Modified viral capsids and uses thereof

Abstract

The present disclosure concerns disclosed gene therapy vectors and associated capsid proteins and / or cells and methods relating to determination of deamidation state thereof. The disclosure further provides methods, compositions, and therapeutic uses of the same for treating genetic disorders, including Duchenne muscular dystrophy and limb girdle muscular dystrophy.

Description

MODIFIED VIRAL CAPSIDS AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 702,708, filed October 3, 2024, the disclosure of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 29, 2025, is named 8208_WO00_SequenceListing_ST26.xml and is 173,810 bytes in size.FIELD OF DISCLOSURE

[0003] The present disclosure relates to methods for characterizing the VP1, VP2 and VP3 capsid proteins in an adeno-associated virus (AAV) particle and the purity of an AAV composition using peptide mapping and mass spectrometry and to associated compositions, including, but not limited to, modified recombinant AAV capsid proteins.BACKGROUND

[0004] Adeno- Associated viruses (AAVs) have become one of the most widely used vehicles for delivering gene therapy. These vectors combine an excellent safety profile with high-efficiency transduction of a broad range of target tissues. AAVs are small non-enveloped viruses belonging to the Parvoviridae family that feature an icosahedral capsid containing a linear single stranded DNA genome of about 4.7 kilobases. AAVs are commonly expressed recombinantly in suitable host cells.However, for recombinant AAVs, several factors can impact quality of these gene therapy vectors, including protein deamidation.

[0005] Deamidation in AAVs is a critical quality attribute (CQA), as it impacts AAV infectivity, yet this post-translational modification (PTM) remains poorly understood. Extensive deamidation has been reported for multiple AAV serotypes (Giles,A. R., et al. (2018). Molecular Therapy 26 (12), 2848-2862.). However, these previous analyses of AAV deamidation may suffer from over-estimation due to analytical artifacts generated during sample preparation, particularly denaturation and proteolysis (peptide mapping). As a spontaneous and non-enzymatic process, deamidation can easily be generated during sample preparation (such as proteolysis), as the resulting peptides lack higher-order structures of proteins that typically reduces deamidation, causing analytical artifacts. As such, commonly practiced proteolysis protocols under neutral and mildly basic conditions likely overestimate deamidation due to these artifacts (see, e.g., US2020 / 0407750). Therefore, there is a need for methods to characterize AAV capsid deamidation.BRIEF SUMMARY OF THE DISCLOSURE

[0006] The present disclosure concerns methods and compositions related to a low pH process for deamidation analysis of AAV capsid proteins. Said process is also applicable for identification of several other protein post-translational modifications, , such as N-terminal methionine loss, acetylation, phosphorylation and oxidation.

[0007] In one aspect, the present disclosure concerns a method for using a pH buffer to detect deamidated amino acid residues in a viral capsid, the method comprising the steps of (a) denaturation and reduction of the capsid, (b) proteolytic digestion of the capsid with one or more proteases to generate several polypeptide fragments, and (c) mass spectrometry analysis of the polypeptide fragments. In some embodiments, the pH buffer is an acidic buffer. In some embodiments, the pH buffer is pH 5. In some embodiments, the pH buffer is a basic buffer. In some embodiments, the pH buffer is pH 7.5. In some embodiments, the pH buffer is pH 7.8. In some embodiments, the capsid is denatured and reduced in step (a) with 6M guanidine HC1 and lOmM TCEP. In some embodiments, the method further comprises an optional buffer exchange step to reduce the concentration of guanidine HC1 to IM. In some embodiments, the one or more proteases in step (b) comprises recombinant Lys-C. In some embodiments, the one or more proteases in step (b) comprises trypsin. In some embodiments, the protease:capsid (w / w) ratio is selected from 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, and 1 : 1. In someembodiments, the mass spectrometry analysis in step (c) is Quadropole Time-of-Flight (QTOF) mass spectrometry.

[0008] In one aspect, the present disclosure concerns a modified adeno- associated virus (AAV) capsid protein comprising one or more deamidated asparagine residues. In some embodiments, the AAV capsid protein is selected from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, MyoAAV-4A, and MyoAAV-4E. In some embodiments, said deamidated asparagine residues in an AAV8 capsid protein or its equivalent conserved residue are selected from N56, N254, N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N516, N539, N598, N629, N652, N664, and N717. In some embodiments, said AAV8 deamidated asparagine residues are selected from N254, N478, N513, N516, and N539. In some embodiments, said deamidate asparagine residues in an AAVrh74 capsid protein or its equivalent conserved residue are selected from N56, N254, N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N598, N629, N652, N664, and N717. In some embodiments, said deamidated asparagine residues in an AAV9 capsid protein or its equivalent conserved residue are selected from N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N627, N650, N662, and N715. In some embodiments, said deamidated asparagine residues in an MyoAAV9-4E capsid protein or its equivalent conserved residue are selected from N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N637, N660, N672, and N725. In some embodiments, said deamidate asparagine residues in an AAV5 capsid protein or its equivalent conserved residue are selected from N55, N243, N292, N374, N399, N462, N481, N585, and N639. In some embodiments, the modified AAV capsid protein is 95% identical or greater to the capsid protein of any one of the aforementioned capsid proteins. In some embodiments, the modified AAV capsid protein is 99% identical or greater to the capsid protein of any one of the aforementioned capsid proteins. In one aspect, the present disclosure concerns a vector expressing any one of the proceeding capsid proteins. In some embodiments, the vector comprises at least one bidirectional promoter. In some embodiments, the vector comprises a ubiquitous promoter or a tissue-specific promoter operably linked to one or more encoded therapeutic polynucleotides. In some embodiments, the tissue-specificpromoter is a muscle specific promoter. In some embodiments, the tissue-specific promoter is a neuron specific promoter. In some embodiments, the one or more encoded therapeutic polynucleotide is selected from the group consisting of microdystrophin, alpha-sarcoglycan, beta-sarcoglycan, gamma-sarcoglycan, FKRP, SMN1, SMN2, a guide RNA (gRNA), a CRISPR Cas nuclease, dysferlin, calpain, and acid alpha-glucosidase (GAA). In one aspect, the present disclosure concerns a cell comprising any of the preceding capsid proteins or any of the preceding vectors. In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a yeast cell, an insect cell, a mammalian cell, or a bacterial cell. In some embodiments, the cell is a muscle cell, a heart cell, a stem cell, a satellite cell, and / or a liver cell. In some embodiments, the cell is a muscle cell is a HeLa cell, a 293 cell, a PerC.6 cell, or a Sf9 cell. In one aspect, the present disclosure concerns a kit comprising any one of the preceding capsid proteins or any one of the preceding vectors.

[0009] In one aspect, the present disclosure concerns a method of treating a disease in a patient in need thereof, the method comprising administering to the patient any one of the preceding capsid proteins or any one of the preceding cells. In some embodiments, the disease is Duchenne muscular dystrophy (DMD). In some embodiments, the disease is Limb Girdle muscular dystrophy (LGMD). In some embodiments, the disease is Pompe disease. In some embodiments, the protein, the vector, or the cell is administered to the patient intramuscularly, intravenously, or a combination thereof.BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 illustrates asparagine deamidation resulting in the formation of a mixture of aspartic acid (Asp, D) and isoaspartic acid (isoAsp, isoD) via the formation of an aspartyl succinimide intermediate (Asu).

[0011] Figure 2 illustrates exemplary embodiments of AAV peptide mapping. Particularly, the low pH workflow (pH 5) shows significantly lower levels of deamidation, as compared to the peptide mapping workflows that utilize slightly basic buffer systems (pH 7 - 8).

[0012] Figures 3A, 3B, and 3C collectively illustrate extracted ion chromatograms (EIC) of the model peptide YHLNGR (SEQ ID NO: 9) (unmodified, asparaginyl (black trace) and deamidated species, Asp and isoAsp (red trace)) from different processes, A) pH 5.0; B) pH 7.5 (Tris) and C) pH 7.8 (Ammonium Bicarbonate).

[0013] Figures 4A, 4B, and 4C collectively illustrate extracted ion chromatograms of the peptide YHLNGR (SEQ ID NO: 9) (unmodified, asparaginyl (black trace) and deamidated species, Asp and isoAsp (red trace)) generated after proteolysis of AAV8 from different processes, A) pH 5.0; B) pH 7.5 (Tris) and C) pH 7.8 (Ammonium Bicarbonate).

[0014] Figure 5 illustrates that 95% sequence coverage is obtained for AAV8 VP1 capsid protein (SEQ ID NO: 14) using the low pH workflow (pH 5.0) with Trypsin / Lys-C digestion. Sequence coverage from the AspN digestion is shown to ensure 100% sequence coverage was obtained for the AAV8 capsid proteins. The 2 asparagine residues covered by AspN digestion are highlighted / denoted with an asterisk.

[0015] Figures 6A, 6B, 6C, and 6D collectively illustrate pairwise alignments between AAV8 VP1 (SEQ ID NO: 15) and the capsid proteins of A) AAVrh74 (SEQ ID NO: 16), B) AAV9 (SEQ ID NO: 17), C) MyoAAV9-4E (SEQ ID NO: 18), and D) AAV5 (SEQ ID NO: 19). Highlighting / denotation with an asterisk marks conserved asparagine residues between AAV8 VP1 and the aligned capsid protein, for which deamidation has been detected.DETAILED DESCRIPTION OF THE DISCLOSURE

[0016] Disclosed herein are compositions and methods related to peptide mapping and deamidated viral capsid proteins. In some aspects, methods are provided for determining the deamidation state of asparagine residues in an AAV capsid protein using low pH peptide mapping and mass spectrometry. The disclosure also provides compositions related to the process. In some aspects, these compositions include AAV capsid proteins with deaminated asparagine residues.1. Definitions

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Unless expressly stated to the contrary herein, any term, as used in this application, shall have the meaning set forth in this application. While not explicitly defined below, such terms should be interpreted according to their common meaning. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0018] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0019] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0020] Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.

[0021] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of + / - 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2% and such ranges are included. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0022] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

[0023] The articles “a,” “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0024] As used herein, the terms “about” and / or “approximately” shall 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 (i.e., the limitations of the measurement system). For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 181%, 17%, 16%, 15%, 14%, 13'%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0025] The terms “ Adeno-associated virus” or “AAV” as used interchangeably herein refer to any virus belonging to the Parvoviridae family (genus Dependovirus) that endemically infects humans and some other primate species. AAV is not currently known to cause disease and consequently causes a very mild immune response. In addition to the naturally occurring serotypes of the virus (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAV rhlO), these terms shall expressly include any and all “recombinant variants” (e.g., engineered versions) of an AAV virus, including, but not limited to, AAVs with RGD insertions (see, e.g., Manini, A., et al. Frontiers in Neurology, 12, 814174 (2022).). Additional non-limiting examples of contemplated recombinant AAV variants include MyoAAV variants (e.g., Myo AAV2 and MyoAAV4E), and AAV-MYO variants (see, e.g., Weinmann, J., et al. Nat Commun 11, 5432 (2020)). In some aspects, the rAAV is of the serotype AAVrh.74. Production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692, which is incorporated by reference in its entirety. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).

[0026] As used herein, the term “AAV particle,” “AAV vector,” “AAV virion,” “AAV viral particle,” or “AAV vector particle” is used to refer to a viral particle composed of an AAV capsid and an encapsidated AAV genome. The AAV particle, in some aspects, comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell). Production of AAV viral particles, in some aspects, includes production of AAV vector, as such a vector is contained within an AAV vector particle. For example, a wild-type (wt) AAV virus particle comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat. The AAV virion can be either a singlestranded (ss) AAV or self-complementary (SC) AAV. In some aspects, a single-stranded AAV nucleic acid molecules of either complementary sense, e.g., “sense” or “antisense” strands, can be packaged into an AAV virion and both strands are equally infectious.

[0027] As used herein, the term “ AAVrh74” refers to an AAV particle having AAVrh74 VP1, VP2 and VP3 capsid proteins or variants thereof. An exemplary AAVrh74 VP1 capsid protein sequence is set forth in U.S. patent number 9,434,928, which is hereby incorporated by reference in its entirety. Exemplary variants of AAVrh74 VP1 capsid proteins are also set forth in U.S. patent number 9,434,928.

[0028] As used herein, the term “capsid protein” refers to a protein that forms the coat or shell of a virus. The term “AAV capsid protein” refers to the protein that forms the coat of an adeno-associated virus (AAV), which is composed of a total of 60subunits; each subunit is an amino acid sequence, e.g., viral protein 1 (VP1), VP2 or VP3.

[0029] The terms “coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized.

[0030] The terms “complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.“Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0031] The terms “comprise(s),” “include(s),” “having,” “has,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts, structures or components. The singular forms of articles, such as “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0032] The term “directional promoter” refers to two or more promoters that are capable of driving transcription of two separate sequences in both directions. In one embodiment, one promoter drives transcription from 5' to 3' and the other promoter drives transcription from 3' to 5'. In one embodiment, bidirectional promoters are double-strand transcription control elements that can drive expression of at least two separate sequences, for example, coding or non-coding sequences, in opposite directions. Such promoter sequences may be composed of two individual promoter sequences acting in opposite directions, such as one nucleotide sequence linked to the other(complementary) nucleotide sequence, including packaging constructs comprising the two promoters in opposite directions, for example, by hybrid, chimeric or fused sequences comprising the two individual promoter sequences, or at least core sequences thereof, or else by only one transcription regulating sequence that can initiate the transcription in both directions. The two individual promoter sequences, in some embodiments, may be juxtaposed or a linker sequence can be located between the first and second sequences. A promoter sequence may be reversed to be combined with another promoter sequence in the opposite orientation. Genes located on both sides of a bidirectional promoter can be operably linked to a single transcription control sequence or region that drives the transcription in both directions. In other embodiments, the bidirectional promoters are not juxtaposed. For example, one promoter may drive transcription on the 5' end of a nucleotide fragment, and another promoter may drive transcription from the 3' end of the same fragment. In another embodiment, a first gene can be operably linked to the bidirectional promoter with or without further regulatory elements, such as a reporter or terminator elements, and a second gene can be operably linked to the bidirectional promoter in the opposite direction and by the complementary promoter sequence, again with or without further regulatory elements.

[0033] The term “expression cassette” as used herein refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a competent host cell, such that a particular gene product (e.g., RNA or protein) is expressed. Expression of any gene product may be dependent upon presence of cellular factors or additional gene products from other expression cassettes. An expression cassette or vector may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette or vector includes a polynucleotide to be transcribed, operably linked to a promoter. In one embodiment, an expression cassette comprises a regulatory element operably linked to a polynucleotide sequence encoding a Cas protein or a gRNA. In another embodiment, an expression cassette comprises a nucleotide sequence flanked by a 5’ inverted reverse repeat (ITR) and a 3’ ITR.

[0034] The terms “functional” and “fully functional” as used herein describe protein that has biological activity. A “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional protein.

[0035] The term “gene” as used herein refers to the segment of a DNA molecule that codes for a polypeptide chain (e.g., the coding region). In some embodiments, a gene is positioned by regions immediately preceding, following, and / or intervening the coding region that are involved in producing the polypeptide chain (e.g., regulatory elements such as a promoter, enhancer, polyadenylation sequence, 5 '-untranslated region, 3 '-untranslated region, or intron).

[0036] The terms “genetic construct” or “construct” as used herein refer to the nucleic acid molecules that comprise a nucleotide sequence encoding a protein. The coding sequence may be DNA or RNA and includes initiation and termination signals operably linked to regulatory elements, such as a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to genetic constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that when present in the cell of an individual, the coding sequence will be expressed.

[0037] The term “genetic disease” as used herein refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, especially a condition that is present from birth. The abnormality may be a mutation, an insertion or a deletion. The abnormality may affect the coding sequence of the gene or its regulatory sequence. The genetic disease may be, but not limited to DMD, BMD, hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited disorders of hepatic metabolism, sickle cell anemia, thalassaemias, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and Tay-Sachs disease.

[0038] The terms “identity,” “identical,” “percent identity,” and / or “percent identical,” as used herein as applicable to one or more particular polynucleotide or amino acid sequences, refer to the proportion of identical residues between a particularreference sequence and another sequence, as calculated by a pairwise alignment using the Needleman-Wunsch algorithm using a generally available alignment program, e.g., the Needle (EMBOSS) program. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator for the purposes of calculating identity. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent.

[0039] As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique that measures the mass-to-charge (m / z) ratio of ions to identify and quantify molecules in simple and complex mixtures. MS technology generally includes: (1) ionizing the compounds to form charged compounds; and (2) detecting the mass-to- charge ratio of the charged compounds and calculating the molecular weight. The compounds may be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer, a mass analyzer, and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”). In some mass spectrometry methods, ions may be separated from one another using time-of-flight (TOF), an orbitrap, a Fourier transform ion cyclotron resonance spectrometer, a quadrupole or an ion trap, for example, and then detected using an ion detector.

[0040] The term “mutant gene" or "mutated gene" as used interchangeably herein refers to a gene that has undergone a detectable mutation. A mutant gene has undergone a change, such as the loss, gain, or exchange of genetic material, which affects the normal transmission, expression, and / or functionality of the gene. A “disrupted gene” as used herein refers to a mutant gene that has a mutation that causes a premature stop codon. The disrupted gene product is truncated relative to a full-length undisrupted gene product.

[0041] The term “normal gene” as used herein refers to a gene that has not undergone a change, such as a loss, gain, or exchange of genetic material. The normalgene undergoes normal gene transmission and gene expression and is sufficiently functional to not cause symptomatic disease. For the avoidance of doubt, wildtype genes and asymptomatic variants of a wildtype gene, such as those containing single-nucleotide polymorphisms (SNPs), are considered normal genes.

[0042] The terms “nucleic acid,” “oligonucleotide” or “polynucleotide” as used herein refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral- methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Any combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine are expressly contemplated by this application.

[0043] The term “operably linked” as used herein means that expression of a gene is under the control of a promoter or regulatory element with which it is spatially connected. For example, a promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

[0044] The term “partially functional" as used herein describes a protein that is encoded by a mutant gene and has less biological activity than a fully functional protein but more than a non-functional protein.

[0045] The term “peptide mapping” as used herein refers to the process of preparing a protein for mass spectrometry analysis. Exemplary protocols of this process include the Promgea AccuMap system.

[0046] The terms “promoter or “promoter element,” which may be used interchangeably, refer to a nucleotide sequence that assists with controlling expression ofa coding sequence. Generally, promoters are located 5' (i.e., upstream) of the translation start site of a gene. However, in certain embodiments, a promoter element may be located within an intron sequence, or 3' of the coding sequence. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. In some embodiments, one of a plurality of well-characterized promoter elements is used with a vector described herein. Non-limiting examples of well-characterized promoter elements include a SV40 early promoter, a SV40 late promoter, a human U6 (hU6) promoter, a CMV early promoter, a P-actin promoter, and a methyl CpG binding protein 2 (MeCP2) promoter. In some embodiments, the promoter is a constitutive promoter, which drives substantially constant expression of the target protein. In other embodiments, the promoter is tissue-specific promoter, which drives expression of the target protein in response to presence in a particular tissue or cell type. In some embodiments, the promoter is a muscle-specific promoter. Non-limiting examples of muscle-specific promoters include a MHCK7 promoter, a CK8 promoter, and a Spc512 promoter.

[0047] A promoter may comprise one or more transcriptional regulatory elements to further enhance expression and / or to alter the spatial expression and / or temporal expression of the same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.

[0048] The terms “protospacer,” “targeting sequence” or “crRNA sequence,” which may be used interchangeably refer to a component of a functional gRNA in a CRISPR system that has complementarity to a targeted polynucleotide or targeted gene.

[0049] The terms “Protospacer Adjacent Motif’ or “PAM,” which may be used interchangeably herein, refer to the region of a targeted gene or targeted polynucleotide sequence that is recognized and bound by a CRISPR-associated (Cas) protein, such as Cas9. In some embodiments, the PAM is no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 bases from a protospacer sequence. Naturally occurring Cas9 molecules recognize specific PAM sequences. It is understood that PAMs may be degenerate in nature such that multiple sequences are recognized by a particular protein (e.g., NGG for SpCas9 or NNGRRV / NNGRRT for SaCas9, wherein N means any nucleotide and Rmeans any purine nucleotide, and V means any one of guanine, cytosine, and adenine). In some embodiments, the PAM is NNGRRV. In other embodiments, the PAM is NNGRRT.

[0050] The term “recombinant AAV,” or “rAAV” is defined herein as an infectious, replication-defective virus composed of an AAV protein shell, encapsulating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs. A rAAV, in some aspects, is produced in a suitable host cell which has an AAV vector, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0051] The term “regulatory element” as used herein refers to nucleotide sequences, such as promoters, enhancers, terminators, polyadenylation sequences, introns and the like, that provide for the expression of a coding sequence in a cell or otherwise control said expression.

[0052] The terms “subject” or “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate— such as, a monkey (e.g., a cynomolgus or rhesus monkey), a chimpanzee,— and a human). In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.

[0053] The term “variant” as used herein encompasses, but is not limited to, proteins (including fusion proteins) which comprise an amino acid sequence that differs from the amino acid sequence of a reference protein by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference protein. A variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference protein. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. A variant retains the biological activity ascribed to the reference protein. Regarding nucleic acids encoding proteins, a variant may comprise one or more conservative substitutions in its sequence as compared to thesequence of a reference nucleic acid. Conservative nucleic acid substitutions may involve substitution at positions that do not alter the resultant encoded amino acid sequence.

[0054] The term “vector” as used herein refers to any vehicle used to transfer a nucleic acid (e.g., encoding a CRISPR-Cas9 system construct) into a host cell. In some embodiments, a vector includes a replicon, which functions to replicate the vehicle, along with the target nucleic acid. Non-limiting examples of vectors useful for therapeutic purposes include plasmids, phages, cosmids, artificial chromosomes, and viruses, which function as autonomous units of replication in vivo. In some embodiments, the vector is a viral vehicle for introducing a target nucleic acid (e.g., a CRISPR-Cas9 system construct). Many modified eukaryotic viruses useful for genetic construct delivery are known in the art. For example, adeno-associated viruses (AAVs) are particularly well-suited for use in human gene therapy because humans are a natural host for the virus, the native viruses are not known to contribute to any diseases, and the viruses illicit a mild immune response. In certain embodiments, the vector is a lipid nanoparticle.

[0055] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.2. Capsid Characterization Methods

[0056] Provided herein are methods for characterization of AAV capsids. In one aspect, the method determines the deamidation state of AAV capsid proteins. a. Characterization of AA V capsid deamidation state

[0057] In one aspect, the present disclosure provides methods to characterize any one of VP1, VP2 and / or VP3 capsid proteins in an adeno-associated virus (AAV) particle through peptide mapping, which comprises subjecting the AAV particle or the capsid protein to denaturation and reduction, proteolytic digestion with one or more proteases(thereby generating several polypeptide fragments), and mass spectrometry analysis of the polypeptide fragments. In some embodiments, the capsid proteins are buffer exchanged into 6M guanidine hydrochloride in any one of lOOmM ammonium bicarbonate buffer, 20mM Tris buffer, and / or IX Promega AccuMAP reaction buffer. In some embodiments, the method includes further subjecting the denatured capsid proteins to reduction (e.g., by incubation with a reducing agent such as TCEP or betamercaptoethanol). In some embodiments, the incubation occurs at room temperature. In a preferred embodiment, the incubation occurs at 37 degrees Celsius. In some embodiments, the denatured and reduced capsid proteins are further buffer exchanged into IM guanidine hydrochloride in any one of lOOmM ammonium bicarbonate buffer, 20mM Tris buffer, and / or 1.5X Promega AccuMAP reaction buffer. In some embodiments, the denatured capsid proteins are digested by any combination of Asp-N, Lys-C and / or trypsin at an enzyme: protein ratio of about 1 : 100, about 1 :50, about 1 :25, 1 : 10, about 1 :9, about 1 :8, about 1 :7, about 1 :6, about 1 :5, about 1 :4, about 1 :3, about 1 :2, or about 1 : 1. In some embodiments, digestion with multiple enzymes occurs concurrently. In some embodiments, digestion with multiple enzymes is serial. In some embodiments, mass spectrometry analysis consists of a liquid chromatography system and a QTOF spectrometer. In some embodiments, the denaturing, reduction, and digestion steps above are all conducted in buffers at pH 5.0. In some embodiments, the denaturing, reduction, and digestion steps above are all conducted in buffers at pH 7.5. In some embodiments, the denaturing, reduction, and digestion steps above are all conducted in buffers at pH 7.8.

[0058] In some embodiments, the relative amounts of the capsid proteins are determined by comparing the total ion chromatogram (TIC) of the VP1, VP2 and VP3 capsid proteins. In some embodiments, the high-performance liquid chromatography system is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. In some embodiments, the liquid chromatography is reverse phase liquid chromatography. In some embodiments, the liquid chromatography is high-pressure liquid chromatography (HPLC). In some embodiments, the liquid chromatography is ultra-high pressure liquid chromatography (UHPLC).

[0059] In some embodiments, the liquid chromatography is performed using a Cl 8 column, a C8 column, or a C4 column. In some embodiments, the liquid chromatography is performed using a Cl 8 column. In some embodiments, the stationary phase of the liquid chromatography is comprised within a chromatography column that is about 50-300 mm long and has an internal diameter of about 1-4.6 mm. In some embodiments, the column is a BEH column. In some embodiments, the column has an internal diameter of 1, 2.1, 3, or 4.6 mm. In some embodiments, the column has a length of 50, 75, 100, 150, or 300 mm. In some embodiments, the column size is 1 mm x 50 mm, 2.1 mm x 50 mm, 3 mm x 50 mm , 4.6 mm x 50 mm, 1 mm x 75 mm, 2.1 mm x 75 mm, 3 mm x 75 mm , 4.6 mm x 75 mm, 1 mm x 100 mm, 2.1 mm x 100 mm, 3 mm x 100 mm, 4.6 mm x 100 mm, 1 mm x 150 mm, 2.1 mm x 150 mm, 3 mm x 150 mm , 4.6 mm x 150 mm, 1 mm x 300 mm, 2.1 mm x 300 mm, 3 mm x 300 mm , or 4.6 mm x 300 mm. In some aspects, the column size is 1.6 x 50 mm, 1.6 x 60 mm, 1.6 x 70 mm, 1.6 x 80 mm, 1.6 x 90 mm, 1.6 x 100 mm, 1.6 x 110 mm, 1.6 x 120 mm, 1.6 x 130 mm, 1.6 x 140 mm, 1.6 x 150 mm, 1.7 x 50 mm, 1.7 x 60, 1.7 x 70 mm, 1.7 x 80 mm, 1.7 x 90 mm, 1.7 x 100 mm, 1.7 x 110 mm, 1.7 x 120 mm, 1.7 x 130 mm, 1.7 x 140 mm, 1.7 x 150 mm, 1.8 x 50 mm, 1.8 x 60, 1.8 x 70 mm, 1.8 x 80 mm, 1.8 x 90 mm, 1.8 x 100 mm, 1.8 x 110 mm, 1.8 x 120 mm, 1.8 x 130 mm, 1.8 x 140 mm, 1.8 x 150 mm, 1.9 x 50 mm, 1.9 x 60 mm, 1.9 x 70 mm, 1.9 x 80 mm, 1.9 x 90 mm, 1.9 x 100 mm, 1.9 x 110 mm, 1.9 x 120 mm, 1.9 x 130 mm, 1.9 x 140 mm, 1.9 x 150 mm, 2.0 x 50 mm, 2.0 x 60 mm, 2.0 x 70 mm, 2.0 x 80 mm, 2.0 x 90 mm, 2.0 x 100 mm, 2.0 x 110 mm, 2.0 x 120 mm, 2.0 x 130 mm, 2.0 x 140 mm, 2.0 x 150 mm, 2.1 x 50 mm, 2.1 x 60 mm, 2.1 x 70 mm, 2.1 x 80 mm, 2.1 x 90 mm, 2.1 x 100 mm, 2.1 x 110 mm, 2.1 x 120 mm, 2.1 x 130 mm, 2.1 x 140 mm, 2.1 x 150 mm, 2.2 x 50 mm, 2.2 x 60 mm, 2.2 x 70 mm, 2.2 x 80 mm, 2.2 x 90 mm, 2.2 x 100 mm, 2.2 x 110 mm, 2.2 x 120 mm, 2.2 x 130 mm, 2.2 x 140 mm, 2.2 x 150 mm, 2.3 x 50 mm, 2.3 x 60 mm, 2.3 x 70 mm, 2.3 x 80 mm, 2.3 x 90 mm, 2.3 x 100 mm, 2.3 x 110 mm, 2.3 x 120 mm, 2.3 x 130 mm, 2.3 x 140 mm, 2.3 x 150 mm, 2.4 x 50 mm, 2.4 x 60 mm, 2.4 x 70 mm, 2.4 x 80 mm, 2.4 x 90 mm, 2.4 x 100 mm, 2.4 x 110 mm, 2.4 x 120 mm, 2.4 x 130 mm, 2.4 x 140 mm, 2.4 x 150 mm, 2.5 x 50 mm, 2.5 x 60, 2.5 x 70 mm, 2.5 x 80 mm, 2.5 x 90 mm, 2.5 x 100 mm, 2.5 x 110 mm, 2.5 x 120 mm, 2.5 x 130 mm, 2.5 x 140 mm, 2.5 x 150 mm, 2.6 x 50 mm, 2.6 x 60 mm, 2.6 x 70 mm,2.6 x 80 mm, 2.6 x 90 mm, 2.6 x 100 mm, 2.6 x 110 mm, 2.6 x 120 mm, 2.6 x 130 mm, 2.6 x 140 mm, or 2.6 x 150 mm. In some embodiments, the stationary phase of the reverse phase liquid chromatography is comprised within a chromatography column that is about 150 mm long and has an internal diameter of about 2.1 mm.

[0060] In some embodiments, the stationary phase of the reverse phase liquid chromatography comprises particles sized between about 1.2 pm-2.5 pm. In another aspects, the stationary phase of the reverse phase liquid chromatography comprises particles sized at about 1.7 pm, 1.8 pm or 2.1 pm. In some embodiments, the particle size is about 1.2 pm, 1.3 pm, 1.4 pm, 1.5 pm, 1.6 pm, 1.7 pm, 1.8 pm, 1.9 pm, 2.0 pm, 2.1 pm, 2.2 pm, 2.3 pm, 2.4 pm, or 2.5 pm. In some embodiments, the stationary phase of the reverse phase liquid chromatography is comprised of particles of about 1.7 pm.

[0061] In some embodiments, the chromatography uses a first mobile phase including formic acid (optionally fluoro-substituted) in water. In some embodiments, the first mobile phase includes from about 0.05 to about 0.15% of formic acid by volume. In some embodiments, the first mobile phase comprises about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.2% of formic acid by volume. In some embodiments, the first mobile phase includes about 0.1% of formic acid by volume.

[0062] In some embodiments, the chromatography uses a second mobile phase including formic acid (optionally fluoro-substituted) in acetonitrile. In some embodiments, the chromatography uses a second mobile phase including formic acid in the mixture of acetonitrile and water. In some embodiments, the second mobile phase includes about 0.05-0.2% of formic acid by volume. In some embodiments, the second mobile phase includes about 0.05-0.15% of formic acid by volume. In some embodiments, the second mobile phase includes about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.2% of formic acid by volume. In some embodiments, the second mobile phase comprises about 0.1% formic acid. In some embodiments, the second mobile phase includes about 75-95% of acetonitrile by volume. In some embodiments, the second mobile phase includes about 75%, 80%, 85%, 90%, or 95% of acetonitrile by volume. Insome embodiments, the second mobile phase includes about 90% acetonitrile and 10% water by volume.

[0063] In some embodiments, the percentage of the second mobile phase, in the combination of the first mobile phase and the second mobile phase, in the chromatography is increased over time. In some embodiments, the percentage of the second mobile phase is increased from about 1% to about 50% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 45% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 70% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 100% by volume. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 45% in about 40-90 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 2% to about 45% in about 80 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 45% to about 70% by volume in about 1-20 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 45% to about 70% by volume in about 5 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 70% to about 100% by volume in about 1- 10 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 70% to about 100% by volume in about 2 minutes.

[0064] In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 40% by volume in about 5-10 minutes, from about 40% to about 45% in about 25-35 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 45% to about 100% in about 0.5-2 minutes. In some embodiments, the percentage of the second mobile phase is decreased from about 100% to about 10% in about 0.5-2 minutes.

[0065] In some embodiments, the percentage of the second mobile phase is increased from about 10% to about 40% by volume in about 6 minutes, from about 40% to about 45% in about 29 minutes. In some embodiments, the percentage of the second mobile phase is increased from about 45% to about 100% in about 1 minute. In someembodiments, the percentage of the second mobile phase is decreased from about 100% to about 10% in about 1 minute.

[0066] In some embodiments, the mass spectrometry analysis may use any ionization modes, particularly those modes suitable for analyzing biological molecules including, but not limited to, direct infusion-mass spectrometry, electrospray ionization (ESI)-MS, desorption electrospray ionization (DESI)-MS, direct analysis in real-time (DART)-MS, atmospheric pressure chemical ionization (APCI)-MS, electron impact (El) or chemical ionization (CI), matrix-assisted laser desorption / ionization (MALDI)-MS, and Atmospheric Pressure lonization-Electrospray (API-ES). In some embodiments, the mass spectrometry analysis uses API-ES ionization mode. In some embodiments, the mass spectrometry scans signals over a range of 100-5000 m / z. In some embodiments, the mass spectrometry scans signals over a range of 300-3000 m / z.

[0067] In some embodiments, the scan type of the mass spectrometry is positive polarity. In some embodiments, the data acquisition time of the mass spectrometry is about 5-35 minutes. In some embodiments, the data acquisition time of the mass spectrometry is about 17-28 minutes.

[0068] In some embodiments, the capillary voltage of the mass spectrometry is about 500-6000 V. In some embodiments, the capillary voltage of the mass spectrometry is about 4000 V. In some embodiments, the fragmentor voltage of the mass spectrometry is about 50-1000 V. In some embodiments, the fragmentor voltage of the mass spectrometry is about 175 V. In some embodiments, the skimmer voltage of the mass spectrometry is about 60-70 V. In some embodiments, the skimmer voltage of the mass spectrometry is about 65 V.

[0069] In some embodiments, the drying gas temperature of the mass spectrometry is about 200-350 °C. In some embodiments, the drying gas temperature of the mass spectrometry is about 275 °C. In some embodiments, the drying gas flow rate of the mass spectrometry is about 5-13 L / min. In some embodiments, the drying gas flow rate of the mass spectrometry is about 12 L / min.

[0070] In some embodiments, the AAV particle is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVrh74 or any naturally occurring, recombinant, or synthetic variant thereof. In someembodiments, the AAV particle is a recombinant AAV (rAAV) particle. In some embodiments, the AAV particle is AAV8. In some embodiments, the AAV particle is AAV5. In some embodiments, the AAV particle is AAV9. In some embodiments, the AAV particle is AAVrh74. In some embodiments, the AAV particle is MyoAAV9-4E.

[0071] In one aspect, the present disclosure includes determining post- translational modification of at least one of VP1, VP2 and VP3 capsid proteins. In some embodiments, the post-translational modification is glycosylation, sialylation, acetylation, amidation, phosphorylation, formylation, hydroxylation, methylation, and / or sulfation of at least one of VP1, VP2 and VP3 capsid proteins. In some embodiments, the post-translational modification is de-glycosylation, de-sialylation, de-acetyl ati on, deamidation, dephosphorylation, de-formyl ati on, de-hydroxylation, demethylation, desulfation, and / or loss of a N-terminal methionine of at least one of VP1, VP2 and VP3 capsid proteins. In preferred embodiments, the post-translational modification is deamidation of one or more VP1, VP2, or VP3 capsid proteins. In some embodiments, deamidation of asparagine residues of AAV capsid proteins are characterized. In some embodiments, the method of characterizing deamidated asparagine residues comprises determining the ratio of VP1, VP2 and VP3 capsid proteins and / or the masses of one or more of the VP1, VP2 and VP3 capsid proteins in an AAV particle.

[0072] Deamidation is a common post-translational modification resulting in the conversion of an asparagine residue to a mixture of isoaspartate and aspartate.Deamidation of glutamine residues also occurs, but at a much slower rate. Oxidation is also a common post-translational modification which is a result of the reaction of proteins with a variety of free radicals and reactive oxygen species. Methionine oxidation is most common, however oxidation of several other amino acid residues, such as cysteine and tryptophan, have also been observed. Deamidation and / or oxidation are also common degradation pathways for proteins occurring during manufacturing and storage. Deamidation can have an impact on the activity and stability of proteins. Oxidation can cause conformational changes in proteins and therefore impact protein activity and stability. Oxidation can also impact the immunogenicity of proteins. Therefore, monitoring such post-translation modifications is a necessary component of a critical quality assessment for AAV particles.

[0073] Characterization methods with ammonium bicarbonate likely generated false signals or overestimated the deamidation in AAV capsid protein (Table 1) as compared with the methods of the present disclosure at pH 5.0. In some embodiments, the post-translation modification comprises deamidation at one or more of N56, N254, N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N516, N539, N598, N629, N652, N664, and N717 of AAV8 or its equivalent residue at AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV 13, AAVrhlO, AAVrh74, or MyoAAV-4E. In some embodiments, the post-translation modification comprises deamidation at one or more of N56, N254, N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N598, N629, N652, N664, and N717 of AAVrh74. In some embodiments, the post-translation modification comprises deamidation at one or more of N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N627, N650, N662, and N715 of AAV9. In some embodiments, the post-translation modification comprises deamidation at one or more of N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N637, N660, N672, and N725 of MyoAAV9-4E. In some embodiments, the post-translation modification comprises deamidation at one or more of N55, N243, N292, N374, N399, N462, N481, N585, and N639 of AAV5.3. Genetic Constructs for Gene Therapy a. Deamidated capsid proteins and therapeutic uses thereof

[0074] Provided herein are genetic constructs for therapeutic use. In one aspect, one or more AAV vectors comprises a capsid protein with one or more deamidated asparagine residues. In some embodiments, the AAV vector further comprises at least one heterologous or non-naturally occurring polynucleotide (e.g., a transgene). In some embodiments, the polynucleotide encodes any one of a microdystrophin protein, an alpha-sarcoglycan protein, a beta-sarcoglycan protein, a gamma-sarcoglycan protein, a FKRP protein, a SMN1 protein, a SMN2 protein, a guide RNA (gRNA), a CRISPR Cas nuclease, a dysferlin protein, a calpain protein, and / or an acid alpha-glucosidase (GAA) protein.(1) Deamidated capsid proteins

[0075] In one aspect, the present disclosure is directed to a recombinant AAV (rAAV) particle comprising a heterogeneous group of capsid proteins that contain a subpopulation with an amino acid modification. In some embodiments, the modification can be deamidation, acetylation, isomerization, phosphorylation, or oxidation. In preferred embodiments, the modification is deamidation.

[0076] In some embodiments, the rAAV capsid contain subpopulations of VP1, VP2 and VP3 having at least 1, at least 2, at least 3, at least 4, at least 5 to at least about 25 deamidated amino acid residues, of which at least about 0.1% to about 1%, at least about 1% to about 25%, at least about 25% to about 50%, at least about 50% to about 70%, at least about 70% to about 100%, at least about 75% to about 100%, at least about 80% to about 100% or at least about 90% to about 100% are deamidated as compared to the encoded amino acid sequence of the VP proteins. In some embodiments, the majority of the deamidated residues are asparagine residues. In some embodiments, the deamidated residues are glutamine residues.

[0077] In some embodiments, the AAV composition comprises an AAV8 capsid protein comprising one or more deamidated residues at positions of N56, N254, N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N516, N539, N598, N629, N652, N664, and N717, or at the equivalent residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV 13, AAVrhlO, AAVrh74, or MyoAAV, as measured by mass spectrometry and / or ultraviolet (UV)- visible spectroscopy. In some embodiments, the AAV composition comprises an AAVrh74 capsid protein comprising one or more deamidated residues at positions N56, N254, N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N598, N629, N652, N664, and N71. In some embodiments, the AAV composition comprises an AAV9 capsid protein comprising one or more deamidated residues at positions N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N627, N650, N662, and N715. In some embodiments, the AAV composition comprises a MyoAAV9-4E capsid protein comprising one or more deamidated residues at positions N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N637, N660, N672, andN725 of MyoAAV9-4E. In some embodiments, the AAV composition comprises an AAV5 capsid protein comprising one or more deamidated residues at positions N55, N243, N292, N374, N399, N462, N481, N585, and N639. In some embodiments, the deamidation is measured by any of the methods disclosed herein.

[0078] In some embodiments, the heterogeneous group comprises less than about 75%, less than about 50%, less than about 25%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05% of capsid proteins with deamidation at N254 of AAV8 capsid.

[0079] In some embodiments, the heterogeneous group comprises less than about 75%, less than about 50%, less than about 25%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05% of capsid proteins with deamidation at N478 of AAV8 capsid.

[0080] In some embodiments, the heterogeneous group comprises less than about 75%, less than about 50%, less than about 25%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05% of capsid proteins with deamidation at N513 of AAV8 capsid.

[0081] In some embodiments, the heterogeneous group comprises less than about 75%, less than about 50%, less than about 25%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05% of capsid proteins with deamidation at N516 of AAV8 capsid.

[0082] In some embodiments, the heterogeneous group comprises less than about 75%, less than about 50%, less than about 25%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.9%, less than about 0.8%,less than about 0.7%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05% of capsid proteins with deamidation at N539 of AAV8 capsid.

[0083] Though described in considerable detail, nothing written here should be read as to limit the spirit and scope of the appended claims with reference to any particular aspect not herein expressly contained. As such, the present disclosure fully contemplates additional aspects and / or inventions supported by the description contained herein.(2) Pharmaceutical compositions targeting a mutant gene

[0084] Further disclosed herein are one or more AAV vectors that comprise at least one therapeutic composition and deamidated asparagine residues as part of a viral gene delivery system. In some embodiments, the therapeutic composition comprises an encoded protein that treats a subject with a mutant gene. In some embodiments, the encoded protein is a normal gene to supplement expression of the mutant gene within a subject. In some embodiments, the encoded protein is selected from any one of a microdystrophin protein, an alpha-sarcoglycan protein, a beta- sarcoglycan protein, a gamma-sarcoglycan protein, a FKRP protein, a SMN1 protein, a SMN2 protein, a guide RNA (gRNA), a CRISPR Cas nuclease, a dysferlin protein, a calpain protein, and an acid alpha-glucosidase (GAA) protein. In some embodiments, the therapeutic composition comprises a guide RNA.

[0085] In some embodiments, the encoded protein is a Cas nuclease and, as part of a CRISPR / Cas system with at least one guide RNA (gRNA), effectuates the alteration of a targeted gene or locus in a eukaryotic cell by effecting an alteration of the sequence at a target position (e.g., by creating an insertion or deletion (collectively, an indel) resulting in loss-of-function of (i.e., knocking out) the affected gene or allele; e.g., a nucleotide substitution resulting in a truncation, nonsense mutation, or other type of loss-of-function of an encoded gene product; a of loss-of-function of, for example, an encoded gene product; e.g., loss-of- function of the encoded mRNA or protein by a single nucleotide, double nucleotide, or other frame-shifting deletion, or a deletion resulting in a premature stop codon; or an insertion resulting in a truncation, nonsense mutation, or other type of loss-of-function of an encoded gene product, such as theencoded mRNA or protein; e.g., a single nucleotide, double nucleotide, or other frameshifting insertions, or an insertion resulting in a premature stop codon.

[0086] In some embodiments, the Cas nuclease is a deactivated Cas nuclease (a dCas protein). dCas proteins have been mutated such that at least one nuclease domain (e.g., HNH and / or RuvC domains) are inactive. dCas proteins retain all other capabilities associated with Cas nucleases, such as association with gRNAs and ability to recognize specific PAMs. In some embodiments, localization of a dCas protein to a particular genomic locus (as part of a CRISPR / dCas system with one or more gRNAs) can interfere with gene expression (e.g., due to blockade of RNA polymerases or covalent linkage to one or more effector proteins, such as repressors and / or epigenetic modulators). In some embodiments, dCas localization can enhance gene expression (e.g., when covalently linked to effector proteins, such as transcriptional activators, and / or epigenetic modulators).

[0087] In certain embodiments, the one or more AAV vectors is a recombinant AAV variant vector. In some embodiments, the recombinant AAV variant vector has enhanced cardiac and skeletal muscle tissue tropism. In some embodiments, the recombinant AAV variant vector is capable of delivering and expressing the CRISPR / Cas9-based gene editing system in the cell of a mammal. For example, the recombinant AAV variant vector may be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23 :635-646). The recombinant AAV variant vector may deliver nucleases to skeletal and cardiac muscle in vivo. The recombinant AAV variant vector may be based on one or more of several capsid types, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrh74 and MyoAAV. The recombinant AAV variant vector may be based on AAV2 pseudotype with alternative muscle-tropic AAV capsids, such as AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, and AAV / SASTG vectors that efficiently transduce skeletal muscle or cardiac muscle by systemic and local delivery (Seto et al. Current Gene Therapy (2012) 12:139-151). The recombinant AAV variant vector may be AAV2i8G9 (Shen et al., J. Biol. Chem. (2013) 288:28814-28823).

[0088] The one or more AAV vectors may further comprise an initiation codon, and a stop codon. The initiation and termination codons may be in frame with the therapeutic composition coding sequence. The vector may also comprise a promoter that is operably linked to the therapeutic composition coding sequence. The promoter that is operably linked to the therapeutic composition coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, a U6 promoter, such as the human U6 promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human ubiquitin C (tlUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication Nos. US20040175727 and US20040192593, the contents of which are incorporated herein in their entirety. Examples of muscle-specific promoters include a Spc512 promoter (described in US Patent Application Publication No. US 20040192593), which is incorporated by reference herein in its entirety; Hakim et al. Mol. Ther. Methods Clin. Dev. (2014) 1:14002: and Lai et al. Hum Mol Genet. (2014) 23(12): 3189-3199), a MHCK7 promoter (described in Salva et al., Mol. Ther. (2007) 15:320-329), a CK8 promoter (described in Park et al. PLoS ONE (2015) 10(4): e0124914), and a CK8e promoter (described in Muir et al., Mol. Ther. Methods Clin. Dev. (2014) 1:14025). In some embodiments, the expression of the gRNA and / or Cas9 protein is driven by tRNAs.

[0089] The one or more AAV vectors may also comprise a polyadenylation signal, which may be downstream of the therapeutic composition coding sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (HGH) polyadenylation signal, or human P-globin polyadenylationsignal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).

[0090] The one or more AAV vectors may also comprise an enhancer upstream of the therapeutic composition coding sequence. The enhancer may be necessary for DNA and / or protein expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, RSV or EBV Functional polynucleotide enhancers are described in U.S. Patent Nos. 5,593,972; 5,962,428; and WO / 94 / 016737, the contents of each are hereby fully incorporated by reference for all purposes. The vector may also comprise a mammalian origin of replication in order to maintain the vector extrachromosomally and produce multiple copies of the vector within a cell. The vector may also comprise a regulatory sequence, which may be well suited for gene expression in a mammalian or human cell into which the vector is administered. The vector may also comprise a reporter gene, such as green fluorescent protein ("GFP") and / or a selectable marker, such as hygromycin (Hygro).

[0091] The presently disclosed subject matter provides for compositions comprising the above-described genetic constructs as part of one or more pharmaceutical compositions. Said pharmaceutical compositions as detailed herein can be formulated according to the mode of administration to be used. In some embodiments, pharmaceutical compositions are injectable, and are preferably sterile, pyrogen-free and particulate-free isotonic formulations. In some embodiments, additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some embodiments, isotonic solutions (e.g., phosphate buffered saline) are preferred. In some embodiments, said pharmaceutical compositions further comprise stabilizers, such as gelatin and / or albumin. In some embodiments, a vasoconstriction agent is added to the formulation.

[0092] In some embodiments, said pharmaceutical compositions may further comprise a pharmaceutically acceptable excipient (e.g., functional molecules used as vehicles, adjuvants, carriers, or diluents). In some embodiments, the pharmaceutically acceptable excipient is a transfection facilitating agent (e.g., surface active agents, such as immune-stimulating complexes (ISCOMS), Freumis incomplete adjuvant,LPS analog including monophosphoryl lipid A, muramyl peptides, or other known transfection facilitating agents).(3) Delivery and administration routes

[0093] Provided herein is a method for delivering the presently disclosed AAV vