 polypeptide, or the nucleic acid molecule encoding it. In some embodiments, the term "corresponding to" can be used in reference to a region or domain of a capsid polypeptide. For example, a position corresponding to a position within the VP1 section of a reference capsid polypeptide may correspond to the VP1 portion of the polypeptide of a variant capsid polypeptide. Thus, when aligning two sequences to determine whether one position corresponds to another, the full-length polypeptide can be used, or a domain (region) can be used to determine whether one position corresponds to a particular position. In some embodiments, the region is the VP1 polypeptide. In some embodiments, the region is the VP2 polypeptide. In some embodiments, the region is the VP3 polypeptide. In some embodiments, if the reference polypeptide is the wild-type sequence (e.g., full length or region) of a particular serotype of AAV, the variant polypeptide can be of the same serotype as the reference sequence (e.g., full length or region) and have the mutation produced at such corresponding positions. In some embodiments, the variant capsid polypeptide is of a different serotype compared to the reference sequence.

[0017] Dependoparvovirus capsid: As used herein, the term "dependoparvovirus capsid" refers to an assembled viral capsid that includes dependoparvovirus polypeptides. In some embodiments, the dependoparvovirus capsid is a functional dependoparvovirus capsid, e.g., fully folded and / or assembled and having the ability to infect a target cell or remaining stable for at least a threshold time (e.g., folded / assembled and having the ability to infect a target cell).

[0018] Dependoparvovirus particle: As used herein, the term "dependoparvovirus particle" refers to an assembled viral capsid that includes dependoparvovirus polypeptides and packaged nucleic acids, e.g., a payload, one or more components of a dependoparvovirus genome (e.g., the entire dependoparvovirus genome), or both. In some embodiments, the dependoparvovirus particle is a functional dependoparvovirus particle, e.g., containing a desired payload, fully folded and / or assembled and having the ability to infect a target cell or remaining stable for at least a threshold time (e.g., folded / assembled and having the ability to infect a target cell).

[0019] Depend Parvovirus X Particles / Capsids: As used herein, the term “Depend Parvovirus X Particles / Capsids” refers to dependent parvovirus particles / capsids containing at least one polypeptide or polypeptide encoding a nucleic acid sequence derived from a naturally occurring depend Parvovirus X species or serotype. For example, depend Parvovirus B particles refer to depend Parvovirus particles containing at least one polypeptide or polypeptide encoding a nucleic acid sequence derived from a naturally occurring depend Parvovirus B sequence. As used in this context, “derived from” means having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the sequence in question. Correspondingly, as used herein, AAVX particles / capsids refer to AAV particles / capsids containing at least one polypeptide or polypeptide encoding a nucleic acid sequence derived from a naturally occurring AAV X serotype. For example, an AAV9 particle refers to an AAV particle containing at least one polypeptide or polypeptide encoding a nucleic acid sequence derived from a naturally occurring AAV9 sequence. A dependent parvovirus X capsid may be referred to as “wild-type” or “wt” if such a capsid contains a capsid polypeptide from a specific sequence identifier associated with such a dependent parvovirus X capsid. Thus, for example, the terms wild-type AAV9 capsid or wtAAV9 capsid (or simply wtAAV9) are used interchangeably and refer to a capsid containing the capsid polypeptide of SEQ ID NO: 1 (e.g., the VP1 capsid of SEQ ID NO: 1, as well as its VP2 and VP3 portions).

[0020] 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 sequences) or a single nucleotide (for nucleotide sequences) that transform one sequence into another is the edit distance between the two sequences. The term “edit distance” is often used interchangeably with the term “Levenshtein distance.”

[0021] Exogenous: As used herein, the term “exogenous” means a feature, sequence, or component that is present in a context (e.g., in a nucleic acid, polypeptide, or cell) that does not exist naturally in that context. For example, a nucleic acid sequence encoding a polypeptide may include an exogenous codon (e.g., a codon encoding an amino acid that does not exist naturally in the reference sequence) as provided herein. Using the term exogenous in this way means that the codon in question does not exist naturally in that position, for example, it does not exist in AAV9, for example, it does not exist in Sequence ID No. 1. In some embodiments, the codon replaces an endogenous codon. In some embodiments, the exogenous codon is inserted into the nucleic acid sequence, for example, relative to a reference sequence. Those skilled in the art will readily understand that a sequence (e.g., a codon) may be exogenous when provided to a particular sequence (e.g., the codon is not naturally present at the site in question), but may not be exogenous in a second sequence (e.g., the particular codon is naturally present at the site in question).

[0022] Functionality: As used herein in reference to polypeptide components of dependent parvovirus capsids (e.g., Cap (e.g., VP1, VP2, and / or VP3) or Rep), the term “functional” means a polypeptide that provides at least 50, 60, 70, 80, 90, or 100% of the activity of the naturally occurring version of that polypeptide component (e.g., when present in host cells). For example, a functional VP1 polypeptide can be stably folded and assembled into a dependent parvovirus capsid (e.g., suitable for packaging and / or secretion). As used herein in reference to dependent parvovirus capsids or particles, “functional” means a capsid or particle that contains a desired payload, is fully folded and / or assembled, and has the ability to infect target cells or remains stable for at least a threshold time (e.g., folded / assembled and / or has the ability to infect target cells).

[0023] Mutational difference: As used herein in relation to polypeptide sequences, this means a single amino acid mutation (e.g., substitution, insertion, or deletion) present in the polypeptide sequence of interest compared to a reference polypeptide sequence. In various embodiments, the reference polypeptide sequence is one of the polypeptides from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NOs. 12-73, and SEQ ID NOs. 136-252, or their VP2 or VP3 portions. In some embodiments, the reference polypeptide is one of the polypeptides from SEQ ID NO: 1, SEQ ID NOs. 12-73, and SEQ ID NOs. 136-252, or their VP2 or VP3 portions. In preferred embodiments, the reference polypeptide is the polypeptide of SEQ ID NO: 1. In various embodiments, the polypeptide of the present invention is one of the polypeptides from SEQ ID NOs. 12-73, or their VP2 or VP3 portions.

[0024] Mutation Set: As used herein, the term “mutation set” refers to a complete set of single-amino acid mutations (substitutions, deletions, and / or insertions) in a variant capsid polypeptide sequence (e.g., any one polypeptide sequence from SEQ ID NOs. 12 to SEQ ID NOs. 73, or their VP2 or VP3 portions) relative to a reference sequence (e.g., a wild-type reference sequence). In some embodiments, the reference sequence is the wild-type AAV9 VP1 capsid polypeptide (SEQ ID NO: 1), or its VP2 or VP3 portion. In some cases, parts of a mutation set (i.e., more than one single-amino acid mutation) are expressed collectively, but even when referred to in this way, it will be understood that a mutation set is an aggregation of single-amino acid mutations. For example, the insertion of amino acids 1, 2, and 3 between amino acid N at position nn and amino acid W at position ww in the reference sequence may be expressed as “Nnn_3aa_Www_123”, and it will be understood that each of amino acids 1, 2, and 3 represents a distinct single-amino acid mutation within the mutation set. The specific variant capsid polypeptide mutation sets described herein are found, for example, in Tables 13 and 27. In some embodiments, the variant capsid polypeptides of this disclosure include mutation sets that do not consist solely of the mutation sets shown in Table 13 (for example, the mutation sets present in any one of the capsid polypeptides from SEQ ID NOs. 135 to 252).

[0025] Nucleic Acid: As used herein, the term “nucleic acid” in its broadest sense refers to any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be apparent from the context, in some embodiments, “nucleic acid” refers to individual nucleic acid monomers (e.g., nucleotides and / or nucleosides), in some embodiments, “nucleic acid” refers to an oligonucleotide chain containing individual nucleic acid monomers, or a longer polynucleotide chain containing many individual nucleic acid monomers. In some embodiments, “nucleic acid” is or contains RNA, and in some embodiments, “nucleic acid” is or contains DNA. In some embodiments, a nucleic acid is one or more native nucleic acid residues, or contains or consists of them. In some embodiments, a nucleic acid is one or more nucleic acid analogs, or contains or consists of them. In some embodiments, a nucleic acid is one or more modified, synthetic, or naturally occurring nucleotides, or contains or consists of them. 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, the nucleic acid is, contains, or consists of one or more "peptide nucleic acids" known in the art, having peptide bonds instead of phosphodiester bonds in the backbone, and is considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate bonds and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or a protein. In some embodiments, the nucleic acid is partially or completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded.

[0026] Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in a choice (choice A is mutually exclusive from choice B, A or B) and the selection of combined features (both A and B are selected, A or B). In some parts of the text, the terms "and / or" are used for the same purpose and should not be interpreted as meaning that "or" is used to refer to mutually exclusive choices.

[0027] Identity Percentage: Sequences disclosed herein may be described in terms of “identity percentage” (identity %). To calculate the identity percentage 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 / ) with the following parameters: Matrix: BLOSUM62 (for amino acid sequences) or DNAfull (for DNA sequences), Gap open: 10, Gap extended: 0.5, End Gap penalty: false, End Gap open: 10, and End Gap extended: 0.5. The identity percentage 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 the 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.

[0028] When calculating the identity percentage of two capsid polypeptides, one or both of which contain one or more targeted peptide insertions, the identity percentage can be determined without removing the targeted peptide insertion sequence(s) from the capsid polypeptide sequence(s), or alternatively, after removing the targeted peptide insertion sequence(s) from the capsid polypeptide sequence(s). For example, if a first capsid polypeptide has the same sequence as a second capsid polypeptide, and the identity percentage is determined without removing the targeted peptide insertion sequence(s) from the first capsid polypeptide sequence, except that the first capsid polypeptide has a heptameric targeted peptide insertion, the two capsid polypeptides will have less than 100% sequence identity. However, if the targeted peptide insertion sequence(s) are removed from the first capsid polypeptide sequence before calculating the identity percentage, the two capsid polypeptides will have 100% sequence identity. In this specification, where the identity percentage of a capsid polypeptide is referred to without reference to the targeted peptide, it refers to the identity percentage of the capsid polypeptide determined after removing any targeted polypeptide insertion sequences present in both capsid polypeptides, unless otherwise required by context. References herein to identity percentages calculated "considering targeted peptide insertions" mean calculating the identity percentage without removing any targeted polypeptide insertion sequences present in both capsid polypeptides, if they exist. References herein to identity percentages calculated "without considering targeted peptide insertions" mean calculating the identity percentage after removing any targeted polypeptide insertion sequences present in both capsid polypeptides, if they exist.

[0029] PNS: As used herein, “PNS” means one or more regions of the peripheral nervous system that do not include the CNS. In embodiments, the PNS includes dorsal root ganglia. In embodiments, the PNS includes sensory neurons and motor neurons.

[0030] Polypeptides, peptides, and proteins: The terms “polypeptides,” “peptides,” and “proteins” are used interchangeably herein to refer to polymers of amino acids of any length. These polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids.

[0031] Targeted peptide: As used herein, “targeted peptide” refers to a peptide inserted into or conjugated to a capsid polypeptide in order to alter the tropism of the capsid polypeptide. Targeted peptides can be inserted into an AAV capsid sequence, for example, to enhance targeting to a desired cell type, tissue, or organ, to enhance targeting to the CNS. Targeted peptides are typically 3 to 20 amino acids long, e.g., 3 to 12 amino acids, 5 to 12 amino acids, 5 to 10 amino acids, or 7 to 10 amino acids.

[0032] Treatment: As used herein, the term “treatment of a disease or condition” means treating an apparent disease or condition, for example, when the subject already has one or more symptoms of the disease or condition, or treating a pre-emergency disease or condition, for example, when the subject has been identified as having a disease or condition but has not yet exhibited one or more symptoms of the disease or condition. A pre-emergency condition may be identified, for example, by genetic testing.

[0033] Variant: As used herein, “variant capsid polypeptide” refers to a polypeptide that differs from the reference sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, preferably SEQ ID NO: 1, or its sequence subunits, e.g., the VP2 or VP3 portion). A variant capsid polypeptide may include, for example, mutations (e.g., substitutions, deletions, or insertions). In some embodiments, the variant is approximately, 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. Those skilled in the art will see from this disclosure that any capsid polypeptide, for example, any capsid polypeptide disclosed herein, for example, any one of SEQ ID NOs: 12 to 73, is a variant capsid polypeptide having a different amino acid sequence, for example, a variant capsid polypeptide to another capsid polypeptide having the aforementioned reference sequence. Therefore, the term “variant capsid polypeptide” as used herein means “capsid polypeptide” and is used interchangeably without requiring any comparison to a specific reference sequence. In some embodiments, the reference sequence is a polypeptide comprising SEQ ID NO: 1. In some embodiments, the reference sequence comprises, for example, the VP1 polypeptide, VP2 polypeptide, or VP3 polypeptide of SEQ ID NO: 1. In some contexts as used herein, the term “variant” refers to a viral particle comprising, for example, a variant capsid polypeptide as described herein.

[0034] 5.2. Capsid polypeptides and nucleic acids encoding them This disclosure covers, in part, variant capsid polypeptides and nucleic acids comprising a sequence encoding a variant capsid polypeptide, wherein the variant capsid polypeptide contains mutations (insertions, deletions, or substitutions) compared to a wild-type sequence. In some embodiments, the wild-type sequence is Sequence ID No. 1. This disclosure covers, in part, variant capsid polypeptides comprising Sequence ID No. 1 having one or more mutations compared to Sequence ID No. 1, and nucleic acid molecules encoding a variant capsid polypeptide. The mutations may be, for example, insertions, deletions, or substitutions compared to a wild-type sequence. In some embodiments, the wild-type sequence is Sequence ID No. 1.

[0035] In some embodiments, the variant capsid polypeptide contains a mutation that corresponds to the position of the mutation present in any one of SEQ ID NOs: 12 to 73, compared to SEQ ID NO: 1.

[0036] In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the variant capsid polypeptide) comprising at least one mutation related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or at least one mutation corresponding to any variant capsid polypeptide of SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the capsid polypeptide) comprising at least two mutations related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or at least two mutations corresponding to two mutations related to any variant capsid polypeptide of SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the capsid polypeptide) comprising at least three mutations related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or at least three mutations corresponding to three mutations related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the capsid polypeptide) comprising at least four mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or comprising at least four mutations corresponding to four mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the capsid polypeptide) comprising at least five mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or comprising at least five mutations corresponding to five mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73.In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding such capsid polypeptide) comprising at least six mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or comprising at least six mutations corresponding to six mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding such capsid polypeptide) comprising at least seven mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or comprising at least seven mutations corresponding to seven mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding such capsid polypeptide) comprising at least eight mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or comprising at least eight mutations corresponding to eight mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the capsid polypeptide) comprising at least nine mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or comprising at least nine mutations corresponding to nine mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 73. In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the capsid polypeptide) comprising at least ten mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73, or comprising at least ten mutations corresponding to ten mutational differences related to any variant capsid polypeptide of SEQ ID NOs. 12 to SEQ ID NOs. 73.

[0037] Mutations associated with VAR-1 to VAR-62 (corresponding to the capsid polypeptides of SEQ ID NOs. 12 to 73, respectively) are shown in Table 1 in relation to the VP1 polypeptide of SEQ ID NO. 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0038] In some embodiments, the Disclosure provides a variant capsid polypeptide (and a nucleic acid encoding the said capsid polypeptide) that includes all the mutational differences associated with any variant capsid polypeptide of SEQ ID NOs. 12 to 73, or a mutation corresponding to all the mutational differences associated with any variant capsid polypeptide of SEQ ID NOs. 12 to 73.

[0039] In any of the embodiments described above, in a variant capsid polypeptide for which the number of variant differences or corresponding variant differences associated with any variant capsid polypeptide of SEQ ID NOs. 12 to 73 has been identified, it will be understood that the mutation may be selected from any of the variant differences associated with that variant capsid polypeptide. Therefore, for example, with respect to the mutations of a variant having mutations #1, #2, #3, and #4 compared to a reference polypeptide, if a variant capsid polypeptide contains one mutation, it is #1 or #2 or #3 or #4; similarly, if a variant capsid contains two mutations, those two are #1 and #2, #1 and #3, #1 and #4, #2 and #3, #2 and #4, or #3 and #4; similarly, if a variant contains three mutations, those three are #1 and #2 and #3, #1 and #2 and #4, #1 and #3 and #4, or #2 and #3 and #4; similarly, if a variant contains all four mutations, those four are #1, #2, #3, and #4. All possible combinations of the number of variant differences for any variant capsid polypeptide from SEQ ID NOs. 12 to 73 (up to the total number of variant differences for that variant capsid polypeptide) can be generated using conventional techniques, and it will be understood by those skilled in the art that each such table for each variant capsid polypeptide from SEQ ID NOs. 12 to 73 is incorporated herein by reference. Such tables can be generated, for example, using the "combination" method from the Python "itertools" package, and such a method is incorporated herein by reference in its entirety.

[0040] In some embodiments, the variant capsid polypeptide comprises one or more variant differences related to the capsid polypeptide described herein, for example, related to any of SEQ ID NOs. 12 to 73, and has sequence identity of 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% with respect to a reference AAV serotype, for example, SEQ ID NO. 1 as described herein. In embodiments, the variant capsid polypeptide comprises one or more variant differences related to the capsid polypeptide described herein, for example, related to any of SEQ ID NOs. 12 to 73, or corresponding to one or more variant differences related to the capsid polypeptide described herein, for example, related to any of SEQ ID NOs. 12 to 73. In embodiments, the variant capsid polypeptide is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the reference AAV serotype described herein, except for a variant difference related to the capsid polypeptide described herein, for example, related to any of SEQ ID NOs. 12 to 73, or a variant capsid polypeptide containing such variant capsid polypeptides, except for a variant difference related to the capsid polypeptide described herein, for example, related to any of SEQ ID NOs. 12 to 73, or a variant capsid polypeptide related to the capsid polypeptide described herein, for example, related to any of SEQ ID NOs. 12 to 73, or a variant capsid polypeptide containing such variant capsid polypeptides, except for a variant difference corresponding to a variant difference related to the capsid polypeptide described herein, for example, related to any of SEQ ID NOs. 12 to 73.In embodiments, the variant capsid polypeptide described herein is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the capsid polypeptide of SEQ ID NO: 3 (e.g., the VP1, VP2, or VP3 sequence of SEQ ID NO: 3), except for variant differences that correspond to variant differences related to the capsid polypeptide described herein, such as those related to any of SEQ ID NOs: 12 to 73, or those related to the capsid polypeptide described herein, such as those related to any of SEQ ID NOs: 12 to 73. In embodiments, the variant capsid polypeptide described herein is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the capsid polypeptide of SEQ ID NO: 5 (e.g., the VP1, VP2, or VP3 sequence of SEQ ID NO: 5), except for mutations related to any of SEQ ID NOs: 12 to 73 contained within the variant capsid polypeptide, or corresponding to mutations related to any of SEQ ID NOs: 12 to 73. In embodiments, the variant capsid polypeptide described herein is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the capsid polypeptide of SEQ ID NO: 7 (e.g., the VP1, VP2, or VP3 sequence of SEQ ID NO: 7), except for mutations related to any of SEQ ID NOs: 12 to 73 contained within the variant capsid polypeptide, or corresponding to mutations related to any of SEQ ID NOs: 12 to 73. In embodiments, the variant capsid polypeptide described herein is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the capsid polypeptide of SEQ ID NO: 9 (e.g., the VP1, VP2, or VP3 sequence of SEQ ID NO: 9), except for variant differences related to any of SEQ ID NOs: 12 to 73 contained within the variant capsid polypeptide, or corresponding to variant differences related to any of SEQ ID NOs: 12 to 73.In embodiments, the variant capsid polypeptide described herein is at least 90%, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the capsid polypeptide of SEQ ID NO: 11 (e.g., the VP1, VP2, or VP3 sequence of SEQ ID NO: 1), except for variant differences related to any of SEQ ID NOs: 12 to 73 contained within the variant capsid polypeptide, or corresponding to variant differences related to any of SEQ ID NOs: 12 to 73.

[0041] In some embodiments, variant capsid polypeptides are provided that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variant capsid polypeptide provided herein.

[0042] In some embodiments, variant capsid polypeptides are provided that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical (without insertion of a targeted peptide) to the variant capsid polypeptide provided herein.

[0043] In some embodiments, the variant capsid polypeptide comprises VP1, VP2, VP3, or any combination thereof, each of which is at least, or about 95, 96, 97, 98, or 99% identical to any one polypeptide from SEQ ID NOs: 12 to SEQ ID NOs: 73, and optionally comprises at least one, e.g., all, of such variant differences from SEQ ID NOs: 12 to SEQ ID NOs: 73.

[0044] In some embodiments, the variant capsid polypeptide comprises VP1, VP2, VP3, or any combination thereof, each of which is at least, or about 95, 96, 97, 98, or 99% identical (without target peptide insertion) to any one polypeptide from SEQ ID NOs: 12 to 73, and optionally comprises at least one, e.g., all, of such variant differences from SEQ ID NOs: 12 to 73.

[0045] In some embodiments, the variant capsid polypeptide comprises VP1, VP2, VP3, or any combination thereof, each having about 1 to about 20 mutations compared to any one polypeptide from SEQ ID NOs: 12 to SEQ ID NOs: 73, and optionally comprises at least one, for example, all, of such mutational differences from SEQ ID NOs: 12 to SEQ ID NOs: 73.

[0046] In some embodiments, the variant capsid polypeptide comprises VP1, VP2, VP3, or any combination thereof, each having about 1 to about 10 mutations compared to any one polypeptide from SEQ ID NOs: 12 to SEQ ID NOs: 73, and optionally comprising at least one, for example, all, of such mutational differences from SEQ ID NOs: 12 to SEQ ID NOs: 73.

[0047] In some embodiments, the variant capsid polypeptide comprises VP1, VP2, VP3, or any combination thereof, each having 1 to 5 mutations compared to any one polypeptide from SEQ ID NOs. 12 to SEQ ID NOs. 73, and optionally comprises at least one, e.g., all, of such variant differences from SEQ ID NOs. 12 to SEQ ID NOs. 73.

[0048] In some embodiments, what is provided herein is a nucleic acid molecule encoding a variant capsid polypeptide provided herein. In some embodiments, the nucleic acid molecule comprises a sequence encoding one of the variant capsid polypeptides from SEQ ID NOs: 12 to 73 (e.g., VP1 capsid polypeptide, VP2 capsid polypeptide, or VP3 capsid polypeptide), or a fragment thereof. In some embodiments, the nucleic acid molecule comprises one of the SEQ ID NOs: 74 to 135, or a fragment thereof (e.g., a fragment encoding VP1, VP2, or VP3).

[0049] In some embodiments, the nucleic acid molecule 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 the variant capsid polypeptide provided herein.

[0050] In some embodiments, the nucleic acid molecule, or the nucleic acid molecule encoding a reference polypeptide for identity %, contains the nucleotide sequences of SEQ ID NOs. 74 to 135, respectively.

[0051] In some embodiments, the nucleic acid molecule, or the nucleic acid molecule encoding a reference polypeptide for identity purposes, includes a nucleotide sequence encoding, for example, any one of SEQ ID NOs: 12 to 73, which encodes a variant capsid polypeptide sequence, as described herein.

