Enhanced AAV9 capsid protein

Abstract

Provided herein are engineered adeno-associated viruses (e.g., AAV9) with enhanced infectivity and manufacturability. Also provided are methods of treatment of disorders / diseases using the disclosed engineered AAVs.

Description

ENHANCED AAV9 CAPSID PROTEINACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with support under Grant No. NS095867 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No: 63 / 556,948, filed on February 23, 2024, and entitled “ENHANCED AAV9 CAPSID PROTEIN,” the entire contents of which is hereby incorporated by reference herein in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The Sequence Listing submitted February 21, 2025 as an XML file named “UTSD_4342_Seq_Listing”, created on February 21, 2025 and having a size of 20,480 bytes is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUNDField

[0004] The present disclosure relates to enhanced recombinant adeno-associated virus serotype 9 (AAV9) capsid proteins and viral vectors and methods of making and using the same.Background

[0005] Adeno-Associated Virus (AAV) is a promising gene delivery vehicle for treatment of disease with known underlying genetic abnormalities. Many AAVs have been identified in nature. AAV9 is one of these naturally identified serotypes and is a popular gene delivery vehicle due to its ability to efficiently transduce a wide range of tissues and its low immunogenicity compared to other viral vectors. However, like any gene delivery system, AAV9 has certain limitations. For example, high levels of transduction are needed for gene therapy trials as there are physical limitations to how much AAV can be delivered in a single injection. If an AAV hadsuperior infectivity, a lower dose and fewer injections would be needed to achieve therapeutic relevance. Similarly for use as a vaccine delivery tool, high efficiency transduction and stability is needed to achieve robust secretion of antigens encoded within the AAV to achieve the therapeutic levels of circulating antibodies in the blood. While AAV9 can transduce a wide range of tissues, its tissue tropism may not always match the desired target tissue for gene therapy. This can limit its effectiveness in specific therapeutic applications. Additionally, Large-scale production of AAV9 vectors for clinical applications can be challenging and expensive, which may limit their widespread use and accessibility for gene therapy.

[0006] Thus, there remains a need for new AAV vectors and efficient methods of manufacturing the same. In particular, there is a need for AAV based vectors that can be used efficiently with select cell types and tissues and that do not react with a pre-existing anti -AAV human immunity that could neutralize or inactivate the vectors. There also remains a need for vectors that transduce different cell types in vivo and in vitro and that offer a more restricted biodistribution or a more promiscuous biodistribution, depending on what may be required.SUMMARY

[0007] In one aspect, the current disclosure encompasses a recombinant adeno-associated virus serotype 9 (rAAV9) comprising a capsid protein sequence with an amino acid substitution at one or more of positions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. In an aspect, the rAAV9 comprises the capsid protein sequence with the amino acid substitution F535Y. In an aspect, the engineered AAV9 capsid may comprise a substitution at H584, for example from a histidine (H) to a polar charged or uncharged amino acid, e.g., arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glutamine (Q), lysine (K), serine (S), threonine (T), or tyrosine (Y) (e.g., H584R, H584N, H584D, H584E, H584Q, H584K, H584S, H584T, or H584Y). In one embodiment, the engineered AAV9 capsid comprises a histidine (H) to an arginine (R) substitution or a histidine (H) to a threonine (T) at position 584; thus, the rAAV9 comprises the capsid protein sequence with the amino acid substitution H584R or the amino acid substitution H584T. In an aspect, the rAAV9 comprises the capsid protein sequence with the amino acid substitution L382I. In one aspect, the rAAV9 comprises a capsid protein sequence with two amino acid substitutions at positions F535 andH584. In another aspect, the rAAV9 comprises a capsid protein sequence with two amino acid substitutions at positions L382 and H584. In another aspect, the rAAV9 comprises a capsid protein sequence with three amino acid substitutions at positions L382, F535, and H584. In one embodiment, the rAAV9 comprises a capsid protein sequence with two amino acid substitutions F535Y and H584R or two amino acid substitutions F535Y and H584T. In another embodiment, the rAAV9 comprises a capsid protein sequence with two amino acid substitutions H584R and L382I or two amino acid substitutions H584T and L382I. In another embodiment, the rAAV9 comprises a capsid protein sequence with three amino acid substitutions F535Y, H584R, and L382I or three amino acid substitutions F535Y, H584T, and L382I. The capsid protein sequence may be at least 80% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. The capsid protein sequence may be any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO 8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. The capsid protein sequence may comprise additional mutations, wherein the capsid protein sequence is at least about 80% identical to any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0008] In an aspect, the rAAV9 disclosed herein may have enhanced infectivity to brain cells in comparison to a AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584. In an aspect, the rAAV9 may have enhanced zeta potential and reduced stability at about pH 7 - about pH 4 in comparison to a AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584. In an aspect, the rAAV9 may have enhanced multispecies infectivity in comparison to a AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584. In an aspect, the rAAV9 may show enhanced packaging capacity in comparison to the AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584. In an aspect, the rAAV9 shows enhanced yield of intact virus particle (manufacturability) in comparison to an AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584.

[0009] In an aspect, the rAAV9 disclosed herein, may further comprise a recombinant nucleic acid. In an aspect, the recombinant nucleic acid encodes a therapeutic protein, a peptide, or an RNA.

[0010] In an aspect, the current disclosure also encompasses an adeno-associated virus serotype 9 (AAV9) capsid protein comprising an amino acid substitution at one or more of positions L382, F535, and H584, with reference to a capsid protein sequence as set forth in SEQ ID NO: 1. In an aspect, the AAV9 capsid protein comprises the amino acid substitution F535Y. In an aspect, the AAV9 capsid protein comprises a substitution at H584, for example from a histidine (H) to a polar charged or uncharged amino acid, e.g., arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glutamine (Q), lysine (K), serine (S), threonine (T), or tyrosine (Y). In one embodiment, the AAV9 capsid protein comprises a histidine (H) to an arginine (R) substitution or a histidine (H) to a threonine (T) at position 584; thus, the AAV9 capsid protein sequence comprises the amino acid substitution H584R or the amino acid substitution H584T. In an aspect, the AAV9 capsid protein sequence comprises the amino acid substitution L382I. In one aspect, the AAV9 capsid protein sequence comprises two amino acid substitutions at positions F535 and H584. In another aspect, the AAV9 capsid protein sequence comprises two amino acid substitutions at positions L382 and H584. In another aspect, the AAV9 capsid protein sequence comprises three amino acid substitutions at positions L382, F535, and H584. In one embodiment, the AAV9 capsid protein sequence comprises two amino acid substitutions F535Y and H584R or two amino acid substitutions F535Y and H584T. In another embodiment, the AAV9 capsid protein sequence comprises two amino acid substitutions H584R and L382I or two amino acid substitutions H584T and L382I. In another embodiment, the AAV9 capsid protein sequence comprises three amino acid substitutions F535Y, H584R, and L382I or three amino acid substitutions F535Y, H584T, and L382I

[0011] In an aspect, the capsid protein sequence may be at least 80% identical to any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In an aspect, the capsid protein sequence may be identical to the sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. In an aspect, the capsid protein furthercomprises additional mutations in the capsid protein sequence, wherein the capsid protein sequence is at least about 80% identical to any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: ! ! .

[0012] A method of manufacturing an adeno-associated virus serotype 9 (AAV9), comprising transfecting a host cell with a nucleic acid encoding an AAV9 capsid protein comprising an amino acid substitution at one or more of positions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO:1. In an aspect, the AAV9 is a recombinant AAV9 (rAAV9), comprising a recombinant nucleic acid.

[0013] In an aspect, the current disclosure also encompasses a method of gene delivery into a host cell, comprising contacting a host cell with a recombinant adeno-associated virus serotype 9 (rAAV9) or a pharmaceutical composition thereof, comprising a capsid protein as disclosed herein and the recombinant nucleic acid as disclosed herein. In an aspect, the host cell is a prokaryotic or eukaryotic cell, such as a mammalian cell. In some aspects, the host cell may be an erythrocyte, a platelet, a bone marrow cell, a vascular endothelial cell, a lymphocyte, a hepatocyte, a neuronal or brain cell (e.g., neurons and neuroglia), a bronchial endothelial cell, an epidermal cell, a respiratory interstitial cell, an epidermal cell, a fat cell, a dermal fibroblast, a muscle cell, or any other mammalian cell type. In one embodiment, the host cell is a neuronal cell. In an aspect of the method of treatment is for treatment of a cancer, a neurological disease, a neuromuscular disease, a liver disease, a kidney or renal disease, an ocular disease, or a muscular disease, a metabolic disease, a pulmonary disease, or any other type of disease afflicting a subject, and may comprise administering to a subject in need thereof a recombinant adeno- associated virus serotype 9 (rAAV9) or a pharmaceutical composition thereof comprising: a disclosed capsid protein and a disclosed recombinant nucleic acid. In an aspect, wherein the recombinant nucleic acid may encode a therapeutic protein, a peptide or an RNA.

[0014] In an aspect, the current disclosure also encompasses the use of the disclosed rAAV9 or the disclosed AAV9 capsid protein for treatment of a cancer, a neurological disease, a neuromuscular disease, a liver disease, a kidney or renal disease, an ocular disease, or a musculardisease, a metabolic disease, a pulmonary disease, or any other type of disease afflicting a subject.

[0015] In an aspect, the current disclosure also encompasses a vector comprising a polynucleotide sequence encoding the disclosed rAAV capsid protein. In an aspect, the vector may be a nucleic acid vector, plasmid vector, or a viral vector. In an aspect, the vector may further comprise a recombinant nucleic acid; wherein the recombinant nucleic acid encodes a therapeutic protein, a peptide, or an RNA.

[0016] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Aspects of the present disclosure are illustrated by way of example in which:

[0018] FIG. 1A is a bar graph showing that H584R and FYHR-AAV (AAV9 with F535Y and H584R mutations) yield more packaged AAV particles at harvest than AAV9. Each AAV capsid was made with triple transfections in suspension HEK293 cells and harvested 72 hours post transfection. At harvest, culture samples were collected for ITR titer quantification.

[0019] FIG. IB is a bar graph showing that most of the H584R and FYHR-AAV packaged virus particles are retained within the cell pellet fraction at harvest.

[0020] FIG. 2A is a bar graph showing the comparison of zeta potential measurements for AAV9, H584R and FYHR-AAV. Zeta potential of the virus is significantly altered when both mutations are present.

[0021] FIG. 2B is a bar graph showing differential scanning fluorimetry (DSF) data for AAV9 and FYHR-AAV at pH 7 - pH 4. DSF data shows that FYHR-AAV has reduced viral stability at pH 7 - pH 4.

[0022] FIG. 2C is a bar graph showing differential scanning fluorimetry (DSF) data for AAV9 and FYHR-AAV at pH 7.

[0023] FIG. 2D is a bar graph showing differential scanning fluorimetry (DSF) data for AAV9 and FYHR-AAV at pH 6.

[0024] FIG. 2E is a bar graph showing differential scanning fluorimetry (DSF) data for AAV9 and FYHR-AAV at pH 5.

[0025] FIG. 2F is a bar graph showing differential scanning fluorimetry (DSF) data for AAV9 and FYHR-AAV at pH 4.

[0026] FIG. 3A is a bar graph providing quantification of GFP positive COS7 cells at an MOI of 1E3 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0027] FIG. 3B is a bar graph providing quantification of GFP positive COS7 cells at an MOI of 1E4 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV

[0028] FIG. 3C is a bar graph providing quantification of GFP positive SH-SY5Y cells at an MOI of 1E3 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0029] FIG. 3D is a bar graph providing quantification of GFP positive SH-SY5Y cells at an MOI of 1E4 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0030] FIG. 3E is a bar graph providing quantification of GFP positive Lec2 cells at an MOI of 1E3 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0031] FIG. 3F is a bar graph providing quantification of GFP positive Lec2 cells at an MOI of 1E4 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0032] FIG. 3G is a bar graph providing quantification of GFP positive HEK cells at an MOI of 1E3 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0033] FIG. 3H is a bar graph providing quantification of GFP positive HEK cells at an MOI of 1E4 vg / cell when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0034] FIG. 31 is a bar graph providing quantification of GFP positive iPSC derived mature cardiomyocytes cells when infected with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV.

[0035] FIG. 3J shows representative immunoblots showing transduction of indicated brain tissue with AAV9, single mutants (F535Y, H584R) or double mutant FYHR-AAV. Mice were injected at 5 weeks of age with 1 x 1010vg / mouse and tissues were collected 3 weeks post injection. 30 pm frozen sections of mouse brains were stained for GFP. H584R and FYHR show increased transduction of the brain compared to AAV9.

[0036] FIG. 4 illustrates 3D structure comparisons of natural AAV serotypes and our cryogenic electron microscopy (Cryo-EM) resolved enhanced AAV9 variant “FYHR”.

[0037] FIG. 5 shows an AAV9 controlled randomization library screening in mice and nonhuman primates (NHP). Based on screening results variants were identified enriched in both NHP and mouse CNS. The plot demonstrates the analysis of the screen with enrichment of or transduction against both species on the x-axis relative to AAV9 and manufacturability relative to AAV9 (see Example 5).

[0038] FIG. 6 is a bar graph showing the results of DNA vector genome biodistribution (see Example 6). FB = forebrain; MB = midbrain; HB = hindbrain; C = cerebellum; SC = spinal cord. Samples were normalized to AAV9 as this serves as the reference control.

[0039] FIG. 7 is a bar graph showing the results of mouse brain vector genome DNA biodistribution.

[0040] FIG. 8 is a bar graph showing the results of vector DNA biodistribution from the peripheral tissues of the adequately injected NHP.

[0041] FIG. 9A and 9B show images of the (A) Mouse and (B) NHP brains with quantification (to the right of the RNAScope images) of the images using automated pixel positive quantification for the RNA stain.

[0042] FIG. 10A displays the manufacturability enrichment analysis relative to AAV9. FIGs. 10B and 10C show head-to-head manufacturability tests to validate the screening manufacturability analysis from FIG. 10A.

[0043] FIG. 11 and FIG. 12 are bar graphs showing that position 584 is not exclusive to benefit from a histidine to an arginine, but can also benefit from threonine, demonstrating that polar charged and uncharged residues at position 584 with respect to the AAV9 VP1 sequence enhance transduction.

[0044] FIG. 13 and FIG. 14 are bar graphs illustrating that enhancement of transduction (e.g., in FIG. 11 and FIG. 12) was not exclusive to immortalized cells.

[0045] FIG. 15 shows the 2-dimensional sequence alignments of our novel variants compared to AAV9 with CLUSTAL omega. The positions of changed amino acids are seen at position 382, 535, and 584 highlighted in grey.DETAILED DESCRIPTION

[0046] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0047] The present disclosure is based, in part, on the surprising discovery that an engineered adeno-associated virus serotype 9 (AAV9) capsid protein (VP1) comprising at least one substitution at positions F535, H584, L382, or a combination thereof, with reference to the wild-type AAV9 capsid protein (SEQ ID NO:1) imparted multiple beneficial features to a packaged virion. Substitutions at these positions in AAV9, or at a position corresponding to these positions in another AAV when the capsid protein sequences (e.g., VP 1) are aligned, for example aligned to SEQ ID NO: 1, have beneficial properties as described herein. A virion or viral particle encapsidated in the engineered capsid protein showed increased infectivity of mammalian cells, for example neuronal and muscle cells, improved tropism to select organs, improved biophysical characteristics, and improved manufacturability compared to a control AAV9. The disclosure is a result of extensive screening and testing of rAAV capsid proteins for desirable characteristics.I. Terminology

[0048] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0049] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface.

[0050] The terms “comprising,” “including” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described.

[0051] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0053] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of’ the disclosed elements, in which additional elements other than the listed elements are not included.

[0054] The term “about” or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, c. ., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the particular value, such as 10% of the value modified by the term “about.” As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0055] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown anddescribed. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0056] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. See, e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991), the disclosure of which is incorporated in its entirety herein.

[0057] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides includeany peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0058] Within the context of the application a protein is represented by an amino acid sequence and correspondingly a nucleic acid molecule or a polynucleotide represented by a nucleic acid sequence. Identity and similarity between sequences: throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID N0:Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID N0:Y. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%.

[0059] Each amino acid sequence described herein by virtue of its identity or similarity percentage with a given amino acid sequence respectively has in a further preferred aspect an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% with the given nucleotide or amino acid sequence, respectively. The terms “homology”,“sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred aspect, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm. A Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps. Default settings can be used and preferred program is Needle for pairwise alignment (in an aspect, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10 , end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment (in an aspect, MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned, Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2, Perform FFTS: none is used).

[0060] “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity may be used for determination of sequence similarity. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met; Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Vai to lie or Leu.

[0061] “Regulatory sequences” or “Regulatory elements” as used interchangeably include, without being limiting, a promoter, an enhancer, a leader, a transcription start site (TSS), a linker, 5' and 3' untranslated regions (UTRs), an intron, a poly adenylation signal, and a termination region or sequence, etc., that are suitable, necessary, or preferred for regulating or allowing expression of the gene or transcribable DNA sequence in a cell. Such additional regulatory element(s) can be optional and used to enhance or optimize expression of the gene or transcribable DNA sequence. A regulatory sequence can, for example, be inducible, noninducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may be a promoter. As used herein, the term “promoter” refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and / or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. In some exemplary aspects the promoter sequence is adapted to enable expression of a polynucleotide in adipocytes. In an aspect, the promoter is a adiponectin promoter or a derivative thereof.

[0062] An “individual” or “subject,” as used interchangeably herein, is a mammal. In certain aspects, the individual or subject is a human.

[0063] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0064] ‘ ‘AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”).

[0065] An “rAAV vector” as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (z.c., a polynucleotide heterologous to AAV), typically a sequence of interest for introducing into a target cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0066] An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (z. ?., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector”. Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.

[0067] “Packaging” refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

[0068] AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”

[0069] A “helper virus” for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0070] “Helper virus function(s)” refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.

[0071] An “infectious” virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. As used herein, an “infectious” virus or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell. Thus, “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome (vg) copies. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as “transduction.” The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and / or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay(ELISA). Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art.

[0072] A “replication-competent” virus (e.g., a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (z.e., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication -incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes.Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector. In general, rAAV vector preparations as described herein are those which contain few if any replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 102 rAAV particles, less than about 1 rcAAV per 104 rAAV particles, less than about 1 rcAAV per 108 rAAV particles, less than about 1 rcAAV per 1012 rAAV particles, or no rcAAV).

[0073] The phrases “tropism” and “transduction” are interrelated, but there are differences. The term “tropism” as used herein refers to the ability of an AAV vector or virion to infect one or more specified cell types, but can also encompass how the vector functions to transduce the cell in the one or more specified cell types; i.e., tropism refers to preferential entry of the AAV vector or virion into certain cell or tissue type(s) and / or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the AAV vector or virion in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequence(s). As used herein, the term “transduction” refers to the ability of an AAV vector or virion to infect one or more particular cell types; i.e., transduction refers to entry of the AAV vector or virion into the cell and the transfer of genetic material contained within the AAV vector or virion into the cell to obtain expression from the vector genome. In some cases, but not all cases, transduction and tropism may correlate.

[0074] The term “tropism profile” refers to the pattern of transduction of one or more target cells, tissues and / or organs. For example, some AAV capsids (variant AAV capsid polypeptides) provide for efficient transduction of skeletal muscle (e.g., quadriceps muscle), diaphragm muscle and / or cardiac muscle tissue. Conversely, some AAV capsids have only low-level transduction of liver, gonads and / or germ cells. The variant AAV capsid polypeptides disclosed herein provide for efficient and / or enhanced transduction of multiple cell types, including but not restricted to muscle and neuronal cells.

[0075] The term “zeta potential” of a virion refers to the net electrical charge on the surface of the viral particle. It is a measure of the potential difference between the surface of the virion and the surrounding fluid medium. Zeta potential is influenced by the presence of charged molecules or groups on the virion's surface, such as proteins, lipids, or carbohydrates.

[0076] The term “administration” and variants thereof e.g., “administering” a composition) in reference to a composition of the disclosure means introducing the composition of the composition into the system of the subject in need of treatment. When a composition of the disclosure thereof is provided in combination with one or more other active agents, “administration” and its variants are each understood to include concurrent and sequential introduction of the composition and other agents. The present disclosure includes within its scope intermediates of the compositions of this disclosure. In general, such intermediates will be functional derivatives of the compositions of this disclosure which are readily convertible in vivo into the required composition. Thus, in the methods of treatment of the present disclosure, the term “administering” shall encompass the treatment of the various conditions described with the composition specifically disclosed or with a composition which may not be specifically disclosed, but which converts to the specified composition in vivo after administration to the patient.

[0077] The term “therapeutically effective amount” as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0078] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who is in need of the treatment, for example, having a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease / disorder includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0079] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and / or recurrence.

[0080] A “neuronal cell,” as used herein, is used interchangeably with “neural cell” and refers to neurons and glia of the central nervous system or peripheral nervous system. The term “neuronal cell” includes cells such as astrocytes, oligodendrocytes, and Schwann cells. The term includes neuronal cells of any brain tissue (e.g., a brain tissue such as cerebral hemisphere, cerebral cortex, subcortex motor cortex, striatum, internal capsule, thalamus, hypothalamus, hippocampus, midbrain, brainstem, and cerebellum). A mature neuron can express one or more markers of a mature neuron, where such markers include, e.g., nestin, NeuroDl, neuron-specificenolase (NSE), neuron-specific nuclear protein (NeuN), neurofilament (NF), 510013, tau, microtubule-associated protein 2 (MAP2), tau, doublecortin (DCX), and the like.

[0081] “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.

[0082] The terms “genetic alteration” and “genetic modification” (and grammatical variants thereof) are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be affected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleoti deliposome complex. Genetic alteration may also be affected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and / or genotype of the cell and its progeny is included in this term.

[0083] A cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is “heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.

[0084] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation,phosphorylation, or conjugation with a labeling component. Polypeptides such as anti -angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti -angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a gene product to a mammalian subject (which may be referred to as “transgenes” to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.

[0085] An “isolated” plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some cases purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.II. Engineered AAV9 Capsid Proteins

[0086] In an aspect, the current disclosure encompasses an engineered adeno-associated virus serotype 9 (AAV9) capsid protein (VP1) comprising one or more mutations. The one or more mutations impart at least one improved characteristic to an AAV9 virion comprising the engineered AAV9 capsid protein, compared to a virion with a wild-type AAV9 capsid protein or a capsid protein without the one or more mutations. The engineered AAV9 capsid protein as provided herein may comprise at least one substitution at positions L382, F535, and H584, or at least two substitutions at positions L382, F535, and H584, or at least three substitutions atpositions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1 (Table 1). The engineered AAV9 capsid may comprise a substitution at F535, for example a phenylalanine (F) to tyrosine (Y) substitution at position 535, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The engineered AAV9 capsid may comprise a substitution at H584, for example from a histidine (H) to a polar charged or uncharged amino acid, e.g., arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glutamine (Q), lysine (K), serine (S), threonine (T), or tyrosine (Y). In one embodiment, the engineered AAV9 capsid comprises a histidine (H) to an arginine (R) substitution or a histidine (H) to a threonine (T) at position 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The engineered AAV9 capsid may comprises a substitution at L382, for example a leucine (L) to an isoleucine (I) at position 382, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The engineered AAV9 capsid may comprise a substitution at position 382 and position 535, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The engineered AAV9 capsid may comprise a substitution at position 535 and position 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The engineered AAV9 capsid may comprise a substitution at position 382 and position 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The engineered AAV9 capsid may comprise a substitution at one, two, or all three of positions 382, 535, and 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1.

[0087] The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO:2, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:2. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:3. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise asequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:4. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO: 5, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:5. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:6. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:7. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO: 8, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:8. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO:9, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO:9. The AAV9 capsid protein of the current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO: 10. The AAV9 capsid protein ofthe current disclosure may comprise an amino acid sequence as set forth in SEQ ID NO: 1 1, or an amino acid sequence at least 80% identical thereto. In an aspect, the AAV9 capsid protein may comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in SEQ ID NO: 11.TABLE 1:

[0088] The AAV9 capsid protein of the current disclosure may further comprise additional mutations in the capsid protein sequence, wherein the capsid protein sequence is at least about 50%, 60%, 70%, or 80% identical to any one of SEQ ID NO:2, SEQ ID NOB, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO 8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11 and possesses the beneficial properties as described herein.

[0089] By way of example, the capsid protein sequence of the current disclosure may include the mutations at one or more of L382, F535, and H584, and also include one or more mutations at any other position(s) in the capsid protein sequence and / or also include amino acid insertions (e.g., 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, or more amino acid fragment insertions) at any other position(s) in the capsid protein sequence. Non-limiting examples of additional mutations that may be included in the capsid protein sequence of the current disclosure can be found, for instance, in Martini et al., Cellular Physiology and Biochemistry (2016) 39 (2): 544-553; lida et al., Biomed Res Int. 2013 May 20;2013:974819; Chen et al., Nature Communications volume 14, Article number: 3345 (2023); each of which is hereby incorporated by reference herein in their entirety. Non-limiting examples of additional amino acid fragments that may be included in the capsid protein sequence of the current disclosure can be found, for instance, in Deverman et al., Nat Biotechnol. 2016 Feb. 34(2):204-9;Huang et al., Science 2024 Jun 14;384(6701): 1220-1227; and Tabebordbar et al., Cell 2021 Sep 16;184(19):4919-4938.e22; each of which is hereby incorporated by reference herein in its entirety.III. Engineered AAV Capsid Proteins Based on AAV9 Mutations

[0090] In an aspect, the current disclosure encompasses an engineered adeno-associated virus (AAV) capsid comprising a capsid protein (e.g., VP1) comprising one or more mutations as described herein at a corresponding position. The AAV capsid protein may be of a serotype other than AAV9 as disclosed herein, wherein the AAV capsid protein comprises one or more mutations analogous to those described herein with respect to AAV9 (e.g., L382, F535, or H584) at a corresponding position on the engineered capsid, wherein the corresponding position is determined by alignment with SEQ ID NO: 1. The serotype other than AAV9 may be selected from any one of AAV1, AAV2, AAV3B, AAV4, AAVS, AAV6, AAV7, AAV8, AAVrhlO, AAV11, AAV12, or AAV13.

[0091] TABLE 2 provides exemplary amino acid identities at corresponding positions for F535 and H584 in AAV1, AAV2, AAV3B, AAV4, AAVS, AAV6, AAV7, AAV8, AAVrhlO, AAV1 1, AAV12, and AAV13. For instance, the engineered AAV capsid may comprise a mutation at one or more positions corresponding to L382, F535, or H584 of AAV9, when the amino acid sequence of the engineered AAV capsid is aligned with the sequence set forth in SEQ ID NO: 1. The engineered AAV capsid may comprise a substitution at a position corresponding to F535 when the amino acid sequence of the engineered AAV capsid (e.g., other than AAV9) is aligned with the sequence set forth in SEQ ID NO: 1. The substitution at the position corresponding to F535 may be substitution to a hydrophilic residue (e.g., tyrosine), for example F535Y. The engineered AAV capsid may comprise a substitution at a position corresponding to H584 when the amino acid sequence of the engineered AAV capsid (e.g., other than AAV9) is aligned with the sequence set forth in SEQ ID NO: 1. The substitution at the position corresponding to H584 may be substitution to a positively charged residue (e.g., arginine (R)). The substitution at the position corresponding to H584 may be substitution to a polar charged or uncharged amino acid, e.g., arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glutamine (Q), lysine (K), serine (S), threonine (T), or tyrosine (Y). Theengineered AAV9 capsid may comprise a substitution at a position corresponding to L382, when the amino acid sequence of the engineered AAV capsid (e.g., other than AAV9) is aligned with the sequence set forth in SEQ ID NO: 1. The substitution at the position corresponding to L382 may be to an isoleucine (I). The engineered AAV9 capsid may comprise a substitution at a combination of positions described herein, e.g., (i) F535 and H584; (ii) F535 and L382; (iii) H584 and L382; or (iv) L382, F535, and H584, when the amino acid sequence of the engineered AAV capsid (e.g. , other than AAV9) is aligned with the sequence set forth in SEQ ID NO: 1.IV. Polynucleotides Encoding Engineered AAV Capsids

[0092] In an aspect, the current disclosure also encompasses a polynucleotide sequence comprising a nucleic acid sequence encoding an engineered capsid protein (e.g., AAV9 capsid protein or other engineered capsid protein) disclosed herein. The polynucleotide may encode an AAV9 capsid or another AAV as described herein (e.g., AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO, AAV11, AAV12, or AAV13). The polynucleotide sequence disclosed herein may be an isolated nucleic acid, a plasmid, a genome, or a viral vector. The AAV cap coding region of the AAV genome encodes the capsid proteins VP1, VP2, and VP3. AAV helper functions comprising these capsid proteins and rep proteins can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV vector comprising an expression cassette. AAV helper constructs are thus used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions that are necessary for productive AAV infection. In an aspect, the current disclosure encompasses a AAV helper construct comprising a polynucleotide sequence encoding a VP1 capsid protein comprising at least one substitution at positions L382, F535, and H584, at least two substitutions at positions L382, F535, and H584, or at least three substitutions at positions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1 (Table 1). In addition to substitutions at one or more of L382, F535, and H584, other amino acid positions may be substituted.

[0093] The encoded capsid may be an AAV9 capsid. The encoded AAV9 capsid may comprise a substitution at F535, for example a phenylalanine (F) to tyrosine (Y) substitution at position 535, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9capsid may comprise a substitution at H584, for example from a histidine (H) to a polar charged or uncharged amino acid, e.g., arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glutamine (Q), lysine (K), serine (S), threonine (T), or tyrosine (Y). In one embodiment, the encoded AAV9 capsid comprises a histidine (H) to an arginine (R) substitution or a histidine (H) to a threonine (T) at position 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at L382, for example a leucine (L) to an isoleucine (I) at position 382, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at position 382 and position 535, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at position 535 and position 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at position 382 and position 584, with reference to the amino acid sequence as set forth in SEQ ID NO:1. The encoded AAV9 capsid may comprise a substitution at positions 382, 535, and 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1.

[0094] The AAV helper construct may lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. (See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936- 2945). A number of other vectors have been described which encode Rep and / or Cap expression products. (See, e.g., U.S. Pat. No. 5,139,941.) In an aspect, the current disclosure encompasses any suitable helper construct encoding the engineered adeno-associated virus serotype 9 (AAV9) capsid protein (VP1) as disclosed herein.

[0095] In an aspect, the polynucleotide sequence may also be a viral vector (e.g., rAAV9 viral vector). In an aspect, the polynucleotide sequence, for example the viral vector (e.g., rAAV9 viral vector) may further comprise a recombinant nucleic acid sequence. Description of suitable recombinant nucleic acid sequences are provided herein below. Additionally, the polynucleotide sequence may comprise additional regulatory sequences non-limiting examples of which include transcription initiation, termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability and sequences that enhance secretion of the encoded product. Regulatory sequences, including promoters may be native, constitutive, inducible and / or tissue-specific and are known in the art.V. Recombinant Adeno-Associated Viruses (rAAV)

[0096] Disclosed herein are recombinant adeno-associated virus (rAAV) virions or particles comprising the engineered capsid protein as described herein. The rAAV may comprise an engineered capsid protein comprising at least one substitution at positions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1.

[0097] Disclosed herein are recombinant adeno-associated virus 9 (rAAV9) virions or particles comprising the engineered AAV9 capsid protein. Thus, in an aspect, the current disclosure encompasses a rAAV9 comprising an engineered capsid protein comprising at least one substitution at positions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The rAAV9 may comprise the engineered AAV9 capsid protein disclosed herein with the amino acid sequence as set forth in any one of SEQ ID NOS:2-11, or an amino acid sequence at least 80% identical thereto.

[0098] The rAAV (e.g., rAAV9) comprising the engineered capsid protein is superior to an AAV particle (e.g., AAV9 particle) comprising a wild-type capsid protein in one or more properties. For example the rAAV (e.g., rAAV9) may exhibit increased in vitro and in vivo viral stability, better packaging and manufacturability, increased infectivity of mammalian cells, for example neuronal and muscle cells, improved tropism to select cell-types and enhanced ability to cross the blood-brain barrier, compared to a control AAV (e.g., control AAV9), wherein the engineered capsid protein comprises one or more mutations with respect to wild-type AAV (e.g., AAV9) capsid protein. As used herein, a “control AAV” virion can comprise a parental AAV capsid protein. A control AAV virion can be an AAV virion comprising wild-type AAV capsid, e.g., comprising only wild-type capsid (and not any engineered AAV capsid of the present disclosure). As used herein, a “control AAV9” virion can comprise a parental AAV9 capsid protein. A control AAV9 virion can be an AAV virion comprising wild-type AAV9 capsid, e.g.,comprising only wild-type capsid (and not any engineered AAV capsid of the present disclosure).

[0099] In an aspect, the disclosed rAAV provide higher titers of fully assembled virions compared to a control AAV (c'.g, AAV9), when manufactured in a suitable host cell. In an aspect, the AAV (c.g, rAAV9) comprising the engineered capsid protein have improved packaging function when assembled in a suitable host cell. Suitable host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an exogenous nucleic acid molecule. Thus, a “host cell” as used herein generally refers to a cell which has been transfected with an exogenous nucleic acid molecule. The host cell includes any eukaryotic cell or cell line. For example, the host cell may be an erythrocyte, a platelet, a bone marrow cell, a vascular endothelial cell, a lymphocyte, a hepatocyte, a neuronal or brain cell (e.g, neurons and neuroglia), a bronchial endothelial cell, an epidermal cell, a respiratory interstitial cell, an epidermal cell, a fat cell, a dermal fibroblast, a muscle cell, or any other mammalian cell type. In one embodiment, the host cell is a neuronal cell. Non-limiting examples include HEK293, HEK293T, CHO, Sf9, myeloma cells like SP2 or NSO. In one aspect, cells from the stable human cell line, 293 (readily available through, e.g, the ATCC under Accession No. ATCC CRL1573). Particularly, the human cell line 293, which is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59) and expresses the adenoviral Ela and Elb genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions.

[0100] Production host cells are rendered capable of providing AAV helper functions in order to replicate and encapsidate the recombinant AAV particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV vectors. Thus, AAV helper functions include one, or both of the major AAV open reading frames (ORFs), namely the rep and cap coding regions, or functional homologues thereof.

[0101] The AAV rep coding region of the AAV genome encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other exogenous) promoters. The Rep expression products are collectively required for replicating the AAV genome. The AAV cap coding region of the AAV genome encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV vector comprising an expression cassette, AAV helper constructs are thus used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions that are necessary for productive AAV infection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. (See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945). A number of other vectors have been described which encode Rep and / or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.

[0102] As disclosed herein above, the current disclosure encompasses a AAV helper construct comprising a polynucleotide sequence encoding a VP1 capsid protein comprising at least one substitution at positions L382, F535, and H584, or at least two substitutions at positions L382, F535, and H584, or at least three substitutions at positions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1 (Table 1). The encoded AAV capsid may be an AAV9 capsid comprising a substitution at F535, for example a phenylalanine (F) to tyrosine (Y) substitution at position 535, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at H584, for example from a histidine (H) to a polar charged or uncharged amino acid, e.g., arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glutamine (Q), lysine (K), serine (S), threonine (T), or tyrosine (Y). In one embodiment, the encoded AAV9 capsid comprises a histidine (H) to an arginine (R) substitution or a histidine (H) to a threonine (T) at position 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprisea substitution at L382, for example a leucine (L) to an isoleucine (I) at position 382, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at position 382 and position 535, with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at position 535 and position 584 with reference to the amino acid sequence as set forth in SEQ ID NO: 1. The encoded AAV9 capsid may comprise a substitution at one, two, or all three positions 382, 535, and 584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1.

[0103] Because of the infection of the host cell with a helper virus, the AAV Rep and / or Cap proteins are produced. The Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into capsids, and the AAV genome is packaged into the capsids. This results the AAV being packaged into recombinant AAV particles comprising the expression cassette. Following recombinant AAV replication, recombinant AAV particles can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. The resulting recombinant AAV particles are then ready for use for gene delivery.

[0104] In an aspect, the rAAV particle assembled in a production host cell comprising the helper construct encoding the disclosed capsid protein, may produce higher virus production yields at harvest. In an aspect, the host cell comprising the helper construct encoding the disclosed capsid protein may yield more packaged AAV particles at harvest, for example about 1.2 fold, 1.3 fold,1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold,2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, or 3 fold higher viral titers compared to control helper constructs encoding wild-type VP1. In an aspect, the production host cell comprising the helper construct encoding the disclosed capsid protein may yield more packaged virus particles that are retained within the cell pellet fraction at harvest. In an aspect, the number of packaged virus particles in the pellet may be at least about 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, or 4 fold higher.

[0105] In an aspect, the packaged rAAV virion of the present disclosure may exhibit at least 2- fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased infectivity of a mammalian cell compared to the infectivity of a control AAV9 virion. In an aspect, the rAAV may exhibit increased infectivity for one or more cell-types. For example, the cell type may be an erythrocyte, a platelet, a bone marrow cell, a vascular endothelial cell, a lymphocyte, a hepatocyte, a neuronal or brain cell (e.g., neurons and neuroglia), a bronchial endothelial cell, an epidermal cell, a respiratory interstitial cell, an epidermal cell, a fat cell, a dermal fibroblast, a muscle cell, or any other mammalian cell type. In one embodiment, the host cell is a neuronal cell. Non-limiting examples include COS-7, Lec2, HEK293T, SH-SY5Y, cardiomyocytes A549, WEHI, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, WI38, HeLa, CHO, 293, Vero, NIH 3T3, PC12, Huh-7 Saos, C2C12, RATI, Sf9, L cells, HT1080, human embryonic kidney (HEK), human embryonic stem cells, human adult tissue stem cells, pluripotent stem cells, induced pluripotent stem cells, reprogrammed stem cells, organoid stem cells, bone marrow stem cells, HLHepG2, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. In an aspect, the rAAV virion of the present disclosure may exhibit at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased infectivity of a neuronal cell compared to the infectivity of a control AAV9 virion.

[0106] In an aspect, the rAAV virion of the present disclosure may exhibit at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased infectivity of a neuronal cell compared to the infectivity of a control AAV9 virion. In an aspect, the rAAV virion exhibits at least 5-fold, at least 10-fold, at least 15- fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased infectivity of a neuronal cell, when administered via intracranial, intranerve, intracerebroventicular, intrathecal, intra-ci sterna magna, or intravenous injection, compared to the infectivity of a control AAV9 virion.

[0107] In some cases, an rAAV virion of the present disclosure exhibits at least 2-fold, at least 5- fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased infectivity of a muscle cell, compared to the infectivity of a control AAV9 virion. In an aspect, the rAAV virion exhibits at least 5-fold, at least 10-fold, at least 15- fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, increased infectivityof a muscle cell, when administered via intramuscular, or intravenous injection, compared to the infectivity of a control AAV9 virion.

[0108] Whether a given rAAV virion exhibits increased infectivity of a cell can be determined in vitro or in vivo by techniques well known in the art and demonstrated herein. Therapeutic effect of a therapeutic gene product encoded by the rAAV virion can also be used. Therapeutic effects can include, e.g., a) an increase in neurogenesis; b) amelioration of a symptom of a neurological disease or disorder; c) amelioration of a symptom of a neuromuscular disease or disorder; etc. For example, an rAAV virion (e.g., rAAV9 virion) of the present disclosure that comprises: a) a engineered capsid of the present disclosure; and b) a recombinant nucleic acid sequence encoding a therapeutic gene product, when administered to an individual (e.g., via intracranial, intramuscular, intracerebroventicular, intrathecal, intranerve, intra-ci sterna magna, or intravenous injection), results in a therapeutic effect of the therapeutic gene product, that is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, greater than the therapeutic effect that results when a control rAAV virion that comprises: a) a control AAV9 capsid (e.g., a wild-type AAV9 capsid); and b) a recombinant nucleic acid sequence encoding a therapeutic gene product is administered via the same route of administration.

[0109] In an aspect, the rAAV of the present disclosure exhibit improved tropism to select cells / organs or tissues. Improved tropism can be determined by reference to a control AAV (e.g., AAV9 . In an aspect, the rAAV (e.g., rAAV9) exhibits at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, or 200% or more of the tropism, respectively, of the control. In an aspect, the rAAV exhibits improved transduction to one or more organs or organoids of the body. Organs of the body include for example but are not limited to skin, hair, nails, sense receptors, sweat gland, oil glands, bones, muscles, brain, spinal cord, nerve, pituitary gland, pineal gland, hypothalamus, thyroid gland, parathyroid, thymus, adrenals, pancreas (islet tissue), heart, blood vessels, lymph nodes, lymph vessels, thymus, spleen, tonsils, nose, pharynx, larynx, trachea, bronchi, lungs, mouth, pharynx, esophagus, stomach, small intestine, large intestine, rectum, anal canal, teeth, salivary glands, tongue, liver, gallbladder, pancreas, appendix, kidneys, ureters, urinary bladder, urethra, testes, ductus (vas) deferens, urethra, prostate, penis, scrotum, ovaries, uterus, uterine (fallopian) tubes,vagina, vulva, and mammary glands (breasts). Organ systems of the body include but are not limited to the integumentary system, skeletal system, muscular system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system, and reproductive system. In some aspects, transduction and / or tropism is increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, 65%, about 70%%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. In some embodiments, transduction and / or tropism is increased by about 5% to about 80%, about 10% to about 70%, about 20% to about 60% or about 30% to about 60%. In some aspects, the rAAV9 exhibits a greater ability to cross the blood-brain barrier.

[0110] In an aspect, the rAAV of the present disclosure may exhibit improved biophysical properties. In an aspect, the rAAV of the present disclosure exhibit a significantly altered zeta potential compared to control AAV (e.g., AAV9). The zeta potential is an important parameter as it can affect the stability, aggregation, infectivity, and interactions of the virion with host cells. The electrostatic forces between the virion and the host cell surface play a role in viral attachment and entry into host cells. A higher zeta potential, whether positive or negative, can influence the repulsion or attraction between virions and host cells. This can impact the efficiency of viral attachment, entry, and subsequent infection. Additionally, changes in the zeta potential may be associated with alterations in the virion's structure or surface properties, which can affect its biological activity. In an aspect, the rAAV (e.g., rAAV9) disclosed herein exhibits a higher negative zeta potential than the control AAV (e.g., AAV9). In an aspect, the rAAV has about a 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, or 3 fold more negative zeta potential in comparison control AAV (e.g., control AAV9). In an aspect, the rAAV of the present disclosure may also exhibit decreased stability at pH 7-4 compared to control AAV (e.g., control AAV9). Decreased capsid stability may enhance its release of vector DNA to a transduced cell following infection, increasing the overall infectivity of the virion. Viral stability can be measured using techniques well known in the art, for example differential scanning fluorimetry (DSF).Recombinant nucleic acid sequences

[0111] As disclosed above, provided herein are rAAV (e. ., rAAV9) comprising a recombinant capsid protein and further comprising a recombinant nucleic acid sequence. Also, provided herein polynucleotide sequence comprising a nucleic acid sequence encoding the engineered AAV capsid protein (e.g., engineered AAV9 capsid protein) and further comprising a recombinant nucleic acid sequence. In an aspect, the recombinant nucleic acid may be a non- therapeutic nucleic acid sequence. Examples of non-therapeutic nucleic acids include nucleic acids encoding a reporter RNA or protein. In an aspect, the non-therapeutic nucleic acid may encode a non-therapeutic gene product. In an aspect, the recombinant nucleic acid may be a therapeutic nucleic acid e.g., that provides clinical benefit, for example, to subjects afflicted with a cancer, a neurological disease, a neuromuscular disease, a liver disease, a kidney or renal disease, an ocular disease, or a muscular disease, a metabolic disease, a pulmonary disease, or any other type of disease afflicting a subject. A therapeutic nucleic acid of the present disclosure may provide therapeutic benefits against a neurological or a neuromuscular disorder. Non limiting examples of neurological and neuromuscular diseases include neurodegenerative diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS); neuromuscular disorders: muscular dystrophy (e.g., Duchenne muscular dystrophy), myasthenia gravis, Charcot-Mari e-Tooth disease, spinal muscular atrophy; neurodevel opmental disorders: autism spectrum disorders, attention-deficit / hyperactivity disorder (ADHD), intellectual disabilities; neuroinflammatory disorders: multiple sclerosis, Guillain-Barre syndrome, transverse myelitis; cerebrovascular diseases: stroke (Ischemic and Hemorrhagic), cerebral aneurysms; epileptic disorders: epilepsy, seizure disorders, movement disorders: essential tremor, dystonia, restless legs syndrome. In an aspect, the therapeutic nucleic acid of the present disclosure may provide therapeutic benefits against a genetic disorder of the nervous system, non-limiting examples of which include adrenoleukodystrophy, Alzheimer disease, amyotrophic lateral sclerosis, Angelman syndrome, ataxia telangiectasia, Charcot- Marie-Tooth syndrome, Cockayne syndrome, deafness, Duchenne muscular dystrophy, epilepsy, essential tremor, fragile X syndrome, Friedreich's ataxia, Gaucher disease, Huntington disease, Leigh syndrome, Lesch-Nyhan syndrome, maple syrup urine disease, Menkes syndrome, myotonic dystrophy, narcolepsy, neurofibromatosis, Niemann-Pick disease, Parkinson disease, phenylketonuria, Prader-Willi syndrome, Refsum disease, Rett syndrome, SLC6A1 deficiencydisorder, spastic paraplegia, spinal muscular atrophy, spinocerebellar ataxia, tangier disease, Tay-Sachs disease, tuberous sclerosis, Von Hippel-Lindau syndrome, Williams syndrome, Wilson's disease, Zellweger syndrome.

[0112] In an aspect, the recombinant nucleic acid may comprise a nucleotide sequence encoding one or more gene products (one or more recombinant gene products). The recombinant gene product may be a polypeptide, an RNA or both. Where the gene product is an RNA, in some aspect, the RNA gene product may encode a polypeptide. Where the gene product is an RNA, in some aspects, the RNA gene product may not encode a polypeptide. In some aspects, the rAAV virion of the present disclosure may comprise a single recombinant nucleic acid comprising a nucleotide sequence encoding a single recombinant gene product. The rAAV virion of the present disclosure may comprise a single recombinant nucleic acid comprising a nucleotide sequence encoding two or more recombinant gene products. Where the single recombinant nucleic acid encodes two or more recombinant gene products, the nucleotide sequences encoding the two or more recombinant gene products may be operably linked to the same promoter or to two or more different promoters. In some cases, an rAAV virion of the present disclosure comprises two recombinant nucleic acids, each comprising a nucleotide sequence encoding a recombinant gene product.

[0113] In some aspects, the gene product may be an RNA. In an aspect the RNA may be a noncoding. Non-limiting examples of non-coding RNA include an interfering RNA, an antisense RNA, a ribozyme, a microRNA (miRNA), an ASO, a siRNA, shRNA, an CRISPR related RNA for example a gRNA, sgRNA, single-stranded RNA (ssRNA), a double stranded RNA, or any combination thereof. In some aspects, the RNA is a non-therapeutic RNA. In some aspects, the RNA is a therapeutic RNA, e.g., a RNA that provides clinical benefit. The therapeutic RNA may provide therapeutic benefits against a neurological or a neuromuscular disorder as provided herein above.

[0114] In some aspects, the gene product is a polypeptide. In some aspects, the polypeptide is a non-therapeutic polypeptide. Examples of non-therapeutic polypeptides include for example reporter polypeptides. Non-limiting examples of reporter polypeptides include fluorescent reporters like GFP, RFP, YFP, BFP, luciferase, renilla luciferase, non-fluorescent reporters likeP-Galactosidase, chloramphenicol acetyl transferase, and alkaline phosphatase. In some aspects, the gene product is a therapeutic polypeptide, e.g., a polypeptide that provides clinical benefit. Examples of therapeutic polypeptides include, but are not limited to antibodies, vaccines, enzymes, receptors, polypeptide inhibitors, or polypeptide activators. The therapeutic polypeptide may provide therapeutic benefits against a range of diseases including neurological or a neuromuscular disorder as provided herein above.

[0115] In some aspects, the gene product is a site-specific nuclease that provide for site-specific knock-down of gene function. In some aspects, the gene product is an RNA-guided endonuclease that provides for modification of a target nucleic acid. In some aspects, the gene products are: i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; and ii) a guide RNA that comprises a first segment that binds to a target sequence in a target nucleic acid and a second segment that binds to the RNA-guided endonuclease. In some aspects, the gene products are: i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; ii) a first guide RNA that comprises a first segment that binds to a first target sequence in a target nucleic acid and a second segment that binds to the RNA-guided endonuclease; and iii) a first guide RNA that comprises a first segment that binds to a second target sequence in the target nucleic acid and a second segment that binds to the RNA-guided endonuclease. In some aspects the site-specific nuclease may be for use in the treatment of a genetic disorder, nonlimiting examples of which include adrenoleukodystrophy, Alzheimer disease, amyotrophic lateral sclerosis, Angelman syndrome, ataxia telangiectasia, Charcot -Marie-Tooth syndrome, Cockayne syndrome, deafness, Duchenne muscular dystrophy, epilepsy, essential tremor, fragile X syndrome, Friedreich's ataxia, Gaucher disease, Huntington disease, Leigh syndrome, Lesch- Nyhan syndrome, maple syrup urine disease, Menkes syndrome, myotonic dystrophy, narcolepsy, neurofibromatosis, Niemann-Pick disease, Parkinson disease, phenylketonuria, Prader-Willi syndrome, Refsum disease, Rett syndrome, SLC6A1 deficiency disorder, spastic paraplegia, spinal muscular atrophy, spinocerebellar ataxia, tangier disease, Tay-Sachs disease, tuberous sclerosis, Von Hippel-Lindau syndrome, Williams syndrome, Wilson's disease, and Zellweger syndrome.

[0116] In some aspects, the gene product is an aptamer. Aptamers may be therapeutic or non- therapeutic, for example suitable for diagnostic use or imaging. Suitable aptamers may includebut are not restricted to aptamers that target amyloid beta, glutamate receptor, brain derived neurotropic factor, VEGF, dopamine, tau protein, neuron specific enolase, glial fibrillary acidic protein, brain tumor markers, or any combination thereof.

[0117] Additionally, the rAAV may comprise regulatory sequences, non-limiting examples of which include a promoter, an enhancer, a leader, a transcription start site (TSS), a linker, 5' and 3' untranslated regions (UTRs), an intron, a polyadenylation signal, and a termination region or sequence, and / or sequences that enhance protein stability and sequences that enhance secretion of the encoded product. Regulatory sequences, including promoters may be native, constitutive, inducible and / or tissue-specific and are known in the art. In some aspects, a cell type-specific or a tissue-specific promoter can be operably linked to the recombinant nucleic acid insert (also referred to as a heterologous nucleotide sequence) encoding the heterologous gene product, and allowing for selectively or preferentially producing a gene product in a particular cell type(s) or tissue(s).

[0118] In some aspects, the recombinant nucleic acid is packaged with the variant AAV9 capsid polypeptides of the present disclosure. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2200. 2400, 2600, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600 or 4800 nucleic acids in length. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 50 nucleic acids to at least 1500 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 100 nucleic acids to at least 1400 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 200 nucleic acids to at least 1100 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 300 nucleic acids to at least 1000 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 100 nucleic acids to at least 900 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 200 nucleic acids to at least 900 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 300 nucleic acids to at least 900 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 100 nucleic acids to at least 600 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 1000 nucleic acids to at least 2000 nucleic acids. In some aspects, the recombinantnucleic acid or packaged nucleic acid is at least 2000 nucleic acids to at least 3000 nucleic acids. In some aspects, the recombinant nucleic acid or packaged nucleic acid is at least 3000 nucleic acids to at least 4000 nucleic acids.VI. Methods of Making

[0119] In an aspect, the current disclosure also encompasses method of making the rAAV virions (e.g., rAAV9 virions) provided herein. In various aspects, the invention provides a method for generating a rAAV virion comprising an engineered capsid protein disclosed herein. A variety of methods for generating AAV virions are known in the art and can be used to generate AAV virions comprising the rAAV vectors (e.g., rAAV9 vectors) described herein. Generally, the methods involve inserting or transducing an AAV vector of the invention into a host cell capable of packaging the AAV vector into an AAV virion. Exemplary methods are described and referenced below; however, any method known to one of skill in the art can be employed to generate the AAV virions of the invention.

[0120] An AAV vector comprising a recombinant nucleic acid and used to generate an AAV virion can be constructed using methods that are well known in the art. For example, the recombinant nucleic acid sequence(s) can be directly inserted into an AAV genome with the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art.

[0121] In order to produce AAV virions, an AAV vector is introduced into a suitable production host cell using known techniques, such as by transfection. Several transfection techniques are generally known in the art, non-limiting examples include calcium phosphate co-precipitation, direct micro-injection into cultured cells, electroporation, liposome-mediated gene transfer, lipid- mediated transduction, and nucleic acid delivery using high-velocity microprojectiles.

[0122] Suitable production host cells for producing AAV virions include any species and / or type of cell that can be, or have been, used as recipients of a heterologous AAV DNA molecule, and can support the expression of required AAV production cofactors from helper viruses. Such hostcells can include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule. The term includes the progeny of the original cell transfected. Thus, a “host cell” as used herein generally refers to a cell transfected with an exogenous DNA sequence. For example, the host cell may be an erythrocyte, a platelet, a bone marrow cell, a vascular endothelial cell, a lymphocyte, a hepatocyte, a neuronal or brain cell (e.g., neurons and neuroglia), a bronchial endothelial cell, an epidermal cell, a respiratory interstitial cell, an epidermal cell, a fat cell, a dermal fibroblast, a muscle cell, or any other mammalian cell type. Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) may be used. The human cell line HEK293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59) and expresses the adenoviral Ela and Elb genes (Aiello et al. (1979) Virology 94:460). The HEK293 cell line is readily transfected and provides a convenient platform in which to produce AAV virions. Methods of producing an AAV virion in insect cells are known in the art and can be used to produce a subject AAV virion. See, e.g., U.S. Patent Publication No. 2009 / 0203071; U.S. Pat. No. 7,271,002; incorporated herein by reference in their entirety.

[0123] In some aspects, the AAV virion or AAV vector is packaged into an infectious virion or virus particle, by any of the methods described herein or known in the art. In some aspects, the engineered AAV capsid polypeptide allows for improved packaging as compared to a control AAV capsid polypeptide (e.g., control AAV9 capsid polypeptide). As described above, the engineered capsid protein may result in higher virus production yields at harvest. In an aspect, the disclosed capsid protein may yield more packaged AAV particles at harvest, for example about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, or 3 fold higher viral titers compared to control AAV capsid polypeptide (e.g., control AAV9 capsid polypeptide). In an aspect, the disclosed capsid protein may yield more packaged virus particles that are retained within the cell pellet fraction at harvest. In an aspect, the number of packaged virus particles in the pellet may be at least about 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, or 4 fold higher. In some aspects, anAAV vector packaged with the engineered AAV capsid polypeptides transduce into cells in vivo better than a vector packaged with control AAV capsid polypeptides (e.g., control AAV9 capsid polypeptides). In some aspects, the AAV vector packaged with the engineered AAV capsid polypeptides transduce into cells in vitro better than a vector packaged with control AAV capsid polypeptides (e.g., control AAV9 capsid polypeptides). In some aspects, the AAV vector packaged with the engineered AAV capsid polypeptides results in recombinant nucleic acid expression higher than a nucleic acid packaged with control AAV capsid polypeptides e.g., control AAV9 capsid polypeptides). In some aspects, the AAV vector packaged with the engineered AAV results in transgene expression better than a transgene packaged with control AAV capsid polypeptides (e.g., control AAV9 capsid polypeptides).VII. Methods of treatment

[0124] In an aspect, the current disclosure also encompasses a method of treatment of a disease / disorder, comprising administering to a subject in need thereof a recombinant adeno- associated virus described herein (e.g., serotype 9 (rAAV9)) or a pharmaceutical composition thereof comprising: the engineered capsid protein; and the recombinant nucleic acid. In an aspect, the current disclosure also encompasses a method of gene delivery into a subject in need thereof, comprising administering to the subject a recombinant adeno-associated virus (e.g., serotype 9 (rAAV9)) or a pharmaceutical composition thereof comprising: the engineered capsid protein; and the recombinant nucleic acid.Pharmaceutical compositions and dosage

[0125] The recombinant AAV of the current disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises the recombinant AAV as disclosed herein and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohol’s such as mannitol, sorbitol, or sodium chloride in thecomposition. Pharmaceutically acceptable carriers may further comprise auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance shelf life or effectiveness.

[0126] The compositions may be in a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Suitable routes of administration may, for example, include intravenous, intracranial, intrathecal, subcutaneous, intranasal route, cranial, transmucosal, trans-nasal, transcranial, intracerebroventricular, intestinal, and / or parenteral delivery. In some aspects, compositions herein can be formulated for parenteral delivery. In some aspects, compositions herein can be formulated for intramuscular, intrathecal, subcutaneous, intramedullary, intravenous, intraperitoneal, intracranial, and / or intranasal injections. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. In one aspect, the vector is administered by intravenous infusion or injection. In another aspect, the vector is administered by intramuscular or subcutaneous injection. In another aspect, the vector is administered periodically. In an aspect, the vector is delivered to a specific location using stereostatic delivery.

[0127] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., rAAV, polynucleotide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0128] Generally, dispersions are prepared by incorporating the active composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0129] The vector of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. In certain aspects, the active composition may be prepared with a carrier that will protect the composition against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. (See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.) The pharmaceutical compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of the vectors of the invention. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the vector may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the vector to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the vector are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0130] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active composition and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0131] Multiple doses of the rAAV virion or a pharmaceutical composition thereof can be administered to a subject in need thereof. Where multiple doses are administered over a period of time, the composition may be administered once a month to about once a year, from about once a year to once every 2 years, from about once every 2 years to once every 5 years, or from about once every 5 years to about once every 10 years, over a period of time. For example, a subject AAV virion is administered over a period of from about 3 months to about 2 years, from about 2 years to about 5 years, from about 5 years to about 10 years, from about 10 years to about 20 years, or more than 20 years. The actual frequency of administration, and the actual duration of treatment, depends on various factors. In some aspects, the administration regimen is part of a vaccination regimen.

[0132] The dose to achieve a therapeutic effect, e.g., the dose in vector genomes / per kilogram of body weight (vg / kg), will vary based on several factors including, but not limited to: route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease treated, any host immune response to the viral vector, a host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the protein expressed. One skilled in the art can readily determine a virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors. Generally, doses will range from at least about, or more, for example, l * 109, l * 1010, 1 011, l * 1012, I O13or l * 1014, 2M014, 3* 1014or more, vector genomes per kilogram (vg / kg) of the weight of the subject, to achieve a therapeutic effect. In an aspect, dosage may range from about 1 x 1010vg total to about 1 x 1016vg total.

[0133] In some aspects, the ph rmaceutical composition can be enclosed in multiple or single dose containers. The enclosed compositions can be provided in kits, for example, including component parts that can be assembled for use. A kit may comprise the rAAV. A kit may comprise the rAAV and a second therapeutic agent for co-administration. The rAAV and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the one or more active agents. The containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical or transdermal administration.

[0134] The pharmaceutical composition may be administered at once or may be divided into a number of smaller doses to be administered at intervals of time. The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. Concentrations and dosage values may also vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.Diseases / Disorders

[0135] Non-limiting examples of diseases treatable in accordance with the invention include those set forth herein as well as a lung disease (e.g., cystic fibrosis), a blood coagulation or bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder (e.g, anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases, a copper or iron accumulation disorders (e.g., Wilson's or Menkes disease) lysosomal acid lipase deficiency, a neurological or neurodegenerative disorder, cancer (e.g., static melanoma, metastatic prostate cancer, metastatic breast cancer, triple negative breast cancer, bladder cancer, brain cancer, esophageal cancer, liver cancer, head and neck cancer, squamous cell lung cancer, non-smalllung cell cancer, Merkel cell carcinoma, sarcoma, hepatocellular cancer, multiple myeloma, leukemia, non-Hodgkin's lymphoma, lymphoma, B cell lymphoma, acute myeloid leukemia, pancreatic cancer, colorectal carcinoma, cervical cancer, gastric carcinoma, kidney cancer, metastatic renal cell carcinoma, leukemia, ovarian cancer, and malignant glioma), type 1 or type 2 diabetes, Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, a metabolic defect (e.g., glycogen storage diseases), a retinal degenerative disease (such as RPE65 deficiency or defect, choroideremia, and other diseases of the eye), and a disease of a solid organ (e.g., brain, liver, kidney, heart), as well as muscle diseases including not limited to Acid Maltase Deficiency (AMD), Amyotrophic Lateral Sclerosis (ALS), Andersen-Tawil Syndrome, Becker Muscular Dystrophy (BMD), Becker Myotonia Congenita, Bethiem Myopathy, Bulbospinal Muscular Atrophy (Spinal-Bulbar Muscular Atrophy), Carnitine Deficiency, Carnitine Palmityl Transferase Deficiency (CPT Deficiency), Central Core Disease (CCD), Centronuclear Myopathy, Charcot-Marie-Tooth Disease (CMT), Congenital Muscular Dystrophy (CMD), Congenital Myasthenic Syndromes (CMS), Congenital Myotonic Dystrophy, Cori Disease (Debrancher Enzyme Deficiency), Debrancher Enzyme Deficiency, Dejerine-Sottas Disease (DSD), Dermatomyositis (DM), Distal Muscular Dystrophy (DD), Duchenne Muscular Dystrophy (DMD), Dystrophia Myotonica (Myotonic Muscular Dystrophy), Emery-Dreifuss Muscular Dystrophy (EDMD), Endocrine Myopathies, Eulenberg Disease (Paramyotonia Congenita), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD), Finnish (Tibial) Distal Myopathy, Forbes Disease (Debrancher Enzyme Deficiency), Friedreich's Ataxia (FA), Fukuyama Congenital Muscular Dystrophy, Glycogenosis Type 10, Glycogenosis Type 11, Glycogenosis Type 2, Glycogenosis Type 3, Glycogenosis Type 5, Glycogenosis Type 7, Glycogenosis Type 9, Gowers-Laing Distal Myopathy, Hauptmann-Thanheuser MD (Emery- Dreifuss Muscular Dystrophy), Hereditary Inclusion-Body Myositis, Hereditary Motor and Sensory Neuropathy (Charcot-Marie-Tooth Disease), Hyperthyroid Myopathy, Hypothyroid Myopathy, Inclusion-Body Myositis (IBM), Inherited Myopathies, Integrin-Deficient Congenital Muscular Dystrophy, Kennedy Disease (Spinal-Bulbar Muscular Atrophy), Kugelberg-Welander Disease (Spinal Muscular Atrophy), Lactate Dehydrogenase Deficiency, Lambert-Eaton Myasthenic Syndrome (LEMS), Leigh syndrome, Limb-Girdle Muscular Dystrophy (LGMD), Lou Gehrig's Disease (Amyotrophic Lateral Sclerosis), McArdle Disease (Phosphorylase Deficiency), Merosin-Deficient Congenital Muscular Dystrophy, Metabolic Diseases of Muscle,Mitochondrial Myopathy, Miyoshi Distal Myopathy, Motor Neurone Disease, Muscle-Eye-Brain Disease, Myasthenia Gravis (MG), Myoadenylate Deaminase Deficiency, Myofibrillar Myopathy, Myophosphorylase Deficiency, Myotonia Congenita (MC), Myotonic Muscular Dystrophy (MMD), Myotubular Myopathy (MTM or MM), Nemaline Myopathy, Nonaka Distal Myopathy, Oculopharyngeal Muscular Dystrophy (OPMD), Paramyotonia Congenita, Pearson Syndrome, Periodic Paralysis, Peroneal Muscular Atrophy (Charcot-Marie-Tooth Disease), Phosphofructokinase Deficiency, Phosphoglycerate Kinase Deficiency, Phosphoglycerate Mutase Deficiency, Phosphorylase Deficiency, Phosphorylase Deficiency, Polymyositis (PM), Pompe Disease (Acid Maltase Deficiency), Progressive External Ophthalmoplegia (PEO), Rod Body Disease (Nemaline Myopathy), SLC6A1 deficiency, spastic paraplegia, Spinal Muscular Atrophy (SMA), Spinal-Bulbar Muscular Atrophy (SBMA), Steinert Disease (Myotonic Muscular Dystrophy), Tarui Disease (Phosphofructokinase Deficiency), Thomsen Disease (Myotonia Congenita), Ullrich Congenital Muscular Dystrophy, Walker-Warburg Syndrome (Congenital Muscular Dystrophy), Welander Distal Myopathy, Werdnig-Hoffmann Disease (Spinal Muscular Atrophy), and ZASP-Related Myopathy.

[0136] Suitable subjects may include, without limit, humans, as well as companion animals such as cats, dogs, rodents, and horses; research animals such as rabbits, sheep, pigs, dogs, primates, mice, rats, and other rodents; agricultural animals such as cows, cattle, pigs, goats, sheep, horses, deer, chickens, and other fowl; zoo animals; and primates such as chimpanzees, monkeys, and gorillas. The subject can be of any age without limitation. In an aspect, the subject may be a human.EXAMPLES

[0137] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1: Screening of AAV9 mutants for brain delivery

[0138] Adeno-Associated Virus (AAV) is a promising gene delivery vehicle for treatments of disease with known underlying genetic abnormalities. Many AAVs have been identified in nature. AAV9 is one of these naturally identified serotypes and has shown to be a dominant vector to get genes to the brain and muscle, albeit having many limitations. Therefore, a library of AAV9 mutants were generated with targeted mutagenesis to engineer and identify better capsids to deliver genes to the brain. The library of AAV9 mutants was screened in mice and non-human primate to select capsids that are compatible across species and can be utilized to develop gene therapies. Using these screens, 3 AAV capsids, which are functionally improved variants of AAV9 were identified. The first one has an F535Y mutation (VP1 numbering, with respect to SEQ ID NO: 1). The second has a H584R mutation (VP1 numbering, with respect to SEQ ID NO: 1). The third mutant “FYHR-AAV”, harbors 2 amino acid mutations (F535Y and H584R) on the capsid viral protein. Basic biology of AAV outlines that a single viral particle is made up of 60 viral proteins (subunits) with symmetrical icosahedral structure, therefore, FYHR- AAV harbors a total of 120 changes on the viral particle surface. FYHR-AAV was characterized, and data provided herein shows that this AAV9 mutant is significantly altered and enhanced with multiple properties including manufacturability, biophysical properties including thermal stability, and functional properties like tropism and infectivity.Example 2: Mutant AAV9 show improved manufacturability

[0139] FIG. 1A and FIG. IB provide data quantifying the production yields at harvest of the mutant AAV9 when compared to wild-type AAV9. Each AAV capsid was made with triple transfections in suspension HEK293 cells and harvested 72 hours post transfection. The triple transfection plasmids were (1) pTRS-ks-Cbh-GFP-BGHpA: a self-complementary AAV transgene vector, (2) Rep-Cap: either AAV9, F535Y-AAV9 (“F535Y”), H584R-AAV9 (“H584R”), or F535Y+H584R-AAV9 (here on referred to as “FYHR-AAV” or “FYHR”, and (3) Ad Helper Plasmid: pALD-X80. At harvest, culture samples were collected for ITR titer quantification. As shown in FIG. 1A, H584R and FYHR-AAV yield more packaged AAV particles at harvest. Additionally, with H584R and FYHR-AAV most of the packaged virusparticles are retained within the cell pellet fraction at harvest, thus confirming fully packaged viral particles that could be easily separated (see FIG. IB).Example 3: Mutant AAV9 show enhanced biophysical properties

[0140] The mutants were compared with wild-type AAV9 for biophysical properties including surface charge and thermal melting characteristics. The zeta potential of the viral particle provides a measure of the overall charge that the particle acquires in a medium. Zeta potential is a measurement of particle repulsion propensity. This can be used to predict the stability of the dispersion and provide insights into the surface chemistry of the particle. Zeta potential of the viral particles were measured using techniques known in the art. FIG. 2A provides a comparison of the zeta potential for the wild-type AAV9 and the mutants. As seen from the graph, the zeta potential, or the surface charge, of the viral particle is significantly altered when both mutations are present.

[0141] Differential scanning fluorimetry (DSF) can be used to measure the thermal stability of viral particles. DSF measures the melting temperature by measuring fluorescence signal at the point of which the virus melts. FIGs. 2B-2F show DSF data at different pH. These results show that FYHR-AAV has reduced viral stability at pH 7 - pH 4.Example 4: Functional properties

[0142] Infectivity assays were conducted in 96 well plates with various cell lines (COS-7, Lec2, HEK293T, and SH-SY5Y and cardiomyocytes) and quantified by measuring the percentage of GFP positive cells. GFP positive cells indicate cells that were transduced or “infected” by the specific viral capsid. FIGS 3A-3I show increased infectivity across multiple cell lines. Data shows that the FYHR-AAV double mutant exhibited higher transduction and hence enhanced infectivity across all cell lines tested.

[0143] Wild-type and mutant AAVs were also tested in vivo in mice by lumbar intrathecal administration for their ability to infect brain tissue. Mice were infected at 5 weeks of age with 1 x 1010vg / mouse and tissues were collected 3 weeks post injection. 30 pm frozen sections of mouse brains were stained for GFP. As seen in FIG. 3 J, H584R and FYHR-AAV both show increased transduction of the brain compared to AAV9.

[0144] Together, these data show that the mutant AAV9 demonstrate better brain infectivity with easier manufacturability, making them a better suited for use in gene therapy.Example 5: 3D Structure Comparisons between AAV Serotypes

[0145] 3D structure comparisons of natural AAV serotypes and our cryogenic electron microscopy (Cryo-EM) resolved enhanced AAV9 variant “FYHR” were generated. Referring to FIG. 4, the left-most image depicts a 3D structural alignment of all major natural AAV serotypes (AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11, AAV12, and AAV13) in light grey with FYHR in dark grey. The magnified portions in the top rectangular box on the right show the amino acid changes in 3 -dimensional space of all serotypes aligned in the left-most 3D structure image at position 584 relative to the VP1 sequence of AAV9. Similarly, the magnified portions in the bottom rectangular box on the right illustrates the position 535 relative to the AAV9 VP1 sequence. In the right-most rectangular boxes, light grey is all major AAVs amino sequences and dark grey displays FYHR’s amino acid. Table 2 below simplifies the table of the 3 -dimensional structure alignments analyzed. All AAVs are similar in length of amino acid sequence but differ at the specific mutations in our novel enhanced AAV9 variant “FYHR” (Table 3). At 535 all naturally occurring serotypes have a preserved hydrophobic residue, predominantly a phenylalanine, except for AAV5 that has a hydrophobic leucine residue. Our novel enhanced variant has a mildly hydrophilic tyrosine residue. At position 584 in respect to the AAV9 VP1 capsid sequence, most serotypes contain a hydrophobic leucine residue (AAV2, 3B, 6, 7, 8, and 11), phenylalanine (AAV1), negatively charged Aspartic Acid (AAV4), or polar no charge asparagine (AAV11, 12, and 13). The only positively charged residue found in nature is the parent AAV9 that has a histidine and has a different chemical characteristic compared to our novel FYHR variant at physiological pH. Amino acid properties of amino acids in different AAV serotypes, which correspond to positions of 535 and 584 of AAV9 are shown below in TABLE 2, which can be used as a guide for identifying corresponding amino acid substitution positions as described herein for engineered capsid proteins for non-rAAV9 vectors.TABLE 2:

[0146] FIG. 15 shows the 2-dimensional sequence alignments of the variants disclosed herein compared to AAV9 using CLUSTAL omega. The positions of changed amino acids are seen at position 382, 535, and 584 highlighted in grey.Example 6: Additional capsid validation transduction data

[0147] An AAV9 controlled randomization library was screened in mice and nonhuman primates (NHP). Based on screening results variants were identified enriched in both NHP and mouse CNS. In the plot above, we demonstrate the analysis of the screen with enrichment of for transduction against both species on the x-axis relative to AAV9 and manufacturability relative to AAV9. From these results we identified FYHR, F535Y, and L382I. See FIG. 5.

[0148] 2 NHPs were injected intrathecal lumbar puncture with a pool of 5 capsids (AAV9 and 4 engineered capsids; 3 are the listed inventions “FYHR”, “HRLI”, and “H584R”). Capsids were validated in a pooled injection format and unique custom DNA barcodes were packaged into each capsid for multiplex identification. Frozen tissues collected from the NHPs were assessed for vector genome DNA biodistribution. 1 NHP was excluded due to poor / inadequate injection. The DNA biodistribution for the CNS tissues of 1 NHP with an acceptable injection is listed above. FB = forebrain; MB = midbrain; HB = hindbrain; C = cerebellum; SC = spinal cord. Samples were normalized to AAV9 as this serves as the reference control. See FIG. 6.

[0149] 6 adult mice (3 male and 3 female) were injected intrathecal lumbar puncture as with a pool of 5 capsids (AAV9 and 4 engineered capsids; 3 are the listed inventions “FYHR”, “HRLI”, and “H584R”). Capsids were validated in a pooled injection format and unique custom DNA barcodes were packaged into each capsid for multiplex identification. Frozen tissues collected from the mice were assessed for vector genome DNA biodistribution. Samples were normalized to AAV9 as this serves as the reference control. See FIG. 7.

[0150] Vector DNA biodistribution from the peripheral tissues of the adequately injected NHP are listed above relative to AAV9. Of note, FYHR and HRLI were not detected in the spleen.See FIG. 8

[0151] Animals previously described were stained for the RNA expressed from each capsid vector barcode with RNAScope and quantified for the amount of RNA expression. Shown above are representative images of the (A) Mouse and (B) NHP brains with quantification (to the right of the RNAScope images) of the images using automated pixel positive quantification for the RNA stain. See FIGs. 9A-9B.Example 7: Manufacturability of Engineered AAV

[0152] FIG. 10A Displays the manufacturability enrichment analysis relative to AAV9 from our screens. FIGs. 10B and IOC show head-to-head manufacturability tests to validate the screening manufacturability analysis from FIG. 10A. In brief, AAV9 or FYHR were produced in small 30mL suspension HEK293 cells with a traditional triple transfection method. After 48 hours, samples of each production vial were collected and quantified for packaged intact viral genomes with qPCR ITR primers.Example 8: Position 584 data

[0153] Capsid variants were tested for transduction activity in mouse neuronal N2A immortalized cells by flow cytometry. Capsid variants were packaged with a GFP containing genome and GFP positive cells indicated the cells transduced by the capsid. 2 MOIs were tested and confirm that position 584 is not exclusive to benefit from a histidine to an arginine, but can also benefit from threonine. This evidence supports that polar charged and uncharged residues at position 584 with respect to the AAV9 VP1 sequence enhances transduction. See FIGs. 11 and 12.

[0154] Enhancement was not exclusive to immortalized cells. Testing transduction in human cardiomyocytes and iPSC derived cortical neurons also showed enhanced gene delivery by changing the histidine at position 584 with an arginine and threonine. See FIGs. 13 and 14.

Claims

CLAIMSWhat is claimed is:

1. A recombinant adeno-associated virus serotype 9 (rAAV9) comprising a capsid protein sequence with an amino acid substitution at one or more of positions L382, F535, and H584, with reference to the amino acid sequence as set forth in SEQ ID NO: 1.

2. The rAAV9 of claim 1, comprising the capsid protein sequence with an amino acid substitution F535Y.

3. The rAAV9 of any one of claims 1 or 2, comprising the capsid protein sequence with an amino acid substitution at H584, wherein the histidine (H) is substituted with a polar charged or uncharged amino acid selected from arginine (R), asparagine (N), aspartic acid (D), glutamic acid (E), glutamine (Q), lysine (K), serine (S), threonine (T), or tyrosine (Y); preferably wherein the amino acid substitution is H584R or H584T.

4. The rAAV9 of any one of claims 1-3, comprising the capsid protein sequence with an amino acid substitution L382I.

5. The rAAV9 of claim 1, comprising the capsid protein sequence with two amino acid substitutions at positions F535 and H584.

6. The rAAV9 of claim 1, comprising the capsid protein sequence with two amino acid substitutions at positions L382 and H584.

7. The rAAV9 of claim 1, comprising the capsid protein sequence with three amino acid substitutions at positions L382, F535, and H584.

8. The rAAV9 of claim 5, comprising the capsid protein sequence with two amino acid substitutions F535Y and H584R or two amino acid substitutions F535Y and H584T.

9. The rAAV9 of claim 6, comprising the capsid protein sequence with two amino acid substitutions H584R and L382I or two amino acid substitutions H584T and L382I.

10. The rAAV9 of claim 7, comprising the capsid protein sequence with three amino acid substitutions F535Y, H584R, and L382I or three amino acid substitutions F535Y, H584T, and L382I.

11. The rAAV9 of claim 1, wherein the capsid protein sequence is at least 80% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO:11.

12. The rAAV9 of claim 11, wherein the capsid protein sequence is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

13. The rAAV9 of any one of claims 1-11, further comprising additional mutations in the capsid protein sequence, wherein the capsid protein sequence is at least about 80% identical to any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

14. The rAAV9 of any one of claims 1-13, wherein the rAAV9 has enhanced infectivity to brain cells in comparison to a AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584.

15. The rAAV9 of any one of claims 1-13, wherein the rAAV9 has enhanced zeta potential and reduced stability at pH 7 - pH 4 in comparison to a AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584.

16. The rAAV9 of claim any one of claims 1-13, wherein the rAAV9 has enhanced multispecies infectivity in comparison to a AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584,.

17. The rAAV9 of any one of claims 1-13, wherein the rAAV9 shows enhanced packaging capacity in comparison to a AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584,.

18. The rAAV9 of any one of claims 1 -13, wherein the rAAV9 shows enhanced yield of intact virus particle (manufacturability) in comparison to an AAV9 lacking the amino acid substitutions at one or more of positions L382, F535, and H584,.

19. The rAAV9 of any one of claims 1-18, further comprising a recombinant nucleic acid.

20. The rAAV9 of claim 19, wherein the recombinant nucleic acid encodes a therapeutic protein, a peptide, or an RNA.

21. An adeno-associated virus serotype 9 (AAV9) capsid protein comprising a capsid protein sequence with an amino acid substitution at one or more of positions L382, F535, and H584, with reference to a capsid protein sequence as set forth in SEQ ID NO: 1.

22. The AAV9 capsid protein of claim 21, comprising an amino acid substitution F535Y.

23. The AAV9 capsid protein of any one of claims 21 or 22, comprising an amino acid substitution H584R or H584T.

24. The AAV9 capsid protein of any one of claims 21-23, comprising the capsid protein sequence with an amino acid substitution L382I.

25. The AAV9 capsid protein of claim 21, comprising the capsid protein sequence with two amino acid substitutions at positions F535 and H584.

26. The AAV9 capsid protein of claim 21, comprising the capsid protein sequence with two amino acid substitutions at positions L382 and H584.

27. The AAV9 capsid protein of claim 21, comprising the capsid protein sequence with three amino acid substitutions at positions L382, F535, and H584.

28. The AAV9 capsid protein of claim 25, comprising the capsid protein sequence with two amino acid substitutions F535Y and H584R or two amino acid substitutions F535Y andH584T.

29. The AAV9 capsid protein of claim 26, comprising the capsid protein sequence with two amino acid substitutions H584R and L382I or two amino acid substitutions H584T and L382I.

30. The AAV9 capsid protein of claim 27, comprising the capsid protein sequence with three amino acid substitutions F535Y, H584R, and L382I or three amino acid substitutions F535Y, H584T, and L382I.

31. The AAV9 capsid protein of claim 21, wherein the capsid protein sequence is at least 80% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NOY, SEQ ID NOY, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

32. The AAV9 capsid protein of claim 31, comprising the amino acid sequence as set forth in SEQ ID NOY, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NOY, SEQ ID NOY, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

33. The AAV9 capsid protein of any one of claims 21-32, further comprising additional mutations in the capsid protein sequence, wherein the capsid protein sequence is at least about 80% identical to any one of SEQ ID NOY, SEQ ID NOY, SEQ ID NO:4,SEQ ID NOY, SEQ ID NOY, SEQ ID NOY, SEQ ID NO:8, SEQ ID NOY, SEQ ID NO: 10, or SEQ ID NO: 11.

34. A method of manufacturing an adeno-associated virus serotype 9 (AAV9), comprising transfecting a host cell with a nucleic acid encoding an AAV9 capsid protein comprising a capsid protein sequence with an amino acid substitution at one or more of positions L382, F535, and H584, with reference to the amino acid sequence as set forth inSEQ ID NO: 1.

35. The method of claim 34, wherein the AAV9 is a recombinant AAV9 (rAAV9), comprising a recombinant nucleic acid.

36. A method of gene delivery into a host cell, comprising contacting a host cell with a recombinant adeno-associated virus serotype 9 (rAAV9) or a pharmaceutical composition thereof, comprising:(a) a capsid protein of any one of claims 21-33; and(b) a recombinant nucleic acid.

37. The method of gene delivery of claim 36, wherein the host cell is a an erythrocyte, a platelet, a bone marrow cell, a vascular endothelial cell, a lymphocyte, a hepatocyte, a neuronal or brain cell (e.g., neurons and neuroglia), a bronchial endothelial cell, an epidermal cell, a respiratory interstitial cell, an epidermal cell, a fat cell, a dermal fibroblast, a muscle cell; preferably a neuronal or brain cell.

38. A method of treatment of a cancer, a neurological disease, a neuromuscular disease, a liver disease, a kidney or renal disease, an ocular disease, or a muscular disease, a metabolic disease, or a pulmonary disease, comprising administering to a subject in need thereof a recombinant adeno-associated virus serotype 9 (rAAV9) or a pharmaceutical composition thereof comprising:(a) a capsid protein of any one of claims 21-33; and(b) a recombinant nucleic acid.

39. The method of gene delivery of any one of claims 36 or 37 or the method of treatment of claim 24, wherein the recombinant nucleic acid encodes a therapeutic protein, a peptide or an RNA.

40. Use of the rAAV9 of any one of claims 1-20 or the AAV9 capsid protein of any one of claims 21-33, for treatment of a cancer, a neurological disease, a neuromuscular disease, a liver disease, a kidney or renal disease, an ocular disease, or a muscular disease, a metabolic disease, or a pulmonary disease.

41. A vector comprising a polynucleotide sequence encoding the rAAV capsid protein of any one of claims 1-20.

42. A vector comprising a polynucleotide sequence encoding the rAAV9 of claims 1-20.

43. The vector of claim 41 or 42, wherein the vector is a nucleic acid, plasmid vector, or a viral vector.

44. The vector of any one of claims 41-43, further comprising a recombinant nucleic acid; wherein the recombinant nucleic acid encodes a therapeutic protein, a peptide, or an RNA.

45. An adeno-associated virus (AAV) capsid protein comprising an amino acid substitution at one or more positions corresponding to L382, F535, and H584, wherein the corresponding position is determined by alignment with SEQ ID NO:1, optionally wherein the AAV capsid protein is of an AAV1, AAV2, AAV3B, AAV4, AAVS, AAV6, AAV7, AAV8, AAVrhlO, AAV11, AAV12, or AAV13 serotype.

46. A recombinant adeno-associated virus (rAAV) comprising the AAV capsid protein of claim 45.

47. Any of the methods or uses as described herein, comprising the AAV of claim 45 or the rAAV of claim 46.

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