[0052] In some embodiments, the variant capsid polypeptide, or reference polypeptide for identity purposes, comprises one of the sequences from SEQ ID NOs: 12 to 73, each encoded by the nucleotide sequences of SEQ ID NOs: 74 to 135.

[0053] In some embodiments, the variant capsid polypeptide includes, for example, a sequence containing all the variant differences associated with any one of sequence numbers 12 to 73 relative to sequence number 1.

[0054] In some embodiments, the variant capsid polypeptide is the VP1 capsid polypeptide. In some embodiments, the variant capsid polypeptide is the VP2 capsid polypeptide. In some embodiments, the variant capsid polypeptide is the VP3 capsid polypeptide. With respect to SEQ ID NO: 1 of the reference sequence, the VP1 capsid polypeptide contains amino acids 1 to 736 of SEQ ID NO: 1. With respect to SEQ ID NO: 1 of the reference sequence, the VP2 capsid polypeptide contains amino acids 138 to 736 of SEQ ID NO: 1. With respect to SEQ ID NO: 1 of the reference sequence, the VP3 capsid polypeptide contains amino acids 203 to 736 of SEQ ID NO: 1.

[0055] With respect to the variant capsid polypeptide sequence, the VP1 capsid polypeptide contains all the amino acids of any one of the sequence numbers from SEQ ID NO: 12 to SEQ ID NO: 73. With respect to the variant capsid polypeptide of any one of the sequence numbers from SEQ ID NO: 12 to SEQ ID NO: 73, the VP2 capsid polypeptide contains, for example, a sequence that begins with a threonine corresponding to the threonine at position 138 of SEQ ID NO: 1 and continues to the C-terminus of any one of the sequence numbers from SEQ ID NO: 12 to SEQ ID NO: 73. With respect to the sequence number from any one of the sequence numbers from SEQ ID NO: 12 to SEQ ID NO: 73, the VP3 capsid polypeptide contains, for example, a sequence that begins with a methionine corresponding to the methionine at position 203 of SEQ ID NO: 1 and continues to the C-terminus of any one of the sequence numbers from SEQ ID NO: 12 to SEQ ID NO: 73.

[0056] Exemplary sequences of variant capsid polypeptides are provided in SEQ ID NOs: 12 to 73, and exemplary nucleic acid molecules encoding them are provided in SEQ ID NOs: 74 to 135, respectively.

[0057] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity with any one of the VP1, VP2, or VP3 sequences from SEQ ID NOs. 12 to 73.

[0058] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity (excluding targeted peptide insertions) with respect to any one of the VP1, VP2, or VP3 sequences from SEQ ID NOs. 12 to 73.

[0059] 5.2.1. Variant capsid polypeptide (corresponding position) Mutations to capsid polypeptide sequences described herein are described in relation to the amino acids at the positions in a reference sequence, e.g., SEQ ID NO: 1. Accordingly, in some embodiments, the capsid polypeptide described herein is a variant capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1, and includes, for example, a capsid polypeptide 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% identity with respect to a reference capsid polypeptide sequence (e.g., reference capsid polypeptide VP1, VP2, and / or VP3 sequence), e.g., SEQ ID NO: 1 (or the VP2 or VP3 sequence contained therein), and includes one or more mutations described herein.

[0060] Those skilled in the art will understand, though not theoretically bound, that each amino acid position in a reference sequence corresponds to a position in the sequence of another reference capsid polypeptide, such as a capsid polypeptide derived from a dependent parvovirus having a different serotype. Such corresponding positions are identified using sequence alignment tools known in the art. A particularly preferred sequence alignment tool is 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 / ). Exemplary reference capsid polypeptide alignments are shown in Figures 1A-1C. Thus, in some embodiments, the variant capsid polypeptide of the present invention comprises a variant of a reference capsid polypeptide containing one or more mutations described herein in such a reference capsid polypeptide at positions corresponding to the mutation positions described herein in relation to a different reference capsid polypeptide. Therefore, for example, with respect to SEQ ID NO: 1, a mutation described as XnnnY (where X is an amino acid located at position nnn in SEQ ID NO: 1, and Y is, for example, an amino acid mutation at that position as described herein), the present disclosure provides a variant capsid polypeptide having 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 with respect to a reference capsid polypeptide sequence other than SEQ ID NO: 1 (e.g., a reference capsid polypeptide VP1, VP2, and / or VP3 sequence) (or a VP2 or VP3 sequence contained therein), and further comprising a mutation disclosed at a position corresponding to position nnn in SEQ ID NO: 1 (e.g., Y at a position in a new variant capsid polypeptide sequence corresponding to position nnn in SEQ ID NO: 1).As described above, such corresponding positions are determined using sequence alignment tools, such as the Cluster Omega Tool mentioned above. Examples of corresponding amino acid positions of exemplary known AAV serotypes are provided in Figures 1A to 1C. In some embodiments, the variant is a variant of the AAV9 capsid polypeptide, which may be referred to as the "AAV9 variant capsid polypeptide" or "variant AAV9 capsid polypeptide".

[0061] Accordingly, in embodiments, the disclosure provides a variant capsid polypeptide sequence that is a reference sequence other than SEQ ID NO: 1, for example, a variant of a reference sequence other than SEQ ID NO: 1 described herein, which includes one or more mutations corresponding to the mutations described herein. In embodiments, such a variant includes mutations corresponding to all of the mutations associated with any one of SEQ ID NOs: 12 to 73.

[0062] The variant capsid polypeptides described herein are, optionally, variants of reference capsid serotypes known in the art. Non-limiting examples of such reference AAV serotypes include AAV1, AAVrh10, 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), and AAVPHP. .N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP .B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT , AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B- STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.eB, AAVPHP.S / G 2A12, AAVG2A15 / G2A3(G2A3), AAVG2B4(G2B4), AAVG2B5(G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9, AAV9 K449R (or K449R AAV9), AAV9.16, AAV9.24, AAV9.45, AABiodisV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-1 5、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4 AV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / rh.6、AAV3.1 / rh.61 hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / rh.7、AAV16.8 / hu.1 0、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu .16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAV hu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12. 3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu. hu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu. AVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43 、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu. u.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu. AVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh. AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37、AAVrh.37、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh. Vrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74(also called AAVrh74)、AAVrh8R、AAVrh8R. A586R mutation、AAVrh8R R533A variant、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36 Er2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-PAEC LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18 、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV 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, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, True type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4、AAV CBr-7.5、AAV CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAV CBr-B7.4、AAV CBr-E1、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-E6、AAV CBr-E6 CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAV CHt-P6、AAV CHt-P8、AAV CHt-P9、AAV CKd-1、AAV CKd-10、AAV CKd-2、AAV CKd-3、AAV CKd-4、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV CLv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D. 5. AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9, 7m8, Spark100, AAVMYO, and their variants are included.

[0063] In some embodiments, the reference AAV capsid sequence includes the AAV2 sequence. In some embodiments, the reference AAV capsid sequence includes the AAV5 sequence. In some embodiments, the reference AAV capsid sequence includes the AAV8 sequence. In some embodiments, the reference AAV capsid sequence includes the AAV9 sequence. In some embodiments, the reference AAV capsid sequence includes the AAVrh74 sequence. While we do not wish to be constrained by theory, it is understood that the reference AAV capsid sequence includes the VP1 region. In certain embodiments, the reference AAV capsid sequence includes the VP1 region, the VP2 region, and / or the VP3 region, or any combination thereof. The reference VP1 sequence may be considered synonymous with the reference AAV capsid sequence.

[0064] The wild-type reference sequence for AAV9, sequence number 1 is as follows: [ka]

[0065] Unless otherwise specified, Sequence ID No. 1 is the reference sequence. In the sequences above, sequences found in VP1, VP2, and VP3 are underlined (for example, the VP3 capsid polypeptide contains, for example, amino acids corresponding to amino acids 203-736 of Sequence ID No. 1), sequences found in both VP1 and VP2 are bolded (for example, the VP2 capsid polypeptide contains, for example, amino acids corresponding to amino acids 138-736 of Sequence ID No. 1), and sequences that are not underlined or bolded are found only in VP1 (for example, the VP1 capsid polypeptide contains, for example, amino acids corresponding to amino acids 1-736 of Sequence ID No. 1).

[0066] The wild-type reference sequence of SEQ ID NO: 1 can be encoded by the reference nucleic acid molecular sequence of SEQ ID NO: 2.

[0067] The exemplary reference sequence for wild-type AAV2, sequence number 3 (wild-type AAV2), is as follows: [ka]

[0068] In the sequence above, sequences found in VP1, VP2, and VP3 are underlined (for example, the VP3 capsid polypeptide contains amino acids corresponding to amino acids 203-735 of SEQ ID NO: 3, e.g., consisting of these), sequences found in both VP1 and VP2 are in bold (for example, the VP2 capsid polypeptide contains sequences corresponding to amino acids 138-735 of SEQ ID NO: 3, e.g., consisting of these), and sequences that are not underlined or in bold are found only in VP1 (for example, the VP1 capsid polypeptide contains amino acids corresponding to amino acids 1-735 of SEQ ID NO: 3, e.g., consisting of these).

[0069] The exemplary nucleic acid sequence encoding SEQ ID NO: 3 is SEQ ID NO: 4.

[0070] The exemplary reference sequence for wild-type AAV5, sequence number 5 (wild-type AAV5), is as follows: [ka]

[0071] In the sequence above, sequences found in VP1, VP2, and VP3 are underlined (for example, the VP3 capsid polypeptide contains amino acids corresponding to amino acids 193-724 of SEQ ID NO: 5, e.g., consisting of these), sequences found in both VP1 and VP2 are in bold (for example, the VP2 capsid polypeptide contains sequences corresponding to amino acids 137-724 of SEQ ID NO: 5, e.g., consisting of these), and sequences that are not underlined or in bold are found only in VP1 (for example, the VP1 capsid polypeptide contains amino acids corresponding to amino acids 1-724 of SEQ ID NO: 5, e.g., consisting of these).

[0072] An exemplary nucleic acid sequence encoding SEQ ID NO: 5 is SEQ ID NO: 6.

[0073] The exemplary reference sequence for wild-type AAV8, sequence number 7 (wild-type AAV8), is as follows: [ka]

[0074] In the sequence above, sequences found in VP1, VP2, and VP3 are underlined (for example, the VP3 capsid polypeptide contains amino acids corresponding to amino acids 204-738 of SEQ ID NO: 7, e.g., consisting of these), sequences found in both VP1 and VP2 are in bold (for example, the VP2 capsid polypeptide contains sequences corresponding to amino acids 138-738 of SEQ ID NO: 7, e.g., consisting of these), and sequences that are not underlined or in bold are found only in VP1 (for example, the VP1 capsid polypeptide contains amino acids corresponding to amino acids 1-738 of SEQ ID NO: 7, e.g., consisting of these).

[0075] An exemplary nucleic acid sequence encoding sequence number 7 is sequence number 8.

[0076] The exemplary reference sequence for wild-type AAVrh74, sequence number 9 (wild-type AAVrh74), is as follows: [ka]

[0077] An exemplary alternative reference sequence (alternative wild-type AAVrh74) for sequence number 11 is as follows: [ka]

[0078] In the above sequence (SEQ ID NO: 9 or SEQ ID NO: 11), sequences found in VP1, VP2, and VP3 are underlined (for example, the VP3 capsid polypeptide contains, for example, amino acids corresponding to amino acids 204-738 of SEQ ID NO: 9), sequences found in both VP1 and VP2 are in bold (for example, the VP2 capsid polypeptide contains, for example, amino acids corresponding to amino acids 137-738 of SEQ ID NO: 9), and sequences that are not underlined or in bold are found only in VP1 (for example, the VP1 capsid polypeptide contains, for example, amino acids corresponding to amino acids 1-738 of SEQ ID NO: 9).

[0079] An exemplary nucleic acid sequence encoding sequence number 9 is sequence number 10.

[0080] This disclosure refers to structural capsid proteins (including VP1, VP2, and VP3) encoded by the capsid (Cap) gene. These capsid proteins form the outer protein structural shell (i.e., capsid) of viral vectors such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally contain methionine (Met1) as the first amino acid in the peptide sequence, which associates with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for the first methionine (Met1) residue, or generally any first amino acid (AA1), to be cleaved after or during polypeptide synthesis by a protein-processing enzyme such as Met-aminopeptidase. This "Met / AA-clipping" process often correlates with the corresponding acetylation of a second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping generally occurs in VP1 and VP3 capsid proteins, but can also occur in VP2 capsid proteins. If Met / AA-clipping is incomplete, it can produce a mixture of one or more (one, two, or three) VP capsid proteins containing the viral capsid, some of which contain the Met1 / AA1 amino acid (Met+ / AA+), and some of which lack the Met1 / AA1 amino acid (Met- / AA-) as a result of Met / AA-clipping.For further discussion on Met / AA clipping in capsid proteins, please refer to Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 Oct. 28(5):255-267 and Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19. 327(5968):973-977, the contents of which are incorporated herein by reference in their entirety. According to this disclosure, references to capsid polypeptides are not limited to either clipped (Met- / AA-) or unclipped (Met+ / AA+), but also, in context, refer to viral capsids consisting of independent capsid polypeptides, mixtures of capsid proteins, and / or polynucleotide sequences (or fragments thereof) that encode, describe, produce, or result in the capsid polypeptides of this disclosure. Direct references to “capsid polypeptides” (such as VP1, VP2, or VP3) also include VP capsid proteins, including the corresponding VP capsid polypeptides with the Met1 / AA1 amino acid (Met+ / AA+) and those lacking the Met1 / AA1 amino acid (Met- / AA-) as a result of Met / AA- clipping. Furthermore, according to this disclosure, any reference to a specific sequence number (whether a protein or nucleic acid) containing or encoding one or more (Met+ / AA+) capsid polypeptides containing the Met1 / AA1 amino acid should be understood to inform VP capsid polypeptides lacking the Met1 / AA1 amino acid during sequence review, and any sequence simply lacking the first listed amino acid (whether or not it is Met1 / AA1) is readily apparent.As a non-restrictive example, a reference to a (Met+)VP1 polypeptide sequence that is 736 amino acids long and contains the "Met1" amino acid encoded by an AUG / ATG start codon is also understood to teach a (Met-)VP1 polypeptide sequence that is 735 amino acids long and does not contain the "Met1" amino acid of the 736-amino acid Met+ sequence. As a second non-restrictive example, a reference to a (AA1+)VP1 polypeptide sequence that is 736 amino acids long and contains the "AA1" amino acid encoded by an arbitrary NNN start codon is also understood to teach a (AA1-)VP1 polypeptide sequence that is 735 amino acids long and does not contain the "AA1" amino acid of the 736-amino acid AA1+ sequence. References to viral capsids formed from VP capsid proteins (e.g., references to specific AAV capsid serotypes) may include VP capsid proteins containing the Met1 / AA1 amino acid (Met+ / AA1+), corresponding VP capsid proteins lacking the Met1 / AA1 amino acid as a result of Met / AA1- clipping (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-). As a non-limiting example, AAV capsid serotypes may include VP1(Met+ / AA1+), VP1(Met- / AA1-), or combinations of VP1(Met+ / AA1+) and VP1(Met- / AA1-). AAV capsid serotypes can also include VP3(Met+ / AA1+), VP3(Met- / AA1-), or combinations of VP3(Met+ / AA1+) and VP3(Met- / AA1-), and similarly, optional combinations of VP2(Met+ / AA1) and VP2(Met- / AA1-).

[0081] In some embodiments, the reference AAV capsid sequence includes an amino acid sequence having 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 above.

[0082] In some embodiments, the reference AAV capsid sequence is encoded by a nucleotide sequence having 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 above. In certain embodiments, the reference sequence is not the AAV capsid sequence, but rather a different vector (e.g., a lentivirus, plasmid, etc.).

[0083] In some embodiments, the nucleic acids of the Disclosure (e.g., encoding an AAV9 variant capsid protein) include conventional regulatory elements or sequences operably ligated to the nucleic acid molecule in such a manner that they enable transcription, translation, and / or expression in cells transfected with the nucleic acid (e.g., a plasmid vector containing the nucleic acid) or infected with a virus containing the nucleic acid. As used herein, “operably ligated” sequences include both expression regulatory sequences adjacent to the gene of interest and expression regulatory sequences acting trans or asynchronously to control the gene of interest.

[0084] Expression regulatory sequences include efficient RNA processing signals such as splicing and polyadenylation (poly-A) signals; appropriate transcription start, termination, promoter, and enhancer sequences; sequences that stabilize cytoplasmic mRNA; sequences that enhance protein stability; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); and, in some embodiments, sequences that enhance the secretion of encoded transgene products. Expression regulatory sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be used in conjunction with the compositions and methods disclosed herein.

[0085] In some embodiments, a native promoter is used for the transgene. While not intended to be constrained by theory, a native promoter may mimic the native expression of the transgene or provide temporal, developmental, or tissue-specific expression, or expression in response to specific transcriptional stimuli. In some embodiments, the transgene is operably ligated to other native expression regulatory elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, for example, to mimic native expression.

[0086] In some embodiments, the transgene is operably linked to a tissue-specific promoter, such as a promoter specifically active in one or more CNS cell types. In some embodiments, the transgene is operably linked to a promoter active in skeletal muscle. A promoter active in skeletal muscle may be specific to skeletal muscle or may be more broadly expressed in other muscle types.

[0087] In some embodiments, the vector carrying the transgene, e.g., a plasmid, includes a selectable marker or reporter gene. Such a selectable reporter or marker gene can be used to signal the presence of the vector, e.g., plasmid, in bacterial cells. Other components of the vector, e.g., a plasmid, include an origin of replication. The selection of these and other promoters and vector elements has been conventional, and many such sequences are available (see, e.g., Sambrook et al. and the references cited therein).

[0088] In some embodiments, viral particles containing a variant capsid polypeptide, such as the variant capsid polypeptide described herein, exhibit increased CNS transduction compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1).

[0089] In some embodiments, viral particles containing a variant capsid polypeptide, such as the variant capsid polypeptide described herein, exhibit increased skeletal muscle transduction compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1).

[0090] In some embodiments, the capsid polypeptide is an isolated or purified polypeptide (e.g., isolated or purified from cells, other biological components, or contaminants). In some embodiments, the variant polypeptide is present within dependent parvovirus particles, for example, as described herein. In some embodiments, the variant capsid polypeptide is present within cells, cell-free systems, or translation systems, for example, as described herein.

[0091] In some embodiments, the capsid polypeptide is present in dependent parvovirus B (e.g., AAV9) particles. In some embodiments, the capsid particles have increased CNS transduction. In some embodiments, the capsid particles have increased skeletal muscle transduction.

[0092] In some embodiments, dependent parvovirus particles include an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with any one of the amino acid sequences provided herein (e.g., any one of SEQ ID NOs. 12 to 73). In some embodiments, a variant capsid polypeptide includes an amino acid sequence that differs from the amino acid sequence of the variant capsid polypeptide provided herein by 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 one or fewer amino acids.

[0093] In some embodiments, additional modifications improve the production characteristics of dependent parvovirus particles or the method of producing them. In some embodiments, additional modifications improve or modify another characteristic of the dependent parvovirus particles, such as tropism.

[0094] 5.2.1. Targeted Peptides The capsid polypeptides of this disclosure may include (but may not necessarily include) targeted peptides to modify the tropism of the capsid polypeptide, for example, to enhance targeting to the CNS or skeletal muscle. Therefore, in some embodiments, the capsid polypeptides of this disclosure include targeted peptides. In other embodiments, the capsid polypeptides of this disclosure do not include targeted peptides.

[0095] 5.2.1.1. CNS-Targeted Peptides Various targeted peptides for enhancing CNS tropism that may be included in the capsid polypeptide of this disclosure are, for example, WO2017 / 197355, WO2019 / 006182, WO2019 / 060454, WO2012 / 145601, WO2018 / 022905, WO2021 / 243085, WO2019 / 076856, WO2015 / 038958, WO2015 / 191508, WO2020 / 068990, WO2020 / 210655, WO2020 / 198737, WO2020 / 028751, WO2019 / 028306, WO2017 / 100671 A1, WO2020 / 028751 A2, WO2020 / 072683 A1, WO2020 / 160337 A1, WO2020 / 223280 A1, WO2021 / 025995 A1, WO2021 / 202651 A1, WO2021 / 230987 A1, WO2022 / 235702 A1, WO2020 / 014471, WO2018 / 189244, WO2019 / 141765, WO2019 / 207132, WO2019 / 210267, WO2018 / 156654, WO2010 / 093784, WO2015 / 048534, WO2017 / 058892, WO2019 / 169132, WO20 21 / 108468, WO2021 / 102234, WO2022 / 173847, WO2021 / 077000, WO2020 / 160337, WO2021 / 050974, WO2021 / 222831, WO2022 / 020616, WO2020 / 193799, WO2021 / 072197, WO2022 / 1261 88, WO2022 / 126189, WO2021 / 165544, WO2021 / 084133, WO2022 / 040527, WO2022 / 221400, WO2022 / 221404, WO2022 / 221420, WO2021 / 216456, WO2021 / 009684, WO2021 / 242909, WO20 The contents of 19 / 158619, WO2021 / 226267, WO2023 / 283962, WO2021 / 219762, WO2022 / 226374, WO2022 / 226375, WO2022 / 229703, and WO2022 / 229702 are described herein, and their contents are incorporated herein by reference in their entirety.The targeted peptide is typically 3 to 20 amino acids long. In some embodiments, the targeted peptide is 3 to 12 amino acids long. In other embodiments, the targeted peptide is 5 to 12 amino acids long. In other embodiments, the targeted peptide is 5 to 10 amino acids long. In other embodiments, the targeted peptide is 7 to 10 amino acids long. In some embodiments, the targeted peptide is 7 amino acids long. In other embodiments, the targeted peptide is 9 amino acids long.

[0096] In some embodiments, the targeted peptide contains at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence PLNGAVHLY (SEQ ID NO: 255). In some embodiments, the targeted peptide contains the amino acid sequence PLNGAVHLY (SEQ ID NO: 255). In some embodiments, the targeted peptide contains at least 3, 4, 5, 6, or 7 consecutive amino acids from the amino acid sequence IVMNSLK (SEQ ID NO: 256). In some embodiments, the targeted peptide contains the amino acid sequence IVMNSLK (SEQ ID NO: 256). In some embodiments, the targeted peptide contains at least 3, 4, 5, 6, or 7 consecutive amino acids from the amino acid sequence RDSPKGW (SEQ ID NO: 257). In some embodiments, the targeted peptide contains the amino acid sequence RDSPKGW (SEQ ID NO: 257). In some embodiments, the targeted peptide contains at least 3, 4, 5, 6, or 7 consecutive amino acids from the amino acid sequence YSTDVRM (SEQ ID NO: 258). In some embodiments, the targeted peptide includes the amino acid sequence YSTDVRM (SEQ ID NO: 258). In some embodiments, the targeted peptide includes at least 3, 4, 5, 6, or 7 consecutive amino acids from the amino acid sequence RESPRGL (SEQ ID NO: 259). In some embodiments, the targeted peptide includes the amino acid sequence RESPRGL (SEQ ID NO: 259). In some embodiments, the targeted peptide includes 4, 5, 6, or 7 consecutive amino acids from GNNTRSV (SEQ ID NO: 260), GNNRDT (SEQ ID NO: 261), or TNSTRPV (SEQ ID NO: 262). In some embodiments, the targeted peptide includes the amino acid sequence GNNTRSV (SEQ ID NO: 260). In some embodiments, the targeted peptide includes the amino acid sequence GNNRDT (SEQ ID NO: 261). In some embodiments, the targeted peptide includes the amino acid sequence TNSTRPV (SEQ ID NO: 262).

[0097] In some embodiments, the CNS-targeting peptide is, for example, located in loop VIII of the capsid polypeptide, for example, and is inserted. In some embodiments, the targeting peptide is comprehensively inserted at any amino acid position corresponding to positions 586-592 of the wild-type capsid polypeptide (SEQ ID NO: 1). For example, the targeting peptide can be inserted between amino acids 588-589 (positions corresponding to the wild-type capsid polypeptide (SEQ ID NO: 1)). In some embodiments, the targeting peptide is, for example, located immediately after positions corresponding to 586, 588, or 589 of the wild-type capsid polypeptide (SEQ ID NO: 1), and is inserted. In some embodiments, the capsid polypeptide further includes a deletion at the position corresponding to 587 and / or 588 of the wild-type capsid polypeptide (SEQ ID NO: 1).

[0098] 5.2.1.2. Muscle-Targeting Peptides Various targeted peptides for enhancing skeletal muscle tropism that may be included in the capsid polypeptide of this disclosure are described in the art, for example, WO2020 / 206189A1, WO2022 / 226374A1, WO2022 / 053630A1, and WO2019 / 207132A1.

[0099] In some embodiments, the muscle-targeting peptide contains at least 3, 4, 5, 6, or 7 consecutive amino acids from the amino acid sequence ASSLNIA (SEQ ID NO: 263).

[0100] In some embodiments, the targeted peptide targets the insulin receptor (INSR). In some embodiments, the INSR-targeting peptide has at least 80%, 87%, 91%, 94%, 97%, or 100% sequence identity with SLEEEWAQVECEVYGRGCPSGSLDESFYDWFERQL (SEQ ID NO: 264), or includes an amino acid sequence having at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 consecutive amino acids from the amino acid sequence SLEEEWAQVECEVYGRGCPSGSLDESFYDWFERQL (SEQ ID NO: 264).

[0101] In some embodiments, the targeted peptide targets muscle-specific kinase (MUSK). In some embodiments, the inserted MUSK-targeted peptide is derived from acetylcholinesterase collagen tail peptide (ColQ), for example, the C-terminal portion of ColQ (ColQ CTD). In some embodiments, the ColQ CTD peptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to TPFYPVGYTVKQPGTCGDGVLQPGEECDDGNPDVSDGCIDCHRAYCGDGYRHQGVEDCDGSDFGYLTCETYLPGSYGDLRCTQYCSIDSTPCRYFT (SEQ ID NO: 265), or contains an amino acid sequence having at least 70, 80, 85, 90, 91, 92, 93, 94, 95, or 96 consecutive amino acids from the amino acid sequence TPFYPVGYTVKQPGTCGDGVLQPGEECDDGNPDVSDGCIDCHRAYCGDGYRHQGVEDCDGSDFGYLTCETYLPGSYGDLRCTQYCSIDSTPCRYFT (SEQ ID NO: 265).

[0102] In some embodiments, the targeted peptide targets integrins, for example, via the RGD motif. In some embodiments, the RGD peptide contains a subsequence Y or F amino acid to produce the RGDY (SEQ ID NO: 266) or RGDF (SEQ ID NO: 267) motif. In some embodiments, the integrin-targeted peptide has the motif RGDX1X2X3X4, where X1-X4 are each any amino acid. In various embodiments of the motif RGDX1X2X3X4, X1, X2, and X3 are each independently selected from L, G, V, and A, and / or X4 is S, V, A, G, or L. In some embodiments, at least one of X2 and X3 is G. In some embodiments, the targeted peptide contains the amino acid sequence RGDLGLS (SEQ ID NO: 269). In some embodiments, the targeted peptide contains the amino acid sequence RGDLSTP (SEQ ID NO: 270). In some embodiments, the targeted peptide contains the amino acid sequence SNSRGDYNSL (SEQ ID NO: 271). In some embodiments, the targeted peptide comprises the amino acid sequence ENRRGDFNNT (SEQ ID NO: 272). In some embodiments, the targeted peptide comprises the amino acid sequence SRGDYNSL (SEQ ID NO: 273). In some embodiments, the targeted peptide comprises the amino acid sequence RGDYNSL (SEQ ID NO: 274). In some embodiments, the targeted peptide comprises the amino acid sequence RGDLST (SEQ ID NO: 275). In some embodiments, the targeted peptide comprises the amino acid sequence RGDYVGL (SEQ ID NO: 276). In some embodiments, the targeted peptide comprises the amino acid sequence RGDAVGV (SEQ ID NO: 277). The RGD peptide can be inserted into a linker, such as a flexible linker like GGGS (SEQ ID NO: 278) or scaffold. Other suitable linker scaffolds can be found in WO2022 / 226374A1, sections 26-28 of which are incorporated herein by reference.

[0103] In some embodiments, the skeletal muscle targeting peptide is, for example, located in loop VIII of the capsid polypeptide, for example, and is inserted. In some embodiments, the targeting peptide is comprehensively inserted at any amino acid position corresponding to positions 586-592 of the wild-type capsid polypeptide (SEQ ID NO: 1). For example, the targeting peptide can be inserted between amino acids 588-589 (positions corresponding to the wild-type capsid polypeptide (SEQ ID NO: 1)). In some embodiments, the targeting peptide is, for example, located immediately after positions corresponding to 586, 588, or 589 of the wild-type capsid polypeptide (SEQ ID NO: 1), and is inserted. In some embodiments, the capsid polypeptide further includes a deletion at the position corresponding to 587 and / or 588 of the wild-type capsid polypeptide (SEQ ID NO: 1).

[0104] 5.2.2. Nucleic acids and polypeptides This disclosure further covers nucleic acids, in part, that include sequences encoding variant capsid polypeptides provided herein. In embodiments, the nucleic acid encodes, for example, a VP1 variant capsid polypeptide, as described herein. In embodiments, the nucleic acid encodes, for example, a VP2 variant capsid polypeptide, as described herein. In embodiments, the nucleic acid encodes, for example, a VP3 variant capsid polypeptide, as described herein. In embodiments, the nucleic acid encodes, for example, VP1, VP2, and VP3 variant capsid polypeptides, as described herein. In some embodiments, the variant capsid polypeptide includes any one sequence from SEQ ID NOs. 12 to SEQ ID NOs. 73. In some embodiments, the nucleic acid includes, respectively, SEQ ID NOs. 74 to SEQ ID NOs. 135.

[0105] 5.3. Depend Parvovirus Particles This disclosure also, in part, covers dependent parvovirus particles (e.g., functional dependent parvovirus particles) comprising nucleic acids or polypeptides, which are produced by or in the manner described herein.

[0106] Depend parvoviruses are single-stranded DNA parvoviruses that grow only inside cells, with specific functions provided, for example, by co-infecting helper viruses. Several species of dependent parvoviruses are known, including dependent parvovirus A and dependent parvovirus B, which include serotypes known in the art as adeno-associated viruses (AAVs). At least 13 AAV serotypes have been characterized. General information and an overview of AAVs can be found, for example, in Carter, Handbook of Parvoviruses, Vol. 1, pp. 169-228 (1989), and Berns, Virology, pp. 1743-1764, Raven Press, (New York, 1990). AAV serotypes, and to some extent dependent parvovirus species, are structurally and functionally remarkably interrelated. (See, for example, Blacklowe, pp. 165-174, Parvoviruses and Human Disease, JRPattison, ed. (1988), and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, and all possess three related capsid proteins. In addition, heteroduplex analysis reveals extensive cross-hybridization between serotypes along genome length, further suggesting interrelationships. Depend parvovirus genomes also contain self-annealing segments at the ends corresponding to “inverted terminal repeat sequences” (ITRs).

[0107] The genomes of naturally occurring dependent parvoviruses, such as AAV serotypes, are remarkably similar. For example, the AAV genome is a linear single-stranded DNA molecule of approximately 5,000 nucleotides (nt) or less in length. Inverted terminal repeats (ITRs) are adjacent to coding nucleotide sequences specific to the unstructured replication (Rep) protein and the structural capsid (Cap) protein. Three distinct viral particle (VP) proteins form the capsid. Approximately 145 nt at the ends of the genome are self-complementary and organized to form energetically stable intramolecular double helixes that form a T-shaped hairpin. These hairpin structures function as the origin of viral DNA replication and act as primers for the cellular DNA polymerase complex. The Rep gene codes for Rep proteins such as Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. The cap gene encodes the VP proteins VP1, VP2, and VP3. The cap gene is transcribed from the p40 promoter.

[0108] In some embodiments, the dependent parvovirus particles of this disclosure comprise nucleic acids comprising variant capsid polypeptides provided herein. In some embodiments, the particles comprise polypeptides provided herein.

[0109] In some embodiments, the dependent parvovirus particles of the present disclosure are AAV9 particles. In some embodiments, the AAV9 particles include a variant capsid polypeptide provided herein, or a nucleic acid molecule encoding it.

[0110] In some embodiments, the dependent parvovirus particle comprises a variant capsid containing a variant capsid polypeptide as described herein. In embodiments, the dependent parvovirus particle comprises a variant capsid polypeptide as described herein and a nucleic acid molecule. In embodiments, the dependent parvovirus particle comprises a variant capsid polypeptide as described herein and a nucleic acid molecule comprising one or more inverted terminal repeat sequences (ITRs), e.g., ITRs derived from AAV9 dependent parvovirus or AAV2 dependent parvovirus, one or more regulatory elements (e.g., promoters), and a payload (e.g., xenotransgenes, as described herein). In embodiments, at least one of the ITRs is modified. In embodiments, the nucleic acid molecule is single-stranded. In embodiments, the nucleic acid molecule is double-stranded, e.g., self-complementary.

[0111] 5.4. Improved in vivo distribution and transduction characteristics This disclosure, in part, relates to nucleic acids, polypeptides, cells, cell-free systems, translation systems, viral particles, and methods relating to using and producing them for producing viral particles having increased distribution into CNS tissues and cells and / or CNS transduction compared to viral particles containing, for example, the capsid polypeptide sequence of SEQ ID NO: 1, compared to viral particles containing a reference sequence that does not contain the mutations described herein (or the corresponding mutations). In some embodiments, the use of viral particles containing the variant capsid polypeptide described in Section 5.2 or any one of the numbered embodiments 1 to 531 (e.g., viral particles described in any one of Sections 5.3, 5.4, or the numbered embodiments 552 to 736) results in increased CNS in vivo distribution of viral particles in CNS cells and / or increased transgene transduction of transgene viral particles, and therefore increased expression of the payload (transgene) in the CNS or brain. In some embodiments, the use of viral particles containing the variant capsid polypeptide described in Section 5.2 or any one of the numbered embodiments 1 to 531 (e.g., viral particles described in Sections 5.3 and 5.4, or any one of the numbered embodiments 552 to 736) results in a reduced (or no increased) in vivo distribution of viral particles and / or reduced (or no increased) transduction of the transgene in one or more peripheral tissues, e.g., the liver, spleen, dorsal root ganglia, or any combination of two or more of the aforementioned peripheral tissue types. In some embodiments, the use of viral particles containing the variant capsid polypeptide described in Section 5.2 or any one of the numbered embodiments 1 to 531 (e.g., viral particles described in Sections 5.3 and 5.4, or any one of the numbered embodiments 552 to 736) results in an increased in vivo distribution of viral particles in skeletal muscle cells and / or increased transgene transduction of viral particles, and therefore further results in increased expression of the payload (transgene) in skeletal muscle.

[0112] In some embodiments, in vivo distribution and transduction (e.g., of the tissue types described in this section) are measured as described herein, for example, as described in Section 7 (e.g., by relative quantification (e.g., via qPCR) of transgene mRNA in one or more samples isolated from the relevant tissue type, e.g., CNS or skeletal muscle, e.g., any of Examples 2, 4, and 5). In some embodiments, the in vivo distribution and / or transduction of viral particles having a variant capsid polypeptide can be measured using viral particles having a transgene operably linked to a promoter that is active in the target cell or tissue type of interest. In some embodiments, the promoter is a ubiquitous promoter. In other embodiments, the promoter is selective or specific to the target cell or tissue type (e.g., CNS and / or muscle (either broad-spectrum muscle expression or skeletal muscle)) and, optionally, less active (or inactive) in cells or tissue types (e.g., liver, spleen, PNS, or any combination of two or all of the aforementioned) where transgene expression is not desired. A promoter that is selective to a first cell or tissue type more than 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. In some embodiments, the promoter is CNS-specific or CNS-selective. In some embodiments, the promoter is muscle-specific, muscle-selective. In some embodiments, the promoter is active in both CNS and muscle tissue. In some embodiments, the in vivo distribution and / or transduction of viral particles having variant capsid polypeptides can be measured using viral particles having transgenes operably linked to a ubiquitous promoter or a CNS-specific promoter. For example, in vivo distribution can be measured using viral particles having transgenes operably linked to a CBh promoter or an hSYN promoter.In various embodiments, the transgene is a transgene encoding a capsid polypeptide, or any other suitable heterologous transgene, such as a synthetic protein, mammalian protein, or human therapeutic protein, or a nucleic acid sequence encoding nucleic acid (e.g., mRNA or RNAi), or a reporter gene such as a nucleic acid encoding GFP or an mCherrry reporter.

[0113] In embodiments, for example, viral particles containing a variant capsid polypeptide as described herein can cross the blood-brain barrier. In embodiments, for example, viral particles containing a variant capsid polypeptide as described herein show increased passage across the blood-brain barrier compared to viral particles containing a reference capsid polypeptide, for example, the reference capsid polypeptide of SEQ ID NO: 1. In embodiments, for example, viral particles containing a variant capsid polypeptide as described herein show increased transduction of neurons, astrocytes, glial cells, or combinations thereof compared to viral particles containing a reference capsid polypeptide, for example, the reference capsid polypeptide of SEQ ID NO: 1.

[0114] In some embodiments, viral particles containing a variant capsid polypeptide, such as the variant capsid polypeptide described herein, exhibit improved properties, such as improved in vivo distribution, transduction, and / or production. Unless otherwise indicated, the improvement rate is presented as a multiplier of improvement relative to the rate shown by viral particles containing the capsid polypeptide of SEQ ID NO: 1. In some embodiments, improvement means, for example, an increase in CNS in vivo distribution or CNS transduction. In other embodiments, improvement means, for example, a decrease in liver in vivo distribution or liver transduction. Viral particles having increased in vivo distribution or transduction in target cells or target tissue types, such as CNS and / or skeletal muscle, and / or decreased in vivo distribution or transduction in off-target cells or off-target tissue types, such as PNS, liver, and / or spleen, may have improved specificity to target cells or target tissue types. This improvement may be beneficial, for example, in the use of viral particles to deliver therapeutic transgenes to target cells or target tissue types in subjects suffering from diseases affecting target cells or target tissue types.

[0115] In some embodiments, one or more improved properties (e.g., increased or decreased biodistribution and / or transduction) are demonstrated in mammals, e.g., primates, e.g., humans. In embodiments, increased or decreased biodistribution and / or transduction are demonstrated when viral particles or a pharmaceutical composition containing viral particles are administered systemically, e.g., intravenously, as described herein.

[0116] In some embodiments, viral particles containing variant capsid polypeptides exhibit improvements in one or more classifications as defined below.

[0117] In some embodiments, the viral particles exhibit improvements in classification A (CNS in vivo distribution) and classification B (CNS transduction). These improvements may be beneficial in the use of viral particles to deliver therapeutic transgenes to the CNS in subjects suffering from diseases affecting the CNS, for example. In some embodiments, the viral particles exhibit improvements in classification A (CNS in vivo distribution) and classification B (CNS transduction), as well as one or more of classifications C (PNS in vivo distribution and / or transduction), D (liver in vivo distribution and / or transduction), and E (spleen in vivo distribution and / or transduction). In some embodiments, the viral particles exhibit improvements in classification A (CNS in vivo distribution), classification B (CNS transduction), and classification D (liver in vivo distribution and / or transduction), and optionally exhibit improvements in classification C (PNS in vivo distribution and / or transduction) and / or E (spleen in vivo distribution and / or transduction). Selectively, viral particles may also show improvement in classification F (in vivo distribution in skeletal muscle) and / or classification G (skeletal muscle transduction), for example, to deliver therapeutic transgenes to both the CNS and muscle in subjects suffering from diseases affecting both CNS and skeletal muscle tissue (e.g., SMA, multiple sclerosis, amyotrophic lateral sclerosis (ALS), ataxia, Becker muscular dystrophy, Charcot-Marie-Tooth disease, dystonia, Friedreich ataxia, glycogen storage disorder II, Kennedy disease, Lambert-Eaton myasthenic syndrome, mitochondrial DNA depletion syndrome, myo-ophthalm-brain disease, neurogenic myotonia, periodic paralysis, juvenile primary lateral sclerosis, progressive extraocular palsy, spastic paraplegia, congenital Stiff-Person syndrome, tardive dyskinesia, Werdnig-Hoffman disease, or X-linked spinal and medullary muscular atrophy).

[0118] In further embodiments, viral particles containing variant capsid polypeptides exhibit improvements in classification F (in vivo distribution in skeletal muscle) and / or classification G (transduction in skeletal muscle). In some embodiments, viral particles exhibit improvements in both classification F (in vivo distribution in skeletal muscle) and classification G (transduction in skeletal muscle). These improvements may be beneficial, for example, in the use of viral particles to deliver therapeutic transgenes to skeletal muscle tissue in subjects suffering from diseases affecting skeletal muscle. In some embodiments, viral particles exhibit improvements in classification F (in vivo distribution in skeletal muscle) and / or classification G (transduction in skeletal muscle) along with one or more of classifications C (in vivo distribution in PNS and / or transduction), D (in vivo distribution in the liver and / or transduction), and E (in vivo distribution in the spleen and / or transduction). In some embodiments, the viral particles exhibit improvements in classification F (in vivo distribution in skeletal muscle) and / or classification G (transduction in skeletal muscle), as well as improvements in classification D (in vivo distribution in the liver and / or transduction), and optionally further exhibit improvements in classification C (in vivo distribution in the PNS and / or transduction) and / or E (in vivo distribution in the spleen and / or transduction).

[0119] Classification A (CNS Biodistribution): In some embodiments of this disclosure, viral particles containing a variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibit increased CNS biodistribution compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, increased CNS biodistribution includes increased brain biodistribution and / or spinal cord biodistribution. In some embodiments, increased brain biodistribution is aggregated brain biodistribution, or biodistribution in a specific brain tissue such as the brainstem, basal ganglia, cerebellum, forebrain, hippocampus, midbrain, or temporal cortex, and in each case is measured as an aggregate of a CNS-specific promoter such as hSyn, a constitutive promoter such as Cbh, or a CNS-specific promoter such as hSyn and a constitutive promoter such as Cbh). In some embodiments, increased biodistribution is aggregated brain biodistribution increased using the Cbh or hSyn promoter and / or as defined by any one of A-1 to A-30.

[0120] Embodiment A-1: ​​In one embodiment of Classification A, the in vivo distribution is about (or at least about) 5 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0121] Embodiment A-2: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 10 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0122] Embodiment A-3: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 15 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0123] Embodiment A-4: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 20 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0124] Embodiment A-5: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 25 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0125] Embodiment A-6: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 30 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0126] Embodiment A-7: In one embodiment of Classification A, the in vivo distribution is approximately (or at least approximately) 35 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0127] Embodiment A-8: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 40 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0128] Embodiment A-9: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 50 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0129] Embodiment A-10: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 60 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0130] Embodiment A-11: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 70 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0131] Embodiment A-12: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 80 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0132] Embodiment A-13: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 90 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0133] Embodiment A-14: In one embodiment of Classification A, the in vivo distribution is about (or at least about) 100 times better than that of viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0134] In some embodiments, the improved in vivo distribution lies within a range defined by any two values ​​shown in embodiments A-1 to A-14. Exemplary ranges are shown in embodiments A-15 to A-30 below.

[0135] Embodiment A-15: In one embodiment of Classification A, the in vivo distribution is approximately 5 to 100 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0136] Embodiment A-16: In one embodiment of Classification A, the in vivo distribution is approximately 5 to 80 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0137] Embodiment A-17: In one embodiment of Classification A, the in vivo distribution is approximately 5 to 90 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0138] Embodiment A-18: In one embodiment of Classification A, the in vivo distribution is approximately 5 to 60 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0139] Embodiment A-19: In one embodiment of Classification A, the in vivo distribution is approximately 10 to 100 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0140] Embodiment A-20: In one embodiment of Classification A, the in vivo distribution is approximately 10 to 80 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0141] Embodiment A-21: In one embodiment of Classification A, the in vivo distribution is approximately 10 to 60 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0142] Embodiment A-22: In one embodiment of Classification A, the in vivo distribution is approximately 15 to 90 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0143] Embodiment A-23: In one embodiment of Classification A, the in vivo distribution is approximately 15 to 70 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0144] Embodiment A-24: In one embodiment of Classification A, the in vivo distribution is approximately 15 to 50 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0145] Embodiment A-25: In one embodiment of Classification A, the in vivo distribution is approximately 20 to 60 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0146] Embodiment A-26: In one embodiment of Classification A, the in vivo distribution is approximately 20 to 40 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0147] Embodiment A-27: In one embodiment of Classification A, the in vivo distribution is approximately 30 to 50 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0148] Embodiment A-28: In one embodiment of Classification A, the in vivo distribution is approximately 10 to 40 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0149] Embodiment A-29: In one embodiment of Classification A, the in vivo distribution is approximately 35 to 60 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0150] Embodiment A-30: In one embodiment of Classification A, the in vivo distribution is approximately 25 to 70 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0151] Classification B (CNS Transduction): In some aspects of this disclosure, viral particles containing a variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibit increased CNS transduction compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, increased CNS transduction includes increased brain transduction and / or spinal cord transduction. In some embodiments, increased brain transduction is aggregated brain transduction, or transduction in a specific brain tissue such as the brainstem, basal ganglia, cerebellum, forebrain, hippocampus, midbrain, or temporal cortex, and in each case is measured as an aggregation of a CNS-specific promoter such as hSyn, a constitutive promoter such as Cbh, or a CNS-specific promoter such as hSyn and a constitutive promoter such as Cbh. In some embodiments, increased transduction is aggregated brain transduction using a Cbh or hSyn promoter and / or as defined in any one of embodiments B-1 to B-32.

[0152] Embodiment B-1: In one embodiment of classification B, transduction is about (or at least about) 10 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0153] Embodiment B-2: In one embodiment of classification B, transduction is approximately (or at least approximately) 25 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0154] Embodiment B-3: In one embodiment of classification B, transduction is about (or at least about) 50 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0155] Embodiment B-4: In one embodiment of classification B, transduction is approximately (or at least approximately) 75 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0156] Embodiment B-5: In one embodiment of Classification B, transduction is approximately (or at least approximately) 100 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0157] Embodiment B-6: In one embodiment of Classification B, transduction is approximately (or at least approximately) 125 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0158] Embodiment B-7: In one embodiment of classification B, transduction is approximately (or at least approximately) 150 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0159] Embodiment B-8: In one embodiment of classification B, transduction is approximately (or at least approximately) 175 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0160] Embodiment B-9: In one embodiment of classification B, transduction is approximately (or at least approximately) 200 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0161] Embodiment B-10: In one embodiment of classification B, transduction is approximately (or at least approximately) 225 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0162] Embodiment B-11: In one embodiment of classification B, transduction is approximately (or at least approximately) 250 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0163] Embodiment B-12: In one embodiment of classification B, transduction is approximately (or at least approximately) 275 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0164] Embodiment B-13: In one embodiment of classification B, transduction is approximately (or at least approximately) 300 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0165] In some embodiments, the improved transduction falls within a range defined by any two values ​​shown in embodiments B-1 to B-13. Illustrative ranges are shown in embodiments B-14 to B-32 below.

[0166] Embodiment B-14: In one embodiment of classification B, transduction is about 10 to 300 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0167] Embodiment B-15: In one embodiment of classification B, transduction is about 25 to 300 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0168] Embodiment B-16: In one embodiment of classification B, transduction is about 50 to 300 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0169] Embodiment B-17: In one embodiment of classification B, transduction is about 75 to 300 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0170] Embodiment B-18: In one embodiment of classification B, transduction is about 100 to 300 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0171] Embodiment B-19: In one embodiment of classification B, transduction is about 10 to 250 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0172] Embodiment B-20: In one embodiment of classification B, transduction is about 25 to 250 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0173] Embodiment B-21: In one embodiment of classification B, transduction is about 50 to 250 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0174] Embodiment B-22: In one embodiment of classification B, transduction is improved by a range of approximately 75 to 250 times compared to viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0175] Embodiment B-23: In one embodiment of classification B, transduction is about 100 to 250 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0176] Embodiment B-24: In one embodiment of classification B, transduction is about 25 to 225 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0177] Embodiment B-25: In one embodiment of classification B, transduction is about 25 to 225 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0178] Embodiment B-26: In one embodiment of classification B, transduction is about 50 to 225 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0179] Embodiment B-27: In one embodiment of classification B, transduction is about 75 to 225 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0180] Embodiment B-28: In one embodiment of classification B, transduction is about 75 to 225 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0181] Embodiment B-29: In one embodiment of classification B, transduction is about 25 to 200 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0182] Embodiment B-30: In one embodiment of classification B, transduction is about 50 to 200 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0183] Embodiment B-31: In one embodiment of classification B, transduction is about 75 to 200 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0184] Embodiment B-32: In one embodiment of classification B, transduction is about 100 to 200 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0185] Classification C (Peripheral Nervous System ("PNS") Biodistribution and / or Transduction): In some aspects of this disclosure, viral particles containing a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit similar or reduced PNS biodistribution and / or transduction compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, PNS biodistribution and / or transduction is measured as an aggregate of a CNS-specific promoter such as hSyn, a constitutive promoter such as Cbh, or a CNS-specific promoter such as hSyn and a constitutive promoter such as Cbh. In some embodiments, PNS biodistribution and / or transduction is DRG biodistribution and / or transduction as defined by any one of C-1 to C-22.

[0186] Embodiment C-1: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is increased by approximately (or approximately ~ less) 10 times compared to viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0187] Embodiment C-2: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is increased by approximately (or approximately ~ or less) 5 times compared to viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0188] Embodiment C-3: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is increased by approximately (or approximately ~ less) 2 times compared to viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0189] Embodiment C-4: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is increased by approximately (or approximately ~ less) 1.5 times compared to viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0190] Embodiment C-5: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is approximately (or approximately ~ less) 1 times that of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0191] Embodiment C-6: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is approximately (or approximately ~ less) 0.9 times that of a variant capsid polypeptide containing a viral particle having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0192] Embodiment C-7: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is approximately (or approximately ~ less) 0.8 times that of a variant capsid polypeptide containing a viral particle having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0193] Embodiment C-8: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is approximately (or less) 0.7 times that of a variant capsid polypeptide containing a viral particle having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0194] Embodiment C-9: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is approximately (or less) 0.6 times that of a variant capsid polypeptide containing a viral particle having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0195] Embodiment C-10: In one embodiment of Classification C, the in vivo distribution and / or transduction of PNS is approximately (or less) 0.6 times that of a variant capsid polypeptide containing a viral particle having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0196] In some embodiments, the improved PNS in vivo distribution and / or transduction falls within a range defined by any two values ​​shown in embodiments C-1 to C-10. Illustrative ranges are shown in embodiments C-11 to C-22 below.

[0197] Embodiment C-11: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of about 0.5 to about 10 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0198] Embodiment C-12: In one embodiment of Classification C, the PNS biodistribution and / or transduction is in the range of approximately 0.5 to approximately 5 times the PNS biodistribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0199] Embodiment C-13: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of about 0.6 to about 10 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0200] Embodiment C-14: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of approximately 0.6 to approximately 5 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0201] Embodiment C-15: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of about 0.7 to about 10 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0202] Embodiment C-16: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of approximately 0.7 to approximately 5 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0203] Embodiment C-17: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of about 0.8 to about 10 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0204] Embodiment C-18: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of approximately 0.8 to approximately 5 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0205] Embodiment C-19: In one embodiment of Classification C, the in vivo distribution and / or transduction of the PNS is in the range of about 0.9 to about 10 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0206] Embodiment C-20: In one embodiment of Classification C, the PNS biodistribution and / or transduction is in the range of approximately 0.9 to approximately 5 times the PNS biodistribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0207] Embodiment C-21: In one embodiment of Classification C, the PNS biodistribution and / or transduction is in the range of about 1 to about 10 times the PNS biodistribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0208] Embodiment C-22: In one embodiment of Classification C, the PNS biodistribution and / or transduction is in the range of about 1 to about 5 times the PNS biodistribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0209] Classification D (Hepatic In vivo distribution and / or transduction): In some aspects of this disclosure, viral particles containing a variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibit similar or reduced hepatic in vivo distribution and / or transduction compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, hepatic in vivo distribution and / or transduction is measured as an aggregation of a CNS-specific promoter such as hSyn, a constitutive promoter such as Cbh, or a CNS-specific promoter such as hSyn and a constitutive promoter such as Cbh. In some embodiments, hepatic in vivo distribution and / or transduction is as defined in any one of embodiments D-1 to D-30.

[0210] Embodiment D-1: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction of viral particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1, is approximately (or approximately ~ less) 1 times that of such particles.

[0211] Embodiment D-2: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.9 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0212] Embodiment D-3: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.7 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0213] Embodiment D-4: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.6 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0214] Embodiment D-5: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.5 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0215] Embodiment D-6: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.4 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0216] Embodiment D-7: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.3 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0217] Embodiment D-8: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.2 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0218] Embodiment D-9: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is approximately (or approximately ~ less) 0.1 times that of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0219] In some embodiments, the improved hepatic in vivo distribution and / or transduction falls within a range defined by any two values ​​shown in embodiments D-1 to D-9. Illustrative ranges are shown in embodiments D-10 to D-23 below.

[0220] Embodiment D-10: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1, is in the range of approximately 0.1 to approximately 1 times.

[0221] Embodiment D-11: In one embodiment of classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 1 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0222] Embodiment D-12: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.1 to approximately 0.9 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0223] Embodiment D-13: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 0.9 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0224] Embodiment D-14: In one embodiment of classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.1 to approximately 0.8 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0225] Embodiment D-15: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 0.8 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of Sequence ID No. 1.

[0226] Embodiment D-16: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.1 to approximately 0.7 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0227] Embodiment D-17: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 0.7 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0228] Embodiment D-18: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.1 to approximately 0.6 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0229] Embodiment D-19: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 0.6 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0230] Embodiment D-20: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.1 to approximately 0.5 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0231] Embodiment D-21: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 0.5 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0232] Embodiment D-22: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.1 to approximately 0.4 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0233] Embodiment D-23: In one embodiment of Classification D, the hepatic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 0.4 times that of the hepatic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of Sequence ID No. 1.

[0234] Classification E (Splenic In vivo distribution and / or transduction): In some aspects of this disclosure, viral particles containing a variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibit similar or reduced splenic in vivo distribution and / or transduction compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, splenic in vivo distribution and / or transduction is measured as an aggregation of a CNS-specific promoter such as hSyn, a constitutive promoter such as Cbh, or a CNS-specific promoter such as hSyn and a constitutive promoter such as Cbh. In some embodiments, splenic in vivo distribution and / or transduction is as defined in any one of embodiments E-1 to E-30.

[0235] Embodiment E-1: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction is about (or about ~ less) 10 times that of the splenic in vivo distribution and / or transduction of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0236] Embodiment E-2: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is about (or about ~ less) 5 times that of the splenic in vivo distribution and / or transduction of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0237] Embodiment E-3: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction is approximately (or approximately ~ less) 3 times that of a variant capsid polypeptide containing a viral particle having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0238] Embodiment E-4: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction is approximately (or approximately ~ less) twice that of the splenic in vivo distribution and / or transduction of viral particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0239] Embodiment E-5: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction is approximately (or approximately ~ less) 1.5 times that of a variant capsid polypeptide containing a viral particle having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0240] Embodiment E-6: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1, is approximately (or approximately ~ less) 1 times that of the splenic in vivo distribution and / or transduction of the virus particle.

[0241] Embodiment E-7: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1, is approximately (or approximately ~ less) 0.8 times that of the splenic in vivo distribution and / or transduction of the virus particle.

[0242] Embodiment E-8: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1, is approximately (or approximately ~ less) 0.6 times that of the splenic in vivo distribution and / or transduction of the virus particle.

[0243] Embodiment E-9: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1, is approximately (or approximately ~ less) 0.4 times that of the splenic in vivo distribution and / or transduction of the virus particle.

[0244] Embodiment E-10: In one embodiment of Classification E, the splenic in vivo distribution and / or transduction of a virus particle containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1, is approximately (or approximately ~ less) 0.2 times that of the splenic in vivo distribution and / or transduction of the virus particle.

[0245] In some embodiments, the improved spleen in vivo distribution and / or transduction falls within a range defined by any two values ​​shown in embodiments E-1 to E-10. Illustrative ranges are shown in embodiments E-11 to E-22 below.

[0246] Embodiment E-11: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of about 0.2 to about 10 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0247] Embodiment E-12: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 5 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0248] Embodiment E-13: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 3 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0249] Embodiment E-14: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 2 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0250] Embodiment E-15: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.2 to approximately 1.5 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0251] Embodiment E-16: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of about 0.2 to about 1 times that of the capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0252] Embodiment E-17: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of about 0.4 to about 10 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0253] Embodiment E-18: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.4 to approximately 5 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0254] Embodiment E-19: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.4 to approximately 3 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0255] Embodiment E-20: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.4 to approximately 2 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0256] Embodiment E-21: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.4 to approximately 1.5 times the splenic in vivo distribution and / or transduction of a capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0257] Embodiment E-22: In one embodiment of classification E, the splenic in vivo distribution and / or transduction is in the range of approximately 0.4 to approximately 1 times that of the capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0258] Classification F (Skeletal Muscle Biodistribution): In some aspects of this disclosure, viral particles containing a variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibit increased skeletal muscle biodistribution compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased skeletal muscle biodistribution is measured by a constitutive promoter such as Cbh. In some embodiments, the increased skeletal muscle biodistribution is as defined in any one of embodiments F-1 to F-10.

[0259] Embodiment F-1: In one embodiment of classification F, the in vivo distribution is about (or at least about) 1.5 times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0260] Embodiment F-2: In one embodiment of classification F, the in vivo distribution is about (or at least about) twice as good as that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0261] Embodiment F-3: In one embodiment of classification F, the in vivo distribution is approximately (or at least approximately) three times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0262] Embodiment F-4: In one embodiment of classification F, the in vivo distribution is about (or at least about) four times better than that of viral particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0263] Embodiment F-5: In one embodiment of Class F, the biodistribution is about (or at least about) 5-fold better compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0264] Embodiment F-6: In one embodiment of Class F, the biodistribution is about (or at least about) 6-fold better compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0265] Embodiment F-7: In one embodiment of Class F, the biodistribution is about (or at least about) 7-fold better compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0266] Embodiment F-8: In one embodiment of Class F, the biodistribution is about (or at least about) 8-fold better compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0267] Embodiment F-9: In one embodiment of Class F, the biodistribution is about (or at least about) 9-fold better compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0268] Embodiment F-10: In one embodiment of Class F, the biodistribution is about (or at least about) 10-fold better compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0269] In some embodiments, the improved in vivo distribution is within a range bounded by any two values shown in Embodiments F-1 to F-10. Exemplary ranges are shown in the following Embodiments F-11 to F-17.

[0270] Embodiment F-11: In one embodiment of Class F, the in vivo distribution is good in the range of about 1.5 to about 10 times as compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0271] Embodiment F-12: In one embodiment of Class F, the in vivo distribution is good in the range of about 2 to about 10 times as compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0272] Embodiment F-13: In one embodiment of Class F, the in vivo distribution is good in the range of about 3 to about 10 times as compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0273] Embodiment F-14: In one embodiment of Class F, the in vivo distribution is good in the range of about 4 to about 10 times as compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0274] Embodiment F-15: In one embodiment of Class F, the in vivo distribution is good in the range of about 5 to about 10 times as compared to virus particles containing a variant capsid polypeptide having a reference sequence, for example, having a wild-type capsid protein, for example, having the capsid polypeptide of SEQ ID NO: 1.

[0275] Embodiment F-16: In one embodiment of classification F, the in vivo distribution is approximately 6 to 10 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0276] Embodiment F-17: In one embodiment of classification F, the in vivo distribution is approximately 7 to 10 times better than that of virus particles containing variant capsid polypeptides having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0277] Classification G (Skeletal Muscle Transduction): In some aspects of this disclosure, viral particles containing a variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibit increased skeletal muscle transduction compared to viral particles having the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased skeletal muscle biodistribution is measured by a constitutive promoter such as Cbh. In some embodiments, the increased skeletal muscle transduction is as defined in any one of embodiments G-1 to G-5.

[0278] Embodiment G-1: In one embodiment of classification G, transduction is about (or at least about) 1.5 times better than viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0279] Embodiment G-2: In one embodiment of classification G, transduction is about (or at least about) twice as good as with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0280] Embodiment G-3: In one embodiment of classification G, transduction is approximately (or at least approximately) three times better than that of viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0281] Embodiment G-4: In one embodiment of classification G, transduction is about (or at least about) four times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0282] Embodiment G-5: In one embodiment of classification G, transduction is about (or at least about) 5 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0283] In some embodiments, the improved transduction falls within a range defined by any two values ​​shown in embodiments G-1 to G-5. Illustrative ranges are shown in embodiments G-6 to G-9 below.

[0284] Embodiment G-6: In one embodiment of classification G, transduction is approximately 1.5 to 5 times better than that of virus particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0285] Embodiment G-7: In one embodiment of classification G, transduction is approximately 2 to 5 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0286] Embodiment G-8: In one embodiment of classification G, transduction is approximately 3 to 5 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0287] Embodiment G-9: In one embodiment of classification G, transduction is approximately 4 to 5 times better than with viral particles containing a variant capsid polypeptide having a reference sequence, for example, a wild-type capsid protein, for example, the capsid polypeptide of SEQ ID NO: 1.

[0288] 5.5. Method for preparing the compositions described herein This disclosure, in part, relates to dependent parvovirus particles, for example, methods for producing dependent parvovirus particles as described herein. In some embodiments, a method for producing dependent parvovirus particles comprises providing a cell, cell-free system, or other translation system containing a variant capsid polypeptide provided herein, or a nucleic acid described herein that encodes a polypeptide provided herein (e.g., a variant capsid polypeptide), and culturing the cell, cell-free system, or other translation system under conditions suitable for the production of dependent parvovirus particles, thereby producing dependent parvovirus particles.

[0289] In some embodiments, providing a cell comprising a nucleic acid described herein includes introducing the nucleic acid into the cell, e.g., transfecting or transforming the cell with the nucleic acid. In embodiments, the nucleic acids of the disclosure are part of any genetic element (vector) that can be delivered to a host cell, e.g., naked DNA, plasmid, phage, transposon, cosmid, episome, protein in a non-viral delivery vehicle (e.g., lipid-based carrier), virus that transfers sequences carried thereon. Such vectors can be delivered by any suitable method including transfection, liposomal delivery, electroporation, membrane fusion techniques, viral infection, high velocity DNA coating pellets, and protoplast fusion. Those skilled in the art have knowledge and skill in nucleic acid manipulation for constructing any embodiment of the invention, and such skill includes 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.

[0290] In some embodiments, the vectors of the Disclosure include a sequence or fragment thereof encoding a dependent parvovirus variant capsid polypeptide, as provided herein. In some embodiments, the vectors of the Disclosure include a sequence or fragment thereof encoding a dependent parvovirus rep protein. In some embodiments, such vectors include both dependent parvovirus cap protein and rep protein. In vectors in which both AAV rep and cap are provided, in embodiments, both the dependent parvovirus rep and dependent parvovirus cap sequences are of the same dependent parvovirus species or serotype origin, e.g., AAV9. Alternatively, these embodiments also provide vectors in which the rep sequence is of a different dependent parvovirus species or serotype than that which provides the cap sequence. In some embodiments, the rep sequence and cap sequence are expressed from separate sources (e.g., separate vectors, or host cell genome and vector). In some embodiments, the rep sequence is fused in-frame to a cap sequence of a different dependent parvovirus species or serotype to form a chimeric dependent parvovirus vector. In some embodiments, the vector of the present invention further contains a minigene comprising a selected transgene adjacent to a payload, for example, dependent parvovirus 5'ITR and dependent parvovirus 3'ITR.

[0291] The vectors described herein, such as plasmids, are useful for a variety of purposes, but are particularly well suited for use in the production of recombinant dependent parvovirus particles, including dependent parvovirus sequences or fragments thereof, and, in some embodiments, payloads.

[0292] In one embodiment, the Disclosure provides a method for producing depend parvovirus particles (e.g., depend parvovirus B particles, e.g., AAV9 particles) or a portion thereof. In some embodiments, the method comprises culturing a host cell containing a nucleic acid sequence or fragment thereof encoding a depend parvovirus variant capsid protein provided herein, a functional rep gene, a payload, e.g., a minigene including depend parvovirus inverted terminal repeats (ITRs) and a transgene, and helper functions sufficient to facilitate the packaging of the payload, e.g., the minigene into a depend parvovirus capsid. In embodiments, the payload, e.g., components that need to be cultured in the host cell to package the minigene into a depend parvovirus capsid, are provided to the host cell in trans. In some embodiments, any one or more of the required components (e.g., payload (e.g., minigene), rep sequence, cap sequence, and / or helper functions) are provided by a host cell that has been manipulated to stably contain one or more of the required components using methods known to those skilled in the art. In some embodiments, a host cell manipulated to stably contain the required component(s) is contained under the control of an inductive promoter. In some embodiments, the required component(s) is contained under the control of a constitutive promoter. Examples of suitable inductive and constitutive promoters are provided herein, and further examples are known to those skilled in the art. In some embodiments, a selected host cell manipulated to stably contain one or more components contains a component contained under the control of a constitutive promoter and another component contained under the control of one or more inductive promoters. For example, a host cell manipulated to stably contain the required component is generated from a 293 cell (e.g., containing helper functions contained under the control of a constitutive promoter) containing rep and / or cap proteins contained under the control of one or more inductive promoters.

[0293] In embodiments, the payload (e.g., a minigene), rep sequence, cap sequence, and helper function required for the production of the dependent parvovirus particles of this disclosure are delivered to a host cell, packaged in the form of any genetic element that introduces the sequences to be supported thereon (e.g., in a vector or a combination of vectors). The genetic element may be delivered by any preferred method, including those described herein. Methods used to construct the genetic elements, vectors, and other nucleic acids of this disclosure are known to those skilled in the art and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the selection of preferred methods does not limit the invention. See, for example, K. Fisher et al, J. Virol, 70:520-532 (1993) and U.S. Patent No. 5,478,745. Unless otherwise specified, dependent parvovirus ITRs and other selected dependent parvovirus components described herein are readily selected from any dependent parvovirus species and serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh74, or AAV9. ITRs or other dependent parvovirus components can be readily isolated from dependent parvovirus species or serotypes using techniques available to those skilled in the art. Dependent parvovirus species and serotypes can be isolated or obtained from academic, commercial, or public resources (e.g., American Type Culture Collection, Manassas, VA). In some embodiments, dependent parvovirus sequences can be obtained by synthesis or other preferred means by referencing publicly available sequences, such as those available in literature or databases, e.g., GenBank or PubMed.

[0294] The dependent parvovirus particles of this disclosure (e.g., including variant capsid polypeptides and, for example, payloads) may be produced using any invertebrate cell type that enables the production of dependent parvoviruses or biological products and may be maintained in culture. In some embodiments, insect cells may be used in the production of the compositions described herein or in the methods for producing dependent parvovirus particles described herein. For example, the insect cell lines used may be those derived from Spodoptera frugiperda, e.g., Sf9, SF21, SF900+, Drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus cell lines, silkworm cell lines, e.g., Bombyxmori cell lines, Trichoplusia ni cell lines, e.g., High Five cells, or Lepidoptera cell lines, e.g., Ascalapha odorata cell lines. In some embodiments, insect cells are susceptible to baculovirus infection and include High Five, Sf9, Se301, SeIZD2109, SeUCR1, SP900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5, and Ao38.

[0295] In some embodiments, the methods of the present disclosure can be carried out using any mammalian cell type that can enable replication of dependent parvovirus or production of biological products and maintain in culture. In other embodiments, the mammalian cells used may be HEK293, HeLa, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, or MRC-5 cells.

[0296] Methods for expressing proteins (e.g., recombinant or heterologous proteins, e.g., dependent parvovirus polypeptides) in insect cells are well documented, including methods for introducing nucleic acids, e.g., vectors, e.g., insect cell-compatible vectors, into such cells, and methods for maintaining such cells in culture. For example, METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, NJ (1995), O'Reilly et al., BACULOVIRUS EXPRESSION VECtoRS, A LABORAtoRY MANUAL, Oxford Univ. Press (1994), Samulski et al., J. Vir.63:3822-8 (1989), Kajigaya et al.,Proc.Nat'l.Acad.Sci.USA 88:4646-50 (1991), Ruffing et al.,J.Vir.66:6922-30 (1992), Kirnbauer et al.,Vir.219:37-44 (1996), Zhao et al.,Vir.272:382-93 (2000), and Samulski See et al., U.S. Patent No. 6,204,059. In some embodiments, nucleic acid constructs encoding dependent parvovirus polypeptides (e.g., dependent parvovirus genomes) in insect cells are insect cytocompatible vectors. As used herein, “insect cytocompatible vector” 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 used as long as it is insect cytocompatible. A vector may be incorporated into the genome of an insect cell or remain extrachromosomally. A vector may exist permanently or transiently, for example, as an episomal vector. A vector may be introduced by any means known in the art, including, but not limited to, chemical treatment of cells, electroporation, or infection.In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid.

[0297] In some embodiments, a nucleic acid sequence encoding a dependent parvovirus polypeptide is operably ligated to a regulatory expression sequence for expression in specific cell types, such as Sf9 or HEK cells. Techniques known to those skilled in the art for expressing exogenous genes in insect or mammalian host cells can be used in conjunction with the compositions and methods of this disclosure. Methods for molecular manipulation and expression of polypeptides in insect cells include, for example, 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, In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152 (1986); King, L.A. and R.D. Prossee, The baculovirus expression system, Chapman and Hall, United Kingdom (1992); O'Reilly, D.R., L.K. Miller, L.V. Luckow, Baculovirus Expression Vectors: A Laboratory Manual, New York (1992); and WH. Freeman and Richardson, C.D., Baculovirus Expression Protocols, Methods in Molecular Biology, volume It is described in 39 (1995), U.S. Patent No. 4,745,051, U.S.2003148506, and WO03 / 074714.Suitable promoters for transcribing nucleotide sequences encoding dependent parvovirus polypeptides include polyhedral, p10, p35, or IE-1 promoters, and further promoters described in the references mentioned above are also considered.

[0298] In some embodiments, providing cells containing nucleic acids as described herein includes obtaining cells containing nucleic acids.

[0299] Methods for culturing cells, cell-free systems, and other translation systems are known to those skilled in the art. In some embodiments, culturing cells includes providing a suitable medium for the cells and incubating the cells and medium for a suitable time to achieve viral particle production.

[0300] In some embodiments, a method for producing dependent parvovirus particles further includes a purification step of isolating the dependent parvovirus particles from one or more other components (e.g., cells or culture medium components).

[0301] In some embodiments, the production of dependent parvovirus particles comprises one or more (e.g., all) of the following for producing dependent parvovirus particles: expression of dependent parvovirus polypeptide, assembly of dependent parvovirus capsid, expression (e.g., replication) of dependent parvovirus genome, and packaging of dependent parvovirus genome into dependent parvovirus capsid. In some embodiments, the production of dependent parvovirus particles further comprises secretion of dependent parvovirus particles.

[0302] In some embodiments and as described elsewhere herein, a nucleic acid molecule encoding a variant capsid polypeptide is placed within the dependent parvovirus genome. In some embodiments and as described elsewhere herein, the nucleic acid molecule encoding the variant capsid polypeptide is packaged within the dependent parvovirus particle together with the dependent parvovirus genome as part of the method for producing the dependent parvovirus particle described herein. In other embodiments, the nucleic acid molecule encoding the variant capsid polypeptide is not packaged within the dependent parvovirus particle produced by the method described herein.

[0303] In some embodiments, the method for producing dependent parvovirus particles described herein produces dependent parvovirus particles comprising a payload (e.g., the payload described herein) and a variant capsid polypeptide. In some embodiments, the payload comprises a second nucleic acid (e.g., in addition to the dependent parvovirus genome), and the production of dependent parvovirus particles comprises packaging the second nucleic acid into dependent parvovirus particles. In some embodiments, the cells, cell-free system, or other translation system for use in the method for producing dependent parvovirus particles comprises the second nucleic acid. In some embodiments, the second nucleic acid comprises an exogenous sequence (e.g., exogenous to dependent parvovirus, cells, or target cells or subjects to which dependent parvovirus particles are administered). In some embodiments, the exogenous sequence encodes an exogenous polypeptide. In some embodiments, the exogenous sequence encodes a therapeutic product.

[0304] In some embodiments, the viral particles of the Disclosure have a production efficiency similar to that of viral particles having a reference capsid polypeptide, e.g., wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the production efficiency of the viral particles of the Disclosure is, for example, (a) at least 0.1 times, at least 0.2 times, at least 0.3 times, at least 0.4 times, at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, or at least 0.9 times compared to viral particles having a reference capsid polypeptide, e.g., wild-type capsid polypeptide (SEQ ID NO: 1), and / or (b) up to 1 time, or the production efficiency is within any range limited by the values ​​of (a) and (b). In some embodiments, the production efficiency is at least 0.5 times compared to viral particles having wild-type capsid polypeptide (SEQ ID NO: 1). Production efficiency can be assessed by transient triple transfection of adherent HEK293T, followed by iodixanol gradient purification, to produce viral particles having variant capsids with a unique barcode-encoding genome and a fluorescent reporter gene, under the control of a ubiquitous (e.g., CBh) or neuronal cell type-specific promoter (e.g., human synapsin).

[0305] In some embodiments, the nucleic acids or polypeptides described herein are produced by methods readily apparent to those skilled in the art. In embodiments, the nucleic acids, polypeptides, and their fragments are produced by any preferred means, including recombinant production, chemical synthesis, or other synthetic means. Such production methods are within the scope of knowledge of those skilled in the art and do not limit the invention.

[0306] 5.6.Applications This disclosure covers, in part, compositions comprising nucleic acids, polypeptides, or particles described herein. This disclosure further covers, in part, methods utilizing the compositions, nucleic acids, polypeptides, or particles described herein. As will become apparent from this disclosure, the nucleic acids, polypeptides, particles, and methods disclosed herein have a variety of uses.

[0307] This disclosure, in part, covers vectors comprising nucleic acids, such as nucleic acids encoding variant capsid polypeptides, as described herein. Many types of vectors are known to those skilled in the art. In some embodiments, the vector comprises a plasmid. In some embodiments, the vector is an isolated vector, such as an isolated vector removed from a cell or other biological component.

[0308] This disclosure, in part, relates to cells, cell-free systems, or other translation systems comprising nucleic acids or vectors described herein, for example, nucleic acid molecules encoding variant capsid polypeptides. In some embodiments, cells, cell-free systems, or other translation systems can produce dependent parvovirus particles containing variant capsid polypeptides. In some embodiments, cells, cell-free systems, or other translation systems comprise a nucleic acid containing a dependent parvovirus genome or components of a dependent parvovirus genome sufficient to facilitate the production of dependent parvovirus particles containing variant capsid polypeptides.

[0309] In some embodiments, the cell, cell-free system, or other translation system further comprises one or more non-depend parvovirus nucleic acid sequences that promote the production and / or secretion of dependent parvovirus particles. These sequences are referred to herein as helper sequences. In some embodiments, the helper sequences comprise one or more genes derived from another virus, e.g., adenovirus or herpesvirus. In some embodiments, the presence of the helper sequences is required for the production and / or secretion of dependent parvovirus particles. In some embodiments, the cell, cell-free system, or other translation system comprises a vector, e.g., a plasmid, containing one or more helper sequences.

[0310] In some embodiments, a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, the first nucleic acid comprising a sequence encoding one or more dependent parvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependent parvovirus genome) and a helper sequence, and the second nucleic acid comprising a payload. In some embodiments, a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, the first nucleic acid comprising a sequence encoding one or more dependent parvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependent parvovirus genome) and a payload, and the second nucleic acid comprising a helper sequence. In some embodiments, a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, the first nucleic acid comprising a helper sequence and a payload, and the second nucleic acid comprising a sequence encoding one or more dependent parvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependent parvovirus genome). In some embodiments, 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 a sequence encoding one or more dependent parvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependent parvovirus genome), the second nucleic acid comprises a helper sequence, and the third nucleic acid comprises a payload.

[0311] In some embodiments, the first nucleic acid, the second nucleic acid, and optionally the third nucleic acid are located in separate molecules, e.g., separate vectors, or in a vector and genomic DNA. In some embodiments, one, two, or all of the first nucleic acid, the second nucleic acid, and optionally the third nucleic acid are integrated into the cell's genome (e.g., stably integrated).

[0312] In some embodiments, the cells of this disclosure are produced by transfecting preferred cells with nucleic acids described herein. In some embodiments, improving a method for producing dependent parvovirus particles containing variant capsid polypeptides provided herein, or a method for producing dependent parvovirus particles, includes providing the cells described herein. In some embodiments, providing cells includes transfecting preferred cells with one or more nucleic acids described herein.

[0313] Many types and varieties of cells suitable for use with the nucleic acids and vectors described herein are known in the art. In some embodiments, the cells are human cells. In some embodiments, the cells are immortalized cells or cells derived from cell lines known in the art. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293T cells.

[0314] 5.7. How to deliver the payload This disclosure, in part, relates to methods for delivering a payload to cells, for example, cells in a subject or sample. In some embodiments, the method for delivering a payload to cells involves contacting the cells with dependent parvovirus particles containing a variant capsid polypeptide (e.g., as described herein) containing the payload. This disclosure also relates to dependent parvovirus particles containing a variant capsid polypeptide (e.g., as described herein) containing the payload, for use in the method for delivering the payload described herein. In some embodiments, the dependent parvovirus particles are dependent parvovirus particles as described herein and contain the payload as described herein. In some embodiments, the cells are CNS cells. In some embodiments, the cells are skeletal muscle cells. Non-limiting examples of skeletal muscle include the biceps, triceps, quadriceps, medial tibia, gastrocnemius, and diaphragm. In some embodiments, the method is performed ex vivo. In some embodiments, the cells are cells in an ex vivo sample obtained from a subject.

[0315] In some embodiments, the payload includes a transgene. In some embodiments, the transgene is a heterogeneous nucleic acid sequence relative to the vector sequence adjacent to the transgene encoding a polypeptide, RNA (e.g., miRNA or siRNA), or other product of interest. In embodiments, the nucleic acid of the transgene is operably ligated to a regulatory component in a manner sufficient to promote the transcription, translation, and / or expression of the transgene in the host cell.

[0316] In some embodiments, the transgene is any polypeptide or RNA coding sequence, and the selected transgene will depend on the intended use. In some embodiments, the transgene includes a reporter sequence that produces a detectable signal upon expression. Such reporter sequences include, but are not limited to, colorimetric reporters (e.g., β-lactamase, β-galactosidase (LacZ), alkaline phosphatase), cell division reporters (e.g., thymidine kinase), fluorescent or luminescent reporters (e.g., green fluorescent protein (GFP) or luciferase), resistance transport sequences (e.g., chloramphenicol acetyltransferase (CAT)), or DNA sequences encoding membrane-bound proteins that can be produced by conventional means, such as the existence of high-affinity antibodies targeting them, or by including antigen tags, e.g., hemagglutinin or Myc.

[0317] In some embodiments, reporter sequences operably linked to regulatory elements that drive their expression provide a signal detectable by conventional means, including enzyme assays, radiographic assays, colorimetric assays, fluorescence assays, or other spectral assays, fluorescence-activated cell sorting assays, and immunological assays including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry. In some embodiments, the transgene encodes a product useful in biology and medicine, such as RNA, protein, peptide, enzyme, or dominant-negative variant. In some embodiments, the RNA includes tRNA, ribosomal RNA, dsRNA, catalytic RNA, small hairpin RNA, siRNA, trans-splicing RNA, and antisense RNA. In some embodiments, the RNA inhibits or disables the expression of a target nucleic acid sequence in the subject being treated (e.g., a human or animal subject).

[0318] In some embodiments, transgenes are used to correct or improve gene defects. In some embodiments, gene defects include defects in which a normal gene is expressed below normal levels, or defects in which a functional gene product is not expressed. In some embodiments, the transgenes encode therapeutic proteins or polypeptides expressed in host cells. In some embodiments, dependent parvovirus particles contain or deliver multiple transgenes to correct or improve gene defects caused, for example, multi-subunit proteins. In some embodiments, different transgenes (e.g., each located in a different dependent parvovirus particle, or delivered to a single dependent parvovirus particle) are used to encode each subunit of a protein, or different peptides or proteins, for example, when the size of the DNA encoding protein subunits is large, such as for immunoglobulins, platelet-derived growth factors, or dystrophin proteins. In some embodiments, different subunits of a protein are encoded by the same transgene, for example, a single transgene encoding each subunit having DNA for each subunit separated by an internal ribozyme entry site (IRES). In some embodiments, the DNA is separated by a sequence encoding a 2A peptide that self-cleaves in a post-translational event. For example, see Donnelly et al, J. Gen. Virol., 78(Pt 1):13-21 (January 1997), Furler, et al, Gene Ther., 8(11):864-873 (June 2001), and Klump et al., Gene Ther 8(10):811-817 (May 2001) (the entire work is incorporated herein by reference).

[0319] In some embodiments, a viral particle containing a genome is provided, the genome comprising a nucleic acid expression construct. The nucleic acid expression construct may include a xenotransgene and one or more regulatory elements.

[0320] In some embodiments, the regulatory element includes a promoter, e.g., a promoter that is active in the target cell or tissue type of interest. In some embodiments, the promoter is a ubiquitous promoter. In other embodiments, the promoter is selective or specific to the target cell or tissue type (e.g., CNS and / or muscle (either broad muscle expression or skeletal muscle)) and, optionally, less active (or inactive) in cell or tissue types where transgene expression is not desired (e.g., liver, spleen, PNS, or any combination of two or all of the aforementioned). A promoter that is selective to a first cell or tissue type more than 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. In some embodiments, the promoter is a CNS-specific or CNS-selective promoter. In some embodiments, the promoter is a muscle-specific, promoter-specific, or muscle-selective promoter. In some embodiments, the promoter is active in both CNS and muscle tissue.

[0321] In some embodiments, the promoter is a ubiquitous or constitutive promoter active in mammalian cells, e.g., human cells, e.g., the human cell type of interest. In some embodiments, the cell type is CNS cells, e.g., neurons, glial cells, endothelial cells, etc. In some embodiments, the cell type is skeletal muscle cells. Examples of ubiquitous promoters include, but are not limited to, the CAG promoter (a hybrid derived from the cytomegalovirus early enhancer element, the chicken-beta-actin promoter, e.g., the first exon and first intron of the chicken-beta-actin gene, and the splice acceptor of the rabbit-beta-globin gene), the chicken-beta-actin promoter, the CBA promoter, the CBh promoter, the CB6 promoter, the CMV promoter, the human EF1-alpha promoter, the PGK promoter, the ubiquitin C (UBC) promoter, and fragments thereof. In some embodiments, the promoter is a tissue-specific promoter, e.g., a promoter specific to CNS tissue or cells of the CNS. Examples of CNS-specific promoters include, but are not limited to, synapsin (SYN or SYN1) promoters, neuron-specific enolase (NSE) promoters, and Ca 2+ / Calmodulin-dependent kinase subunit α (CaMKII) promoter, Synapsin I with minimal CMV sequence (SynI-minCMV) promoter, Glial fibrillary acidic protein (GFAP) promoter, Internexin neuron intermediate filament protein alpha (INA) promoter, Nestin (NES) promoter, Neurofilament light chain (NfL) promoter, Neurofilament heavy chain (NfH) promoter, Myelin-associated oligodendrocyte basic protein (MOBP) promoter, Myelin basic protein (MBP) promoter, Tyrosine hydroxylase (TH) promoter, Forkhead box A2 (FOXA2) promoter, Aldehyde dehydrogenase 1 family member L1 (ALDH1L1) promoter, Glutamate decarboxylase 2 (GAD2) promoter, Riken gene A930098C07Rik (A93) promoter Examples include motors, somatostatin (SST) promoters, platelet-derived growth factor receptor alpha (PDGFRA) promoters, glutamate receptor metabotype 1 (GRM1) promoters, type C natriuretic peptide precursor (NPPC) promoters, adrenomedullin (ADM) promoters, lactosamine alpha-2,3-sialyltransferase type 2 (ST3GAL6) promoters, ras-responsive element-binding protein 1 (RREB1) promoters, deiodinase iodothyronine type II (DIO2) promoters, excitatory amino acid transporter 2 (EAAT2) promoters, nuclear receptor subfamily 2 group F member 2 (NR2F2) promoters, platelet-derived growth factor (PDGF) promoters, methyl-CpG-binding protein 2 (MeCP2) promoters, and any of the mouse, primate, or human homologs described above, as well as any of the aforementioned fragments (e.g., active fragments). In embodiments, the CNS-specific promoter is a neuron-specific promoter. In embodiments, the CNS-specific promoter is an astrocyte-specific promoter. In some embodiments, the promoter is a promoter specific to muscle tissue or muscle cells, such as desmin, MCK, TNNT2, or the smooth muscle 22 (SM22) promoter.Further exemplary muscle-specific promoters are listed below.

[0322] In some embodiments, the promoter is a CBh promoter. An exemplary CBh promoter sequence is shown as SEQ ID NO: 253. In some embodiments, the CBh promoter includes 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 with respect to SEQ ID NO: 253.

[0323] In other embodiments, the promoter is a synapsin promoter, for example, a human synapsin promoter (hSYN). An exemplary hSYN promoter sequence is shown as SEQ ID NO: 254. In some embodiments, the CBh promoter includes 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 with respect to SEQ ID NO: 254.

[0324] In some embodiments, the nucleic acid expression construct includes an intron. In some embodiments, the intron is located between the promoter and the xenogene. In some embodiments, the intron is located 5' relative to the xenogene on the expression construct, for example, immediately 5' relative to the xenogene, or within 100 nucleotides of 5' relative to the xenogene. In some embodiments, the intron is a chimeric intron derived from human β-globin and Ig heavy chain (also known as a β-globin splice donor / immunoglobin heavy chain splice acceptor intron, or β-globin / IgG chimeric intron, Reed, R., et al., Genes and Development, 1989, the whole of which is incorporated herein by reference). In other embodiments, the intron is a VH4 intron or an SV40 intron.

[0325] As provided herein, in some embodiments, a viral particle comprising a payload is provided, wherein the payload comprises a nucleic acid comprising a xenotransgene. In some embodiments, the xenotransgene encodes an RNA interferant, such as siRNA, shRNA, or other interfering nucleic acid.

[0326] In some embodiments, the payload includes a xenotransgene encoding a therapeutic polypeptide. In some embodiments, the xenotransgene is a human gene or a fragment thereof. In some embodiments, the therapeutic polypeptide is a human protein. In some embodiments, the xenotransgene of the viral particle encodes a molecule useful for treating a disease, and the viral particle is administered to a patient who needs it to treat the disease. In some embodiments, the payload includes a molecule effective for treating chronic CNS diseases, such as an RNA interfering nucleotide (e.g., shRNA, siRNA, or miRNA that inhibits APOL-1). Examples of diseases (and xenotransgenes or molecules encoded by such xenotransgenes) covered by this disclosure include MPSI (alpha-L-idulonidase (IDUA)); MPS II - Hunter syndrome (idulonidase-2-sulfatase (IDS)); ceroid lipofuscinosis - Batten disease (CLN1, CLN2, CLN10, CLN13, CLN5, CLN11, CLN4, CNL14, CLN3, CLN6, CLN7, CLN8, CLN12); MPS Illa-Sanfilippo type A syndrome (heparin sulfate sulfatase (also known as N-sulfoglucosamine sulfohydrolase (SGSH))); MPS IIIB - Sanfilippo type b syndrome (N-acetyl-alpha-D-glucosaminidase (NAGLU)); MPS VI - Maroteaux-Lamy syndrome (arylsulfatase B); MPS IV A-Morquio syndrome type A (GALNS); MPS IV; B-Morquio syndrome type B (GLB1); chronic or neuropathic pain; osteogenesis imperfecta type I, II, III, or IV (COL1Al and / or COL1A2); hereditary angioedema (SERPING1, C1NH); osteogenesis imperfecta type V (IFITM5); osteogenesis imperfecta type VI (SERPINF1); osteogenesis imperfecta type VII (CRTAP); osteogenesis imperfecta type VIII (LEPRE1 and / or P3H1); osteogenesis imperfecta type IX (PPIB); Gaucher disease type I, II, and III (glucocerebrosidase; GBAl); Parkinson's disease (glucocerebrosidase; GBAl and / or dopamine decarboxylase);Pompe (acid maltase; GAA; hGAA); metachromatic leukodystrophy (aryl sulfatase A); MPS VII-Sly syndrome (beta-glucuronidase); MPS VIII (glucosamine-6-sulfate sulfatase); MPS IX (hyaluronidase); maple syrup urine disease (BCKDHA, BCKDHB, and / or DBT); Niemann-Pick disease (sphingomyelinase); Parkinson's disease (anti-alpha-synuclein RNAi); Alzheimer's disease (anti-mutant APP) RNAi); Niemann-Pick disease without sphingomyelinase deficiency (NPC1 or NPC gene encoding cholesterol-metabolizing enzymes); Tay-Sachs disease (alpha subunit of beta-hexosaminidase); Sandhoff disease (both alpha and beta subunits of beta-hexosaminidase); Fabry disease (alpha-galactosidase); fucosidosis (fucosidase (FUCAl)); alpha-mannosidosis (alpha-mannosidase); beta-mannosidosis (beta-mannosidase); Wolman disease (cholesterol ester hydrolase); Dravet syndrome (SCN1A, SC N1B, SCN2A, GABRG2); Parkinson's disease (Neurturin); Parkinson's disease (Glial growth factor (GDGF)); Parkinson's disease (Tyrosine hydroxylase); Frontotemporal dementia (Progranulin); Angleman syndrome (Ubiquitin protein ligase 3A (UBE3A), gene editing system targeting UBE3A inhibitory RNA (UBE3A antisense transcript)); Parkinson's disease (Glutamate decarboxylase; FGF-2; BDGF); Spinal muscular atrophy (SMN, including SMN1 or SMN2); Friedreich ataxia (Frataxin); Amyotrophic lateral sclerosis (ALS) (SOD1 inhibitors, e.g., anti-SOD1 RNAi); Glycogen storage disorder type Ia (glucose-6-phosphatase); XLMTM (MTMl); Crigler-Najjar (UGTlAl); CPVT (CASQ2); Spinocerebellar ataxia (ATXN2; ATXN3 or other ATXN genes; Anti-mutant Machado-Joseph disease / SCA3 allele RNAi); Rett syndrome (MECP2 or its fragments);Color blindness (CNGB3, CNGA3, GNAT2, PDE6C); colloideremia (CDM); Danon's disease (LAMP2); cystic fibrosis (CFTR or fragments thereof); Duchenne muscular dystrophy (mini- / micro-dystrophin gene); SARS-CoV-2 infection (anti-SARS-CoV-2 RNAi, SARS-CoV-2 genome fragment or S protein (including variants)); limb-girdle muscular dystrophy type 2C-gamma-sarcoglycanopathy (human-alpha-sarcoglycan); progressive heart failure (SERCA2a); rheumatoid arthritis (TNFR:Fc fusion; anti-TNF antibody or fragments thereof); Leber congenital amaurosis (GAA); X-linked adrenoleukodystrophy (ABCD1); limb-girdle muscular dystrophy type 2C-gamma-sarcoglycanopathy Lycanopathy (gamma-sarcoglycan); Angelman syndrome (UBE3A); Retinitis pigmentosa (hMERTK); Age-related macular degeneration (sFLT01); Phelan-McDermid syndrome (SHANK3; 22q13.3 substitution); Becker muscular dystrophy and sporadic inclusion body myositis (huFollistatin344); Parkinson's disease (GDNF); Metachromatic leukodystrophy - MLD (cuARSA); Hepatitis C (anti-HCV) RNAi); Limb-girdle muscular dystrophy type 2D (hSGCA); Human immunodeficiency virus infection (PG9DP); Acute intermittent porphyria (PBGD); Leber hereditary optic neuropathy (PIND4v2); Alpha-1 antitrypsin deficiency (alphaIAT); X-linked retinoschisis (RS1); Colloideremia (hCHM); Giant axonal neuropathy (GAN); Hemophilia B (factor IX); Homozygous FH (hLDLR); Dyspheric dysphoric disorder (DYSF); Color blindness (CNGA3 or CNGB3); Progressive supranuclear palsy (MAPT; anti-Tau; anti-MAPT RNAi); Ornithine transcarbamylase deficiency (OTC); Hemophilia A (factor VIII); Age-related macular degeneration (AMD), including wet AMD (anti-VEGF antibody or RNAi); X-linked retinitis pigmentosa (RPGR); Myotonic dystrophy type 1 (DMPK; anti-DMPK) RNAi, including anti-CTG trinucleotide repeat RNAi); myotonic dystrophy type 2 (CNBP); facioscapulohumeral muscular dystrophy (D4Z4 DNA);Oculopharyngeal muscular dystrophy (PABPN1; mutated PABPN1 inhibitors (e.g., RNAi)); mucopolysaccharidosis type VI (hARSB); Leber hereditary optic neuropathy (ND4); X-linked myotubular myopathy (MTM1); Crigler-Najjar syndrome (UGT1Al); retinitis pigmentosa (hPDE6B); mucopolysaccharidosis type 3B (hNAGLU); Duchenne muscular dystrophy (GALGT2); Alzheimer's disease (NGF; ApoE4; ApoE2; ApoE3; anti-ApoE RNAi, MAPT, anti-Tau antibodies, anti-amyloid-beta antibodies (e.g., aducanumab); multiple system atrophy; familial lipoprotein lipase deficiency (LPL); alpha-1 antitrypsin deficiency (hAAT); Leber congenital amaurosis 2 (hRPE65v2); Batten disease; late infantile lipofuscinosis (CLN2); Huntington's disease (HTT; anti-HTT RNAi); fragile X syndrome (FMR1); Leber hereditary optic neuropathy (PlND4v2); aromatic amino acid decarboxylase deficiency (hAADC); retinitis pigmentosa (hMERKTK); and retinitis pigmentosa (RLBPl). In some embodiments, the payload contains molecules effective in treating muscle-related diseases. Exemplary, non-limiting muscle-related diseases are listed below.

[0327] In some embodiments, the xenotransgene encodes a therapeutic polypeptide. In some embodiments, the xenotransgene is a human gene or a fragment thereof. In some embodiments, the therapeutic polypeptide is a human protein. In some embodiments, the xenotransgene encodes an antibody or a fragment thereof (e.g., antibody light chain, antibody heavy chain, Fab, or scFv). Examples of antibodies or fragments encoded by xenotransgenes include, but are not limited to, anti-Ab antibodies (e.g., solanezumab, GSK933776, and lecanemab), anti-soltirin (e.g., AL-001), anti-Tau (e.g., ABBV-8E12, UCB-0107, and NI-105), anti-SEMA4D (e.g., VX15 / 2503), and anti-alpha-synuclein (e.g., pracinezumab, NI-202, and MED). -1341), anti-SOD1 (e.g., NI-204), anti-CGRP receptor (e.g., eptinezumab, fremanezumab, or galcanezumab), anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALKl (e.g., askrinbakumab), anti-C5 (e.g., tesidorumab, ravulizumab, and eculizumab), anti-CD105 (e.g., caro Anti-Tunicine (Tuximab), anti-CClQ (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamlevlumab), anti-IL6R (e.g., satralizumab, tocilizumab, and sarilumab), anti-IL6 (e.g., siltuximab, crazakizumab, silukumab, orokizumab) Anti-IL-4R (e.g., dupilumab), anti-IL-17A (e.g., ixekizumab and secukinumab), anti-IL-5R (e.g., reslizumab), anti-IL-5 (e.g., benralizumab and mepolizumab), anti-IL-13 (e.g., tralokinumab), anti-IL-12 / IL-23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-IL-31RA (e.g., nemolizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOSTmAb (e.g., romosozumab), anti-IgE (e.g., omalizumab), anti-TSLP (e.g., nemolizumab), anti-pKal Examples include mAbs (e.g., lanadermab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., denosumab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lamparizumab), or anti-MMP9 (e.g., andecaliximab), and optionally, the heavy chain (Fab and Fc regions) and light chain are separated by autocleaved furin(F) / F2A or furin(F) / T2A, IRES sites, or flexible linkers to ensure, for example, the expression of equal amounts of heavy and light chain polypeptides.

[0328] In some embodiments, the viral particle contains a xenotransgene encoding a genome editing system. Examples include CRISPR genome editing systems (e.g., one or more components of a CRISPR genome editing system, such as a guide RNA molecule and / or an RNA-induced nuclease such as Cas9, Cpf1, etc.), zinc finger nuclease genome editing systems, TALEN genome editing systems, or meganuclease genome editing systems. In embodiments, the genome editing system targets a target sequence in the mammalian genome, e.g., human genome. In embodiments, the viral particle contains a xenotransgene encoding a targetable transcription factor. Examples include CRISPR-based transcription factors (e.g., one or more components of a CRISPR-based transcription factor, such as a guide RNA molecule and / or an enzymatically inactive RNA-induced nuclease / transcription factor ("TF") fusion protein, e.g., dCas9-TF fusion, dCpf1-TF fusion, etc.), zinc finger transcription factor fusion proteins, TALEN transcription factors, or meganuclease transcription factors.

[0329] In some embodiments, the therapeutic molecule or component of the system is delivered by more than one unique viral particle (e.g., an aggregate containing more than one unique viral particle). In other embodiments, the therapeutic molecule, or component of the therapeutic molecule or system, is delivered by a single unique viral particle (e.g., an aggregate containing a single unique viral particle).

[0330] In embodiments, the transgene encodes any biologically active product or other product, such as a product desirable for testing. Suitable transgenes, but not limited to those described herein, can be readily selected by those skilled in the art.

[0331] Other examples of proteins encoded by transgenes include, but are not limited to, colony-stimulating factors (CSF); blood factors such as β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; interleukins; soluble receptors, e.g., soluble TNF-α receptor, soluble VEGF receptor, soluble interleukin receptor (e.g., soluble IL-1 receptor and soluble IL-2 receptor). Examples include ligand-binding fragments of IL-1 receptors (or soluble receptors); growth factors, e.g., keratinocyte growth factor (KGF), stem cell factor (SCF), or fibroblast growth factor (FGF, e.g., basic FGF and acidic FGF); enzymes; chemokines; enzyme activators, e.g., tissue plasminogen activator; angiogenic agents, e.g., vascular endothelial growth factor, glioma-derived growth factor, angiogenin, or angiogenin-2; anti-angiogenic agents, e.g., soluble VEGF receptors; protein vaccines; neuroactive peptides, e.g., nerve growth factor (NGF) or oxytocin; thrombolytic agents; tissue factor; macrophage activators; tissue inhibitors of metalloproteinases; or IL-1 receptor antagonists.

[0332] This disclosure further, in part, relates to methods for delivering a payload to a subject, e.g., an animal or human subject. In some embodiments, the method for delivering a payload to a subject includes, for example, administering to the subject dependent parvovirus particles containing a variant polypeptide (e.g., those described herein) containing the payload in an amount and time sufficient to deliver the payload. In some embodiments, the dependent parvovirus particles are dependent parvovirus particles described herein and contain the payload described herein. In some embodiments, the particles deliver the payload to the CNS. In some embodiments, delivery to the CNS is increased compared to particles without the variant capsid polypeptide or compared to particles having a wild-type capsid polypeptide, e.g., the capsid polypeptide of SEQ ID NO: 1. In some embodiments, the particles deliver the payload to skeletal muscle tissue. In some embodiments, delivery to skeletal muscle tissue is increased compared to particles without the variant capsid polypeptide or compared to particles having a wild-type capsid polypeptide, e.g., the capsid polypeptide of SEQ ID NO: 1.

[0333] In some embodiments, a viral particle containing a genome is provided, the genome comprising a nucleic acid expression construct containing a xenotransgene and one or more regulatory elements, the one or more regulatory elements comprising a muscle (e.g., skeletal muscle) specific promoter. Examples of muscle-specific promoters that can be used to drive the expression of transgenes in skeletal muscle include 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 C5-12), alpha-skeletal actin (ASKA), SP-301, E-syn, myosin light chain (MLC), myosin heavy chain (MHC), 4 and semi-LIM domain protein 1 (FHL1), alpha-2 actinin (ACTN2), filamin-C (FLNC), sarcoplasmic / endoplasmic reticulum calcium ATPase 1 (ATP2A1), and troponin I. Examples include, but are not limited to, type 1 (TNNI1), myosin-1 (MYH1), phosphorylated, skeletal muscle myosin light chain (MYLPF), alpha-3 chain tropomyosin (TPM3), Pitx3, and the ankyrin repeat domain-containing protein 2 (ANKRD2) promoter. Promoters capable of driving expression in skeletal 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 et al., 2007, JBC 282(45):33192-33200, WO2021 / 127655, and WO2023 / 006890, the contents of each of these are incorporated herein by reference in their entirety.

[0334] In some embodiments, the payload includes molecules effective in treating muscle diseases, such as proteins or RNA-interfering nucleotides (e.g., shRNA, siRNA, or miRNA).

[0335] Exemplary muscle tissue-related diseases that can be treated include acid maltase deficiency (AMD), amyotrophic lateral sclerosis (ALS), Andersen-Tawil syndrome, Barth syndrome (TAZ), Becker muscular dystrophy (BMD), congenital Becker myotonia, Bethlem myopathy, spinal and bulbar muscular atrophy (spinal bulbar muscular atrophy), carnitine deficiency, carnitine palmitoyltransferase deficiency (CPT deficiency), central core disease (CCD), central nucleus myopathy, Charcot-Marie-Tooth disease (CMT), congenital muscular dystrophy (CMD), congenital myasthenic syndrome (CMS), congenital myotonic dystrophy, C Ori disease (debranching enzyme deficiency), Danon disease, debranching enzyme deficiency, Dejerine-Sottas disease (DSD), dermatomyositis (DM), distal muscular dystrophy (DD), distal myopathy with anterior tibial onset, Duchenne muscular dystrophy (DMD), myotonic dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), endocrine myopathy, Eulenberg disease (congenital paramyotonia), facioscapulohumeral muscular dystrophy (FSH or FSHD), Finnish type (tibial) distal myopathy, Forbes disease (debranching enzyme deficiency), Friedreich ataxia (FA), Fukuyama congenital muscular dystrophy, glycogen storage disease type 10, glycogen storage disease type 11, glycogen storage disease type 2, glycogen storage disease type 3, glycogen storage disease type 5, glycogen storage disease type 7, glycogen storage disease type 9, Gowers-Laing distal myopathy, Hauptmann-Thanheuser MD (Emery-Dreifuss muscular dystrophy), hereditary inclusion body myositis, hereditary motor sensory neuropathy (Charcot-Marie-Tooth disease), hyperthyroidism myopathy, hypothyroidism myopathy, inclusion body myositis (IBM), hereditary myopathy, integrin-deficient congenital muscular dystrophy, Kennedy disease (spinal muscular atrophy), Kugelberg-Welander disease (spinal muscular atrophy), lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome (LEMS), limb-girdle muscular dystrophy (LGMD), LouGehrig's disease (amyotrophic lateral sclerosis), McArdle's disease (phosphorylase deficiency), merosin-deficient congenital muscular dystrophy, metabolic disorders of muscle, mitochondrial myopathy, Miyoshi myopathy, Miyoshi distal myopathy, motor neuron disease, muscle-ocular-brain disease, myasthenia gravis (MG), myoadenylate deaminase deficiency, myofibrillar myopathy, muscle phosphorylase deficiency, congenital myotonia (MC), myotonic muscular dystrophy (MMD), myotyl tubular myopathy (MTM or MM), nemaline myopathy, mid-to-distal myopathy, oculopharyngeal muscular dystrophy (OPMD), congenital paramyotonia, Pearson syndrome, periodic paralysis, peroneal muscle atrophy (Charcot-Marie-Tooth disease), phosphofructokinase deficiency, phosphoglyceryl kinase deficiency This includes, but is not limited to, ZASP-related myopathy, including ze deficiency, phosphoglycerate mutase deficiency, phosphorylase deficiency, phosphorylase deficiency, polymyositis (PM), Pompe disease (acid maltase deficiency), primary merosin deficiency (LAMA2), progressive extraocular muscle palsy (PEO), rod disease (nemaline myopathy), spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), Steinert disease (myotonic dystrophy), Tarui disease (phosphofructokinase deficiency), Thomsen disease (congenital myotonia), Ullrich-type congenital muscular dystrophy, Walker-Warburg syndrome (congenital muscular dystrophy), distal Welander myopathy, Werdnig-Hoffmann disease (spinal muscular atrophy), and ZASP-related myopathy.

[0336] Suitable payloads for treating muscle-related diseases are known in the art and include combinations of the following diseases (suitable payloads): Barth syndrome (TAZ), primary merosin deficiency (LAMA2), Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Danon disease (LAMP2), and limb-girdle muscular dystrophy (subtypes and affected genes: LGMD1A (TTID), LG MD1B (LMNA), LGMD1C (CAV3), LGMD1D (DNAJB6), LGMD1E (DES), LGMD1F (TNP03), LGMD1G (HNRPDL), LGMD1H, LGMD2A (CAPN 3), LGMD2B (DYSF), LGMD2C (SGCG), LGMD2D (SGCA), LGMD2E (SGCB), LGMD2F (SGCD), LGMD2G (TCAP), LGMD2H (TRIM32), LGM D2I (FKRP), LGMD2J (TTN), LGMD2K (POMT1), LGMD2L (AN05), LGMD2M (FKTN), LGMD2N (POMT2), LGMD20 (POMGNT1), LGMD2Q (PLEC1), Miyoshi myopathy (DYSF), distal myopathy with anterior tibial onset (DYSF), Welander distal myopathy (TIA1), Gowers-Laing distal myopathy (MYH7), facial and shoulder myopathy Gulohumeral muscular dystrophy (subtypes and affected genes: type 1 (DUX4), type 2 (SMCHD1)), oculopharyngeal muscular dystrophy (PABPN1), myotonic dystrophy (subtypes and affected genes: DM1 (DMPK) and DM2 (ZNF9)), congenital myotonia (CLCN1), congenital paramyotonia (SCN4A), myotyl myopathy (MTM1), and glycogen storage disorder type II (Pompe disease) (GAA).

[0337] In some embodiments, the payload is selected from TAZ, LAMA2, DMD, LAMP2, CAPN3, DYSF, SGCA, SGCB, FKRP, PABPN1, MTM1, and GAA.

[0338] Further exemplary diseases that can be treated, and further exemplary xenotransgenes that can be delivered via the viral particles of this disclosure, are provided in Tables 2, 3, and 4. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 4]

[0339] 5.8. Treatment method This disclosure, in part, relates to methods for treating diseases or conditions in subjects, e.g., animals or human subjects. In some embodiments, a method for treating a disease or condition in a subject involves administering dependent parvovirus particles, e.g., a payload, comprising a variant polypeptide as described herein, to the subject. In some embodiments, dependent parvovirus particles, e.g., a payload, comprising a variant polypeptide, comprising a payload as described herein, are administered in an effective amount and / or time to treat the disease or condition. In some embodiments, the payload is a therapeutic product. In some embodiments, the payload is, e.g., a nucleic acid encoding an exogenous polypeptide. This disclosure also relates to dependent parvovirus particles, e.g., a variant polypeptide, comprising a payload as described herein, for use in therapeutic methods as described herein. This disclosure also relates to the use of dependent parvovirus particles, e.g., a payload, comprising a variant polypeptide, comprising a variant polypeptide, comprising a payload as described herein, for the manufacture of pharmaceuticals for treating diseases or conditions as described herein.

[0340] Depend parvovirus particles containing variant polypeptides described herein or produced by the methods described herein can be used to express one or more therapeutic proteins for treating various diseases or disorders. In some embodiments, the disease or disorder is cancer, such as carcinoma, sarcoma, leukemia, lymphoma, or autoimmune disease, such as multiple sclerosis. Non-exclusive examples of carcinomas include esophageal carcinoma; bronchogenic lung 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 and non-small cell lung carcinoma; adrenal cortical 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 or cholangiocarcinoma; choriocarcinoma; seminoma; fetal carcinoma; Wilms' tumor; cervical carcinoma; epithelial carcinoma; and nasopharyngeal carcinoma. Non-exclusive examples of sarcomas include fibrosarcoma, myxosarcoma, liposarcoma, angiosarcoma, endosarcoma, lymphangiosarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Non-exclusive examples of solid tumors include ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Non-exclusive examples of leukemia include chronic myeloproliferative syndromes; T-cell CLL prelymphocytic leukemia, acute myeloid leukemia; chronic lymphocytic leukemia, including B-cell CLL and hairy cell leukemia; and acute lymphoblastic leukemia. Examples of lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt lymphoma and Hodgkin lymphoma. In some embodiments, the disease or disorder is a hereditary disorder.In some embodiments, hereditary disorders include sickle cell disease, impaired glycogen storage (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 I, Tay-Sachs disease, phenylketonuria, mucopolysaccharidosis, galactosemia, muscular dystrophy (e.g., Duchenne muscular dystrophy), hemophilia, e.g., hemophilia A (classical hemophilia) or hemophilia B (Christmas disease), Wilson's disease, Fabry disease, Gaucher disease, hereditary angioedema (HAE), and alpha-1 antitrypsin deficiency. Examples of other diseases or disorders are provided above in Section 5.7.

[0341] In some aspects, a disease or condition is a disease of the CNS. Exemplary diseases of the CNS include septal defect pellucida, acid lipase disorder, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie pupil, Adie syndrome, adrenoleukodystrophy, corpus callosum aplasia, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, AIDS-neurological complications, Alexander's disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, and hematologic dysplasia. Candimatous malformation, Angleman syndrome, anoxia, antiphospholipid antibody syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia, telangiectatic ataxia, ataxia and degeneration of the cerebellum or spinocerebellum, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, back pain, Barth syndrome, Batten's disease, Becker's type of muscle tone, Bechet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscle atrophy, benign cerebrospinal fluid Intracranial hypertension, Bernhardt-Roth syndrome, Binswanger disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus rupture, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal cord tumors, cerebral aneurysm, brain injury, Brown-Sequard syndrome, bulbar palsy, spinal and bulbar muscular atrophy, autosomal dominant cerebral artery disease with subcortical infarction and progressive leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, burning pain, cavernous cyst, cavernous hemangioma, cavernous dysplasia, central cervical myelopathy Syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, head injury, ceramidase deficiency, cerebellar degeneration, cerebellar dysplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation, cerebral gigantism, cerebral hypoxia, cerebral palsy, cerebro-ocular-facial-skeletal syndrome (COFS), Charcot-Marie-Tooth disease, Chiari malformation, cholesterol ester storage disorder, chorea, acanthocyanotic chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, CoffinLowry syndrome, corpus callosum agenesis, coma, complex regional pain syndrome, concentric sclerosis (Balo sclerosis), congenital bilateral facial nerve palsy, congenital myasthenia gravis, congenital myopathy, congenital vascular cavernous malformation, corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob disease, chronic progressive extraocular muscle palsy, cumulative traumatic injury, Cushing's syndrome, giant cell inclusion disease, cytomegalovirus infection, dancing eyes / dancing feet syndrome, Dandy-Walker syndrome, Dawson's disease, DeMorsier syndrome, Dejerine-Klumpke palsy, dementia, multiple infarct dementia, semantic dementia, subcortical dementia, Lewy body dementia, demyelinating disease, dentatecerebellar ataxia, dentatorubral atrophy, dermatomyositis, developmental integrative motor disorder, Devic syndrome, diabetic neuropathy, diffuse sclerosis, distal inherited motor neuropathy, Dravet syndrome, autonomic neuropathy, dysgraphia, dyslexia, dysphagia, dyspragmatics, myoclosis - Nusian cerebellar synkinesis, progressive cerebellar synkinesis, dystonia, early infantile epileptic encephalopathy, empty cell syndrome, encephalitis, encephalitis lethargica, brain herniation, encephalomyelitis, encephalopathy, encephalopathy (familial infantile), trigeminal nerve hemangioma, epilepsy, epileptic hemiplegia, paroxysmal ataxia, Erb's palsy, Erb-Duchenne and Dejerine-Klumpke palsy, essential tremor, extrapontine myelinolysis, Faber's disease, Fabry's disease, Fahr syndrome, syncope, familial autonomic neuropathy, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial convulsive paralysis, Farber disease, febrile seizures, fibromuscular dysplasia, Fisher syndrome, hypotonia syndrome, foot drop, fragility X syndrome, Friedreich ataxia, frontotemporal dementia, Gaucher disease, systemic gangliosidosis (GM1, GM2), Gerstmann syndrome, Gerstmann n-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, persistent hemicalcium, hemifacial spasm, alternating hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, hereditary polyneurotic ataxia, herpes zosterZoster, herpes zoster otophylaxis, Hirayama disease, Holmes-Adie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, Hurler syndrome, hydrocephalus anencephaly, hydrocephalus, normal pressure hydrocephalus, hydroplegia, adrenal cortical hypertonia, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxial dystrophy, infantile phytanate storage, infantile Refsum disease, infantile spasm, inflammatory myopathy, foramenencephaly, enteric lipodystrophy, intracranial cyst, intracranial hypertension, Isaacs syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy's disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Kluever-Bucy syndrome, Korsakoff amnestic syndrome, Krabbe disease, Kugelberg-Welander disease, Kuru disease, Lambert-Eaton myasthenic syndrome, Landau-K leffner syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, Levine-Critchley syndrome, Lewy body dementia, Lichtheim disease, lipid storage disease, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's disease, lupus - neurological sequelae, Lyme disease - neurological complications, lysosomal storage disorders, Machado-Joseph disease, megaencephalopathy, megaencephaly, Melkersson-Rosenthal syndrome, meningitis, meningitis and encephalitis, Menkes disease, paresthesia of femoral pain, metachromatic leukodystrophy, microcephaly, migraine, MillerFisher syndrome, petit mal seizures, mitochondrial myopathy, mitochondrial DNA depletion syndrome, Moebius syndrome, unilateral muscle atrophy, Morvan syndrome, motor neuron disease, moyamoya disease, mucolipidosis, mucopolysaccharidosis, multiple infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, congenital myasthenia gravis, demyelinating diffuse sclerosis, myelitis, infantile myoclonic encephalopathy, myoclonus, myoclonic epilepsy, myopathy, congenital myopathy, thyroid-toxic myopathy, myotonia, congenital myotonia, narcolepsy, NARP (neuropathy, ataxia and retinitis pigmentosa), neuroacidosis, neurodegeneration with cerebral iron accumulation, neurodegenerative diseases, neurofibromatosis, neuroleptic malignant syndrome, AIDS Neurological complications, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological signs of Pompe disease, neurological sequelae of lupus, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorders, neuropathic pain, hereditary neuropathy, neuropathy, neurosarcoidosis, neurosyphilis, neurotoxicity, spongiform nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, pantothenate kinase-associated neurodegeneration, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal hemiparesis, Parry-Romberg disease, Pelizaeus-Merzbacher disease, Pena Shokeir type II syndrome, perineurial cyst, peroneal muscle atrophy, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage, Pick's disease, nerve compression, piriformis syndrome, pituitary tumor, polymyositis, Pompe's disease, porencephaly, post-polio syndrome, postherpetic neuralgia, post-infectious encephalomyelitis, post-orthostatic hypotension, post-orthostatic tachycardia syndrome, post-orthostatic tachycardia syndrome, primary dentate atrophyAtrophy), primary lateral sclerosis, primary progressive aphasia, prion disease, progressive bulbar palsy, progressive hemifacial atrophy, progressive ataxia, progressive multifocal leukoencephalopathy, progressive muscular atrophy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, pseudobulbar palsy, pseudotorch syndrome, pseudotoxoplasmosis, pseudotumor, psychogenic movement disorders, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Rasmussen encephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, Refsum disease in infants, repetitive movement disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye syndrome, rheumatic encephalitis, Riley-Day syndrome, sacral nerve root cyst, St. Vitus chorea, salivary gland disease, Sandhoff disease, Schilder disease, schizencephaly, S Eitelberger disease, paroxysmal disorder, semantic dementia, septal-optic dysplasia, severe myoclonic epilepsy of infants (SMEI), shaken baby syndrome, herpes zoster (Shingles), Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, sporadic ataxia, S Teele-Richardson-Olszewski syndrome, Stiff-Person syndrome, striatonigral degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, short-acting persistent hemiglinal headache (SUNCT), dysphagia, Sydenham's chorea, fainting, syphilitic myelosclerosis, hydromyelopathy of the spinal cord, syringomyelia, systemic lupus erythematosus, spinal fistula, tardive dyskinesia, Tarlov cyst, Tay - Sachs disease, temporal arteritis, tethered cord syndrome, Thomsen myotonia, thoracic outlet syndrome, thyroid toxic myopathy, painful tics, Todd's palsy, Tourette syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraplegia, Troyer syndrome, tuberous sclerosis, vascular erectile neoplasm, vasculitis syndromes of the central and peripheral nervous systems, vitamin B12 deficiency, Von Economo disease, Von Hippel-Lindau disease (VHL), VonThis includes Recklinghausen's disease, Wallenberg syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome, whiplash, Whipple's disease, Williams syndrome, Wilson's disease, Wolman's disease, and X-linked spinal and medullary muscular atrophy. Examples of other diseases or disorders are provided above in Section 5.7.

[0342] In several aspects, the disease or condition is a disease of skeletal muscle. Examples of diseases of skeletal muscle include acid maltase deficiency (AMD), amyotrophic lateral sclerosis (ALS), Andersen-Tawil syndrome, Becker muscular dystrophy (BMD), congenital Becker myotonic syndrome, Bethlem myopathy, spinal and bulbar muscular atrophy (spinal bulbar muscular atrophy), carnitine deficiency, carnitine palmitoyltransferase deficiency (CPT deficiency), central core disease (CCD), central nucleus myopathy, Charcot-Marie-Tooth disease (CMT), congenital muscular dystrophy (CMD), congenital myasthenic syndrome (CMS), congenital myotonic dystrophy, Cori disease (debranching enzyme deficiency), debranching enzyme deficiency, Dejerine-Sottas disease (DSD), dermatomyositis (D M), distal muscular dystrophy (DD), Duchenne muscular dystrophy (DMD), myotonic dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), endocrine myopathy, Eulenberg disease (congenital paramyotonia), facioscapulohumeral muscular dystrophy (FSH or FSHD), Finnish (tibial) distal myopathy, Forbes disease (debranching enzyme deficiency), Friedreich ataxia (FA), Fukuyama congenital muscular dystrophy, glycogen storage disease type 10, glycogen storage disease type 11, glycogen storage disease type 2, glycogen storage disease type 3, glycogen storage disease type 5, glycogen storage disease type 7, glycogen storage disease type 9, Gowers-Laing distal myopathy, Hauptmann-Thanheuser MD (Emery-Dreifuss muscular dystrophy), hereditary inclusion body myositis, hereditary motor sensory neuropathy (Charcot-Marie-Tooth disease), hyperthyroidism, hypothyroidism, inclusion body myositis (IBM), hereditary myopathy, integrin-deficient congenital muscular dystrophy, Kennedy disease (spinal muscular atrophy), Kugelberg-Welander disease (spinal muscular atrophy), lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome (LEMS), limb-girdle muscular dystrophy (LGMD), LouGehrig's disease (amyotrophic lateral sclerosis), McArdle's disease (phosphorylase deficiency), merosin-deficient congenital muscular dystrophy, metabolic disorders of muscle, mitochondrial myopathy, distal myopathy, motor neuron disease, muscle-ocular-brain disease, myasthenia gravis (MG), myoadenylate deaminase deficiency, myofibrillar myopathy, muscle phosphorylase deficiency, congenital myotonia (MC), myotonic muscular dystrophy (MMD), myotyl tubular myopathy (MTM or MM), nemaline myopathy, distal myopathy, oculopharyngeal muscular dystrophy (OPMD), congenital paramyotonia, Pearson syndrome, periodic paralysis, peroneal muscle atrophy (Charcot-Marie-Tooth disease), phosphofructokinase deficiency, This includes phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphorylase deficiency, phosphorylase deficiency, polymyositis (PM), Pompe disease (acid maltase deficiency), progressive extraocular muscle palsy (PEO), rod disease (nemaline myopathy), spinal muscular atrophy (SMA), spinal muscular atrophy (SBMA), Steinert disease (myotonic dystrophy), Tarui disease (phosphofructokinase deficiency), Thomsen disease (congenital myotonia), Ullrich type congenital muscular dystrophy, Walker-Warburg syndrome (congenital muscular dystrophy), distal Welander myopathy, Werdnig-Hoffmann disease (spinal muscular atrophy), and ZASP-associated myopathy.

[0343] In some embodiments, the disease or condition is a disease affecting the CNS and muscles, such as SMA, multiple sclerosis, amyotrophic lateral sclerosis (ALS), ataxia, Becker muscular dystrophy, Charcot-Marie-Tooth disease, dystonia, Friedreich ataxia, glycogen storage disorder II, Kennedy disease, Lambert-Eaton myasthenic syndrome, mitochondrial DNA depletion syndrome, myo-ophthalm-brain disease, neuromyotonia, periodic paralysis, juvenile primary lateral sclerosis, progressive extraocular palsy, spastic paraplegia, congenital Stiff-Person syndrome, tardive dyskinesia, Werdnig-Hoffman disease, or X-linked spinal and medullary muscular atrophy.

[0344] In some embodiments, administration of dependent parvovirus particles containing a variant polypeptide and a payload (e.g., a transgene as described in Section 5.7 or its subparts) to a subject induces the expression of the payload (e.g., a transgene) in the subject. In some embodiments, the expression is induced in the CNS. In some embodiments, production is similar in the CNS compared to particles similar to wild-type capsid protein. In some embodiments, production is increased in the CNS compared to particles similar to wild-type capsid protein, e.g., particles having the capsid protein of SEQ ID NO: 1. In some embodiments, the expression is induced in skeletal muscle. In some embodiments, production is similar in muscle compared to particles similar to wild-type capsid protein, e.g., particles having the capsid protein of SEQ ID NO: 1. In some embodiments, production is increased in skeletal muscle compared to particles similar to wild-type capsid protein (e.g., particles having the capsid protein of SEQ ID NO: 1). The amount of payload expressed in the subject (e.g., the subject's serum), e.g., a transgene, e.g., a heterologous protein, e.g., a therapeutic polypeptide, can vary. For example, in some embodiments, the payload, such as the protein or RNA product of the transgene, can be expressed in the serum of the subject at a concentration of at least about 9 μg / ml, at least about 10 μg / ml, at least about 50 μg / ml, at least about 100 μg / ml, at least about 200 μg / ml, at least about 300 μg / ml, at least about 400 μg / ml, at least about 500 μg / ml, at least about 600 μg / ml, at least about 700 μg / ml, at least about 800 μg / ml, at least about 900 μg / ml, or at least about 1000 μg / ml.In some embodiments, the payload, for example, the protein or RNA product of the transgene, is expressed in the serum of the subject at an amount of approximately 9 μg / ml, approximately 10 μg / ml, approximately 50 μg / ml, approximately 100 μg / ml, approximately 200 μg / ml, approximately 300 μg / ml, approximately 400 μg / ml, approximately 500 μg / ml, approximately 600 μg / ml, approximately 700 μg / ml, approximately 800 μg / ml, approximately 900 μg / ml, approximately 1000 μg / ml, approximately 1500 μg / ml, approximately 2000 μg / ml, approximately 2500 μg / ml, or in a range between any two of these values.

[0345] In some embodiments, for therapeutic use, viral particles containing the capsid polypeptide described herein are prepared as a pharmaceutical composition. As used herein, the term “pharmaceutical composition” means a composition comprising at least one active ingredient (e.g., viral particles) and optionally one or more pharmaceutically acceptable carriers or excipients.

[0346] The relative amounts of the active ingredient, pharmaceutically acceptable carriers or excipients, and / or any additional ingredients in the pharmaceutical compositions described herein may vary. Differences in the composition of the pharmaceutical compositions may depend on the identity, size, and / or condition of the target being treated, the route by which the composition is administered, and / or any other factors. The compositions may contain 0.0001% to 99% (w / w) of the active ingredient. For example, the compositions may contain 0.0001% to 100%, e.g., 5 to 50%, 1 to 30%, 5 to 80%, or at least 80% (w / w) of the active ingredient. Non-limiting examples of carriers and / or excipients include solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, or combinations thereof.

[0347] 6. Numbered Embodiments While various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by the numbered embodiments described below. Unless otherwise specified, any features of the concepts, aspects and / or embodiments described in the detailed description above will apply mutatis mutandis to any of the numbered embodiments described below.

[0348] The following numbered embodiments refer to viral particles, which will be understood to be engineered particles comprising a capsid polypeptide and a nucleic acid, which do not exist together in nature. Furthermore, the nucleic acid may contain components that do not exist together in nature. For example, the nucleic acid may include a payload (e.g., a nucleotide sequence of a transgene that is unnaturally present and may encode a variant polypeptide), one or more regulatory elements (which may include regulatory sequences such as a variant promoter, which are unnaturally present), and an AAV ITR, where two or more of such components do not exist together in nature. In some embodiments, (a) the viral particle comprises an ITR from the genome of a variant capsid polypeptide of a first serotype (e.g., variant AAV9 capsid polypeptide) and an ITR from the genome of a different serotype of AAV (e.g., AAV2 ITR), and / or (b) the payload is a transgene encoding a human polypeptide or a variant thereof. 1. Capsid polypeptide, (a) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) A capsid polypeptide comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 2. In capsid polypeptides, improvements were made. (a) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 3. The capsid polypeptide according to Embodiment 1 or 2, comprising serine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 4. The capsid polypeptide according to Embodiment 1 or 2, comprising threonine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 5. The capsid polypeptide according to any one of Embodiments 1 to 4, wherein the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1 is valine-free. 6. The capsid polypeptide according to any one of Embodiments 1 to 5, wherein the alanine is not present at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1. 7. Capsid polypeptide, (a) Alanine, arginine, serine, threonine, or valine at the position corresponding to T593 of the VP1 capsid polypeptide of SEQ ID NO: 1, (b) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) A capsid polypeptide comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 8. In capsid polypeptides, improvements were observed. (a) Alanine, arginine, serine, threonine, or valine at the position corresponding to T593 of the VP1 capsid polypeptide of SEQ ID NO: 1, (b) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 9. The capsid polypeptide according to Embodiment 7 or 8, comprising alanine at the position corresponding to T593 of the VP1 capsid polypeptide of SEQ ID NO: 1. 10. The capsid polypeptide according to Embodiment 7 or 8, comprising arginine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 11. The capsid polypeptide according to Embodiment 7 or 8, comprising serine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 12. The capsid polypeptide according to Embodiment 7 or 8, comprising threonine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 13. The capsid polypeptide according to Embodiment 7 or 8, comprising valine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 14. The capsid polypeptide according to any one of Embodiments 7 to 13, comprising serine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 15. The capsid polypeptide according to any one of Embodiments 7 to 13, comprising threonine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 16. The capsid polypeptide according to any one of Embodiments 7 to 15, wherein the alanine is not present at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1. 17. Capsid polypeptide, (a) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1, (b) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) A capsid polypeptide comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 18. In capsid polypeptides, improvements were observed. (a) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1, (b) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 19. The capsid polypeptide according to Embodiment 17 or 18, comprising alanine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1. 20. The capsid polypeptide according to Embodiment 17 or 18, comprising tryptophan at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1. 21. The capsid polypeptide according to Embodiment 17 or 18, comprising tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1. 22. The capsid polypeptide according to any one of Embodiments 17 to 21, comprising serine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 23. The capsid polypeptide according to any one of Embodiments 17 to 21, comprising threonine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 24. The capsid polypeptide according to any one of embodiments 17 to 23, wherein the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1 is valine-free. 25. Capsid polypeptide, (a) Alanine, arginine, serine, threonine, or valine at the position corresponding to T593 of the VP1 capsid polypeptide of SEQ ID NO: 1, (b) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1, (c) Leucine is located at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (d) A capsid polypeptide comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 26. In capsid polypeptides, improvements were observed. (a) Alanine, arginine, serine, threonine, or valine at the position corresponding to T593 of the VP1 capsid polypeptide of SEQ ID NO: 1, (b) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1, (c) Leucine is located at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (d) comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 27. The capsid polypeptide according to Embodiment 25 or 26, comprising alanine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 28. The capsid polypeptide according to Embodiment 25 or 26, comprising arginine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 29. The capsid polypeptide according to Embodiment 25 or 26, comprising serine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 30. The capsid polypeptide according to Embodiment 25 or 26, comprising threonine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 31. The capsid polypeptide according to Embodiment 25 or 26, comprising valine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1. 32. The capsid polypeptide according to any one of embodiments 25 to 31, comprising alanine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1. 33. The capsid polypeptide according to any one of embodiments 25 to 31, comprising tryptophan at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1. 34. The capsid polypeptide according to any one of embodiments 25 to 31, comprising tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1. 35. The capsid polypeptide according to any one of embodiments 25 to 34, comprising serine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 36. The capsid polypeptide according to any one of embodiments 25 to 34, comprising threonine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 37. Capsid polypeptide, (a) Valine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) Alanine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) Leucine is located at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (d) A capsid polypeptide comprising serine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 38. In capsid polypeptides, improvements were observed. (a) Valine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) Alanine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) Leucine is located at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (d) comprising serine at the position corresponding to N598 of the VP1 capsid polypeptide of SEQ ID NO: 1. 39. The capsid polypeptide according to any one of Embodiments 1 to 38, wherein the alanine is not present at the position corresponding to I601 of the VP1 capsid polypeptide of Sequence ID No. 1. 40. The capsid polypeptide according to any one of Embodiments 1 to 38, comprising alanine at the position corresponding to I601 of the VP1 capsid polypeptide of Sequence ID No. 1. 41. The capsid polypeptide according to any one of Embodiments 1 to 38, comprising isoleucine at the position corresponding to I601 of the VP1 capsid polypeptide of Sequence ID No. 1. 42. The capsid polypeptide according to any one of Embodiments 1 to 38, comprising valine at the position corresponding to I601 of the VP1 capsid polypeptide of Sequence ID No. 1. 43. Capsid polypeptide, wherein valine is present at the position corresponding to I601 of the VP1 capsid polypeptide of SEQ ID NO: 1, and optionally, (a) Alanine, arginine, serine, threonine, or valine at the position corresponding to T593 of the VP1 capsid polypeptide of SEQ ID NO: 1 (b) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1 (c) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of SEQ ID NO: 1, (d) A capsid polypeptide comprising one, two, three, or all four serine or threonine molecules at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No. 1. 44. In capsid polypeptides, the improvement involves adding valine at the position corresponding to I601 of the VP1 capsid polypeptide of SEQ ID NO: 1, and optionally, (a) Alanine, arginine, serine, threonine, or valine at the position corresponding to T593 of the VP1 capsid polypeptide of SEQ ID NO: 1 (b) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of SEQ ID NO: 1 (c) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of SEQ ID NO: 1, (d) The VP1 capsid polypeptide of Sequence ID No. 1 contains one, two, three, or all four serine or threonine molecules at the position corresponding to N598. 45. The capsid polypeptide according to any one of Embodiments 1 to 44, comprising alanine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No. 1. 46. ​​The capsid polypeptide according to any one of Embodiments 1 to 44, comprising asparagine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No. 1. 47. The capsid polypeptide according to any one of Embodiments 1 to 44, comprising glutamine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No. 1. 48. The capsid polypeptide according to any one of Embodiments 1 to 44, wherein isoleucine is not present at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No. 1. 49. The capsid polypeptide according to any one of Embodiments 1 to 44, comprising isoleucine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No. 1. 50. The capsid polypeptide according to any one of Embodiments 1 to 44, comprising serine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No. 1. 51. The capsid polypeptide according to any one of Embodiments 1 to 44, comprising glycine at the position corresponding to G549 of the VP1 capsid polypeptide of Sequence ID No. 1. 52. The capsid polypeptide according to any one of Embodiments 1 to 44, wherein glycine is not present at the position corresponding to G549 of the VP1 capsid polypeptide of Sequence ID No. 1. 53. The capsid polypeptide according to any one of Embodiments 1 to 44, comprising alanine, phenylalanine, histidine, isoleucine, asparagine, threonine, or tyrosine at the position corresponding to G549 of the VP1 capsid polypeptide of SEQ ID NO: 1. 54. The capsid polypeptide according to Embodiment 54, comprising alanine at the position corresponding to G549 of the VP1 capsid polypeptide of Sequence ID No. 1. 55. The capsid polypeptide according to Embodiment 54, comprising phenylalanine at the position corresponding to G549 of the VP1 capsid polypeptide of Sequence ID No. 1. 56. The capsid polypeptide according to Embodiment 54, comprising histidine at the position corresponding to G549 of the VP1 capsid polypeptide of SEQ ID NO: 1. 57. The capsid polypeptide according to Embodiment 54, comprising asparagine at the position corresponding to G549 of the VP1 capsid polypeptide of SEQ ID NO: 1. 58. The capsid polypeptide according to Embodiment 54, comprising threonine at the position corresponding to G549 of the VP1 capsid polypeptide of Sequence ID No. 1. 59. The capsid polypeptide according to Embodiment 54, comprising tyrosine at the position corresponding to G549 of the VP1 capsid polypeptide of Sequence ID No. 1. 60. The capsid polypeptide according to any one of Embodiments 1 to 59, comprising tyrosine at the position corresponding to G549 of the VP1 capsid polypeptide of Sequence ID No. 1. 61. The capsid polypeptide according to any one of Embodiments 1 to 60, comprising arginine at the position corresponding to R550 of the VP1 capsid polypeptide of Sequence ID No. 1. 62. The capsid polypeptide according to any one of Embodiments 1 to 60, comprising lysine, leucine, or asparagine at the position corresponding to R550 of the VP1 capsid polypeptide of Sequence ID No. 1. 63. The capsid polypeptide according to Embodiment 62, comprising lysine at the position corresponding to R550 of the VP1 capsid polypeptide of Sequence ID No. 1. 64. The capsid polypeptide according to Embodiment 62, comprising leucine at the position corresponding to R550 of the VP1 capsid polypeptide of Sequence ID No. 1. 65. The capsid polypeptide according to Embodiment 62, comprising asparagine at the position corresponding to R550 of the VP1 capsid polypeptide of Sequence ID No. 1. 66. The capsid polypeptide according to any one of Embodiments 1 to 65, comprising aspartic acid at the position corresponding to D551 of the VP1 capsid polypeptide of Sequence ID No. 1. 67. The capsid polypeptide according to any one of Embodiments 1 to 65, comprising glutamic acid at the position corresponding to D551 of the VP1 capsid polypeptide of Sequence ID No. 1. 68. The capsid polypeptide according to any one of Embodiments 1 to 67, comprising asparagine at the position corresponding to N552 of the VP1 capsid polypeptide of Sequence ID No. 1. 69. The capsid polypeptide according to any one of Embodiments 1 to 68, comprising valine at the position corresponding to V553 of the VP1 capsid polypeptide of Sequence ID No. 1. 70. The capsid polypeptide according to any one of Embodiments 1 to 68, comprising serine at the position corresponding to V553 of the VP1 capsid polypeptide of Sequence ID No. 1. 71. The capsid polypeptide according to any one of Embodiments 1 to 70, comprising aspartic acid at the position corresponding to D554 of the VP1 capsid polypeptide of Sequence ID No. 1. 72. The capsid polypeptide according to any one of Embodiments 1 to 71, comprising alanine at the position corresponding to A555 of the VP1 capsid polypeptide of Sequence ID No. 1. 73. The capsid polypeptide according to any one of Embodiments 1 to 72, comprising aspartic acid at the position corresponding to D556 of the VP1 capsid polypeptide of Sequence ID No. 1. 74. The capsid polypeptide according to any one of Embodiments 1 to 73, comprising lysine acid at the position corresponding to K557 of the VP1 capsid polypeptide of Sequence ID No. 1. 75. The capsid polypeptide according to any one of Embodiments 1 to 73, comprising leucine at the position corresponding to K557 of the VP1 capsid polypeptide of Sequence ID No. 1. 76. The capsid polypeptide according to any one of Embodiments 1 to 73, comprising asparagine at the position corresponding to K557 of the VP1 capsid polypeptide of Sequence ID No. 1. 77. The capsid polypeptide according to any one of Embodiments 1 to 76, comprising valine at the position corresponding to V558 of the VP1 capsid polypeptide of Sequence ID No. 1. 78. The capsid polypeptide according to any one of Embodiments 1 to 77, comprising methionine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 79. The capsid polypeptide according to any one of Embodiments 1 to 77, wherein the VP1 capsid polypeptide of Sequence ID No. 1 does not contain methionine at the position corresponding to M559. 80. The capsid polypeptide according to any one of Embodiments 1 to 77, comprising alanine, cysteine, isoleucine, asparagine, glutamine, serine, threonine, or valine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 81. The capsid polypeptide according to Embodiment 80, comprising alanine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 82. The capsid polypeptide according to Embodiment 80, comprising cysteine ​​at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 83. The capsid polypeptide according to Embodiment 80, comprising isoleucine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 84. The capsid polypeptide according to Embodiment 80, comprising asparagine at the position corresponding to M559 of the VP1 capsid polypeptide of SEQ ID NO: 1. 85. The capsid polypeptide according to Embodiment 80, comprising glutamine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 86. The capsid polypeptide according to Embodiment 80, comprising serine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 87. The capsid polypeptide according to Embodiment 80, comprising threonine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 88. The capsid polypeptide according to Embodiment 80, comprising valine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 89. The capsid polypeptide according to any one of Embodiments 1 to 80, comprising alanine or glutamine at the position corresponding to M559 of the VP1 capsid polypeptide of Sequence ID No. 1. 90. The capsid polypeptide according to any one of Embodiments 1 to 89, comprising isoleucine at the position corresponding to I560 of the VP1 capsid polypeptide of Sequence ID No. 1. 91. The capsid polypeptide according to any one of Embodiments 1 to 89, comprising leucine at the position corresponding to I560 of the VP1 capsid polypeptide of Sequence ID No. 1. 92. The capsid polypeptide according to any one of Embodiments 1 to 89, comprising glutamine at the position corresponding to I560 of the VP1 capsid polypeptide of Sequence ID No. 1. 93. The capsid polypeptide according to any one of Embodiments 1 to 92, comprising threonine at the position corresponding to T561 of the VP1 capsid polypeptide of Sequence ID No. 1. 94. The capsid polypeptide according to any one of Embodiments 1 to 92, comprising serine at the position corresponding to T561 of the VP1 capsid polypeptide of Sequence ID No. 1. 95. The capsid polypeptide according to any one of Embodiments 1 to 94, comprising asparagine at the position corresponding to N562 of the VP1 capsid polypeptide of Sequence ID No. 1. 96. The capsid polypeptide according to any one of Embodiments 1 to 95, comprising glutamic acid at the position corresponding to E563 of the VP1 capsid polypeptide of Sequence ID No. 1. 97. The capsid polypeptide according to any one of Embodiments 1 to 96, comprising glutamic acid at the position corresponding to E564 of the VP1 capsid polypeptide of Sequence ID No. 1. 98. The capsid polypeptide according to any one of Embodiments 1 to 97, comprising glutamic acid at the position corresponding to E565 of the VP1 capsid polypeptide of Sequence ID No. 1. 99. The capsid polypeptide according to any one of Embodiments 1 to 98, comprising isoleucine at the position corresponding to I566 of the VP1 capsid polypeptide of Sequence ID No. 1. 100. The capsid polypeptide according to any one of Embodiments 1 to 99, comprising lysine at the position corresponding to K567 of the VP1 capsid polypeptide of Sequence ID No. 1. 101. The capsid polypeptide according to any one of Embodiments 1 to 100, comprising threonine at the position corresponding to T568 of the VP1 capsid polypeptide of Sequence ID No. 1. 102. The capsid polypeptide according to any one of Embodiments 1 to 101, comprising threonine at the position corresponding to T569 of the VP1 capsid polypeptide of Sequence ID No. 1. 103. The capsid polypeptide according to any one of Embodiments 1 to 102, comprising asparagine at the position corresponding to N570 of the VP1 capsid polypeptide of Sequence ID No. 1. 104. The capsid polypeptide according to any one of Embodiments 1 to 103, comprising proline at the position corresponding to P571 of the VP1 capsid polypeptide of Sequence ID No. 1. 105. The capsid polypeptide according to any one of Embodiments 1 to 104, comprising valine at the position corresponding to V572 of the VP1 capsid polypeptide of Sequence ID No. 1. 106. The capsid polypeptide according to any one of Embodiments 1 to 105, comprising alanine at the position corresponding to A573 of the VP1 capsid polypeptide of Sequence ID No. 1. 107. The capsid polypeptide according to any one of Embodiments 1 to 106, comprising threonine at the position corresponding to T574 of the VP1 capsid polypeptide of Sequence ID No. 1. 108. The capsid polypeptide according to any one of Embodiments 1 to 107, comprising glutamic acid at the position corresponding to E575 of the VP1 capsid polypeptide of Sequence ID No. 1. 109. The capsid polypeptide according to any one of Embodiments 1 to 107, comprising serine at the position corresponding to E575 of the VP1 capsid polypeptide of Sequence ID No. 1. 110. The capsid polypeptide according to any one of Embodiments 1 to 107, comprising tryptophan at the position corresponding to E575 of the VP1 capsid polypeptide of Sequence ID No. 1. 111. The capsid polypeptide according to any one of Embodiments 1 to 110, comprising serine at the position corresponding to S576 of the VP1 capsid polypeptide of Sequence ID No. 1. 112. The capsid polypeptide according to any one of Embodiments 1 to 110, comprising tryptophan at the position corresponding to S576 of the VP1 capsid polypeptide of Sequence ID No. 1. 113. The capsid polypeptide according to any one of Embodiments 1 to 112, comprising tyrosine at the position corresponding to Y577 of the VP1 capsid polypeptide of Sequence ID No. 1. 114. The capsid polypeptide according to any one of Embodiments 1 to 112, comprising histidine at the position corresponding to Y577 of the VP1 capsid polypeptide of Sequence ID No. 1. 115. The capsid polypeptide according to any one of Embodiments 1 to 112, comprising threonine at the position corresponding to Y577 of the VP1 capsid polypeptide of Sequence ID No. 1. 116. The capsid polypeptide according to any one of Embodiments 1 to 115, comprising glycine at the position corresponding to G578 of the VP1 capsid polypeptide of Sequence ID No. 1. 117. The capsid polypeptide according to any one of embodiments 1 to 116, comprising threonine at the position corresponding to Q579 of the VP1 capsid polypeptide of Sequence ID No. 1. 118. The capsid polypeptide according to any one of Embodiments 1 to 116, comprising valine at the position corresponding to Q579 of the VP1 capsid polypeptide of Sequence ID No. 1. 119. The capsid polypeptide according to any one of Embodiments 1 to 118, comprising valine at the position corresponding to V580 of the VP1 capsid polypeptide of Sequence ID No. 1. 120. The capsid polypeptide according to any one of Embodiments 1 to 119, comprising alanine at the position corresponding to A581 of the VP1 capsid polypeptide of Sequence ID No. 1. 121. The capsid polypeptide according to any one of Embodiments 1 to 119, comprising cysteine ​​at the position corresponding to A581 of the VP1 capsid polypeptide of Sequence ID No. 1. 122. The capsid polypeptide according to any one of Embodiments 1 to 119, comprising asparagine at the position corresponding to A581 of the VP1 capsid polypeptide of Sequence ID No. 1. 123. The capsid polypeptide according to any one of Embodiments 1 to 122, comprising threonine at the position corresponding to T582 of the VP1 capsid polypeptide of Sequence ID No. 1. 124. The capsid polypeptide according to any one of Embodiments 1 to 122, comprising isoleucine at the position corresponding to T582 of the VP1 capsid polypeptide of Sequence ID No. 1. 125. The capsid polypeptide according to any one of Embodiments 1 to 122, comprising methionine at the position corresponding to T582 of the VP1 capsid polypeptide of Sequence ID No. 1. 126. The capsid polypeptide according to any one of Embodiments 1 to 125, comprising asparagine at the position corresponding to N583 of the VP1 capsid polypeptide of Sequence ID No. 1. 127. The capsid polypeptide according to any one of Embodiments 1 to 126, comprising histidine at the position corresponding to H584 of the VP1 capsid polypeptide of Sequence ID No. 1. 128. The capsid polypeptide according to any one of Embodiments 1 to 126, comprising isoleucine at the position corresponding to H584 of the VP1 capsid polypeptide of Sequence ID No. 1. 129. The capsid polypeptide according to any one of Embodiments 1 to 126, comprising leucine at the position corresponding to H584 of the VP1 capsid polypeptide of Sequence ID No. 1. 130. The capsid polypeptide according to any one of Embodiments 1 to 126, comprising methionine at the position corresponding to H584 of the VP1 capsid polypeptide of Sequence ID No. 1. 131. The capsid polypeptide according to any one of Embodiments 1 to 126, comprising asparagine at the position corresponding to H584 of the VP1 capsid polypeptide of Sequence ID No. 1. 132. The capsid polypeptide according to any one of Embodiments 1 to 126, comprising glutamine at the position corresponding to H584 of the VP1 capsid polypeptide of Sequence ID No. 1. 133. The capsid polypeptide according to any one of Embodiments 1 to 132, comprising glutamine at the position corresponding to Q585 of the VP1 capsid polypeptide of Sequence ID No. 1. 134. The capsid polypeptide according to any one of Embodiments 1 to 133, comprising serine at the position corresponding to S586 of the VP1 capsid polypeptide of Sequence ID No. 1. 135. The capsid polypeptide according to any one of Embodiments 1 to 133, comprising glutamine at the position corresponding to S586 of the VP1 capsid polypeptide of Sequence ID No. 1. 136. The capsid polypeptide according to any one of Embodiments 1 to 135, comprising alanine at the position corresponding to A587 of the VP1 capsid polypeptide of Sequence ID No. 1. 137. The capsid polypeptide according to any one of Embodiments 1 to 135, comprising histidine at the position corresponding to A587 of the VP1 capsid polypeptide of Sequence ID No. 1. 138. The capsid polypeptide according to any one of Embodiments 1 to 135, comprising serine at the position corresponding to A587 of the VP1 capsid polypeptide of Sequence ID No. 1. 139. The capsid polypeptide according to any one of Embodiments 1 to 135, comprising threonine at the position corresponding to A587 of the VP1 capsid polypeptide of Sequence ID No. 1. 140. The capsid polypeptide according to any one of Embodiments 1 to 139, comprising glutamine at the position corresponding to Q588 of the VP1 capsid polypeptide of Sequence ID No. 1. 141. The capsid polypeptide according to any one of Embodiments 1 to 139, comprising glycine at the position corresponding to Q588 of the VP1 capsid polypeptide of Sequence ID No. 1. 142. The capsid polypeptide according to any one of Embodiments 1 to 139, comprising asparagine at the position corresponding to Q588 of the VP1 capsid polypeptide of Sequence ID No. 1. 143. The capsid polypeptide according to any one of Embodiments 1 to 139, comprising threonine at the position corresponding to Q588 of the VP1 capsid polypeptide of Sequence ID No. 1. 144. The capsid polypeptide according to any one of Embodiments 1 to 143, comprising alanine at the position corresponding to A589 of the VP1 capsid polypeptide of Sequence ID No. 1. 145. The capsid polypeptide according to any one of Embodiments 1 to 143, comprising serine at the position corresponding to A589 of the VP1 capsid polypeptide of Sequence ID No. 1. 146. The capsid polypeptide according to any one of Embodiments 1 to 143, comprising threonine at the position corresponding to A589 of the VP1 capsid polypeptide of Sequence ID No. 1. 147. The capsid polypeptide according to any one of Embodiments 1 to 146, comprising glutamine at the position corresponding to Q590 of the VP1 capsid polypeptide of Sequence ID No. 1. 148. The capsid polypeptide according to any one of Embodiments 1 to 147, comprising alanine at the position corresponding to A591 of the VP1 capsid polypeptide of Sequence ID No. 1. 149. The capsid polypeptide according to any one of Embodiments 1 to 147, comprising proline at the position corresponding to A591 of the VP1 capsid polypeptide of Sequence ID No. 1. 150. The capsid polypeptide according to any one of Embodiments 1 to 149, comprising glycine at the position corresponding to G594 of the VP1 capsid polypeptide of Sequence ID No. 1. 151. The capsid polypeptide according to any one of Embodiments 1 to 150, comprising glutamine at the position corresponding to Q597 of the VP1 capsid polypeptide of Sequence ID No. 1. 152. The capsid polypeptide according to any one of Embodiments 1 to 151, comprising glutamine at the position corresponding to Q599 of the VP1 capsid polypeptide of Sequence ID No. 1. 153. The capsid polypeptide according to any one of Embodiments 1 to 152, comprising glycine at the position corresponding to G600 of the VP1 capsid polypeptide of Sequence ID No. 1. 154. The capsid polypeptide according to any one of Embodiments 1 to 153, comprising leucine at the position corresponding to L602 of the VP1 capsid polypeptide of Sequence ID No. 1. 155. The capsid polypeptide according to any one of Embodiments 1 to 154, comprising proline at the position corresponding to P603 of the VP1 capsid polypeptide of Sequence ID No. 1. 156. The capsid polypeptide according to any one of Embodiments 1 to 155, comprising glycine at the position corresponding to G604 of the VP1 capsid polypeptide of Sequence ID No. 1. 157. The capsid polypeptide according to any one of Embodiments 1 to 156, comprising methionine at the position corresponding to M605 of the VP1 capsid polypeptide of Sequence ID No. 1. 158. The capsid polypeptide according to any one of Embodiments 1 to 157, comprising valine at the position corresponding to V606 of the VP1 capsid polypeptide of Sequence ID No. 1. 159. The capsid polypeptide according to any one of Embodiments 1 to 158, comprising tryptophan at the position corresponding to W607 of the VP1 capsid polypeptide of Sequence ID No. 1. 160. The capsid polypeptide according to any one of Embodiments 1 to 159, comprising glutamine at the position corresponding to Q608 of the VP1 capsid polypeptide of Sequence ID No. 1. 161. The capsid polypeptide according to any one of Embodiments 1 to 160, comprising aspartic acid at the position corresponding to D609 of the VP1 capsid polypeptide of Sequence ID No. 1. 162. The capsid polypeptide according to any one of Embodiments 1 to 160, comprising asparagine at the position corresponding to D609 of the VP1 capsid polypeptide of Sequence ID No. 1. 163. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 70% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 164. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 75% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 165. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 80% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 166. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 85% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 167. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 90% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 168. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 91% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 169. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 92% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 170. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 93% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 171. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 94% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 172. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 95% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 173. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 96% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 174. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 97% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 175. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 98% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 176. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 99% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 1, or its VP2 or VP3 portion. 177. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 70% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 178. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 75% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 179. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 80% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 180. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 85% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 181. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 90% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 182. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 91% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 183. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 92% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 184. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 93% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 185. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 94% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3, or its VP2 or VP3 portion. 186. The capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 95% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3 or its VP2 or VP3 portion. 187. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 96% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3 or its VP2 or VP3 portion. 188. The capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 97% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3 or its VP2 or VP3 portion. 189. The capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 98% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3 or its VP2 or VP3 portion. 190. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 99% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 3 or its VP2 or VP3 portion. 191. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 70% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 192. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 75% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 193. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 80% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 194. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 85% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 195. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 90% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 196. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 91% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 197. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 92% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 198. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 93% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 199. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 94% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 200. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 95% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 201. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 96% sequence identity ((a) calculated considering targeted peptide insertion, or (b) calculated without considering targeted peptide insertion) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 202. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 97% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 203. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 98% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 204. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 99% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 5, or its VP2 or VP3 portion. 205. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 70% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 206. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 75% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 207. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 80% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 208. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 85% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 209. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 90% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 210. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 91% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 211. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 92% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 212. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 93% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 213. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 94% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 214. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 95% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 215. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 96% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 216. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 97% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 217. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 98% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 218. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 99% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 7, or its VP2 or VP3 portion. 219. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 70% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 220. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 75% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 221. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 80% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 222. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 85% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 223. The capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 90% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 224. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 91% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 225. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 92% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 226. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 93% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 227. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 94% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 228. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 95% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 229. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 96% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 230. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 97% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 231. The capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 98% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 232. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 99% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 9, or its VP2 or VP3 portion. 233. A capsid polypeptide according to any one of Embodiments 1 to 162, comprising an amino acid sequence having at least 70% sequence identity ((a) calculated considering the insertion of a targeted peptide, or (b) calculated without considering the insertion of a targeted peptide) with respect to the VP1 capsid polypeptide of Sequence ID No. 11, or its VP2 or VP3 portion. 234. A capsid polypeptide according to any one of Embodiments 1 ...

Claims

1. A capsid polypeptide wherein valine is present at the position corresponding to I601 of the VP1 capsid polypeptide of SEQ ID NO: 1, and optionally, (a) Alanine, arginine, serine, threonine, valine, at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1 (b) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1 (c) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, and (d) A capsid polypeptide comprising one, two, three, or all four serine or threonine molecules at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No.

1.

2. It is a capsid polypeptide, (a) Alanine or serine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) A capsid polypeptide comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No.

1.

3. The capsid polypeptide according to claim 2, wherein serine is contained at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No.

1.

4. The capsid polypeptide according to claim 2, wherein the VP1 capsid polypeptide of Sequence ID No. 1 contains threonine at the position corresponding to N598.

5. It is a capsid polypeptide, (a) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) A capsid polypeptide comprising threonine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No.

1.

6. The capsid polypeptide according to claim 5, wherein the VP1 capsid polypeptide of Sequence ID No. 1 contains alanine at the position corresponding to W595.

7. It is a capsid polypeptide, (a) Alanine or serine at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (d) A capsid polypeptide comprising serine or threonine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No.

1.

8. It is a capsid polypeptide, (a) Alanine, arginine, serine, threonine, and valine are placed at the position corresponding to T593 of the VP1 capsid polypeptide of Sequence ID No. 1, (b) Alanine, tryptophan, or tyrosine at the position corresponding to W595 of the VP1 capsid polypeptide of Sequence ID No. 1, (c) Leucine at the position corresponding to V596 of the VP1 capsid polypeptide of Sequence ID No. 1, (d) A capsid polypeptide comprising threonine at the position corresponding to N598 of the VP1 capsid polypeptide of Sequence ID No.

1.

9. The capsid polypeptide according to any one of claims 1 to 8, wherein glutamic acid is contained at the position corresponding to D551 of the VP1 capsid polypeptide of Sequence ID No.

1.

10. The capsid polypeptide according to any one of claims 1 to 9, wherein the VP1 capsid polypeptide of Sequence ID No. 1 contains threonine at the position corresponding to Q579.

11. The capsid polypeptide according to any one of claims 1 to 9, wherein the VP1 capsid polypeptide of Sequence ID No. 1 contains valine at the position corresponding to Q579.

12. The capsid polypeptide according to any one of claims 1 to 11, comprising alanine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No.

1.

13. The capsid polypeptide according to any one of claims 1 to 11, comprising asparagine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No.

1.

14. The capsid polypeptide according to any one of claims 1 to 11, comprising glutamine at the position corresponding to Q592 of the VP1 capsid polypeptide of Sequence ID No.

1.

15. A capsid polypeptide comprising all six, seven, or eight mutations of the VAR-2 mutation set compared to the VP1 capsid polypeptide of SEQ ID NO: 1 listed in Table 24, preferably wherein the mutations comprise the V596 and N598 mutations, and optionally the Q579 and / or T593 mutations, compared to the VP1 capsid polypeptide of SEQ ID NO:

1.

16. A capsid polypeptide comprising five, six, or seven mutations from the VAR-3 mutation set, compared to the VP1 capsid polypeptide of SEQ ID NO: 1 listed in Table 24, preferably wherein the mutations comprise the V596 and N598 mutations, and optionally the Q579 and / or T593 mutations, compared to the VP1 capsid polypeptide of SEQ ID NO:

1.

17. A capsid polypeptide comprising four, five, or six mutations from the VAR-11 mutation set, compared to the VP1 capsid polypeptide of SEQ ID NO: 1 listed in Table 24, preferably wherein the mutations comprise the V596 and N598 mutations, and optionally the Q579 and / or T593 mutations, compared to the VP1 capsid polypeptide of SEQ ID NO:

1.

18. A capsid polypeptide comprising four, five, or six mutations from the VAR-40 mutation set, compared to the VP1 capsid polypeptide of SEQ ID NO: 1 as described in Table 24, preferably wherein the mutations comprise the V596 and N598 mutations, and optionally the Q579 and / or T593 mutations, compared to the VP1 capsid polypeptide of SEQ ID NO:

1.

19. A capsid polypeptide comprising four, five, or six mutations from the VAR-42 mutation set, compared to the VP1 capsid polypeptide of SEQ ID NO: 1 as described in Table 24, preferably wherein the mutations comprise the V596 and N598 mutations, and optionally the Q579 and / or T593 mutations, compared to the VP1 capsid polypeptide of SEQ ID NO:

1.

20. A capsid polypeptide comprising all six, seven, or eight mutations of the VAR-54 mutation set compared to the VP1 capsid polypeptide of SEQ ID NO: 1 listed in Table 24, preferably wherein the mutations comprise the V596 and N598 mutations, and optionally the Q579 and / or T593 mutations, compared to the VP1 capsid polypeptide of SEQ ID NO:

1.

21. The capsid polypeptide according to any one of claims 1 to 20, wherein the sequence has an edit distance of (a) 12 or less, or (b) 10 or less, to the VP1 capsid polypeptide of sequence number 1, or its VP2 or VP3 portion.

22. The capsid polypeptide according to any one of claims 1 to 20, comprising an amino acid sequence having at least 95% sequence identity with the VP1 capsid polypeptide of Sequence ID No. 1, or with the VP2 or VP3 portion thereof.

23. The capsid polypeptide according to any one of claims 1 to 22, wherein it is a VP1 capsid polypeptide.

24. The capsid polypeptide according to any one of claims 1 to 22, which is a VP2 capsid polypeptide.

25. A capsid polypeptide according to any one of claims 1 to 22, wherein the capsid polypeptide is a VP3 capsid polypeptide.

26. A nucleic acid comprising a nucleotide sequence encoding a capsid polypeptide according to any one of claims 1 to 25.

27. A virus particle comprising a capsid polypeptide according to any one of claims 1 to 25.

28. A viral particle according to claim 27, comprising a nucleic acid, the payload (e.g., a heterologized gene) and one or more regulatory elements.

29. The virus particle according to claim 28, wherein the one or more regulatory elements include a promoter.

30. The virus particle according to claim 29, wherein the promoter is a constitutive promoter.

31. The virus particle according to claim 29, wherein the promoter is a CNS-specific promoter.

32. The virus particle according to claim 29, wherein the promoter is a muscle-specific promoter.

33. A manipulated host cell comprising a nucleic acid encoding a capsid polypeptide according to any one of claims 1 to 25.

34. The host cell according to claim 33, which is a packaged cell line.

35. A host cell according to claim 33 or claim 34, comprising a nucleic acid encoding a rep protein.

36. A host cell according to any one of claims 33 to 35, comprising a nucleic acid containing one or more helper sequences.

37. A host cell according to any one of claims 33 to 36, configured to package the virus particles according to any one of claims 27 to 32.

38. A method for producing viral particles containing a capsid polypeptide, comprising introducing a nucleic acid molecule according to claim 26 into a cell and recovering the viral particles therefrom.

39. A method for producing viral particles containing a capsid polypeptide, comprising culturing cells manipulated to express a capsid polypeptide according to any one of claims 1 to 25, and recovering viral particles therefrom.

40. The method according to claim 38 or claim 39, wherein the cell is a host cell according to any one of claims 33 to 37.

41. A method for delivering a payload (e.g., nucleic acid) to a cell, (a) Contacting the cells with a viral particle comprising the capsid polypeptide described in any one of claims 1 to 25 and a payload, or (b) A method comprising bringing the cells into contact with the virus particles described in any one of claims 27 to 32.

42. The method according to claim 42, wherein the cells are CNS cells.

43. A method for delivering a payload (e.g., nucleic acid) to a target, (a) Administering to the subject a viral particle comprising the capsid polypeptide described in any one of claims 1 to 25 and the payload, or (b) A method comprising administering the viral particles described in any one of claims 27 to 32 to the subject.

44. The method according to claim 43, wherein the virus particle delivers the payload to the CNS.

45. A method for treating a disease or condition in a subject, wherein the subject is given an amount effective for treating the disease or condition. (a) A viral particle comprising a capsid polypeptide according to any one of claims 1 to 25 and a heterogeneous nucleic acid sequence encoding a therapeutic product suitable for treating the disease or condition, (b) A viral particle comprising a capsid polypeptide encoded by the nucleic acid molecule described in claim 26 and a heterogeneous nucleic acid sequence encoding a therapeutic product suitable for treating the disease or condition, or (c) A virus particle according to any one of claims 27 to 32, or (d) A method comprising administering a composition, e.g., a pharmaceutical composition, comprising the virus particles of (a) and optionally, a pharmaceutically acceptable carrier.

46. The method according to claim 45, wherein the disease or condition is a disease or condition of CNS.

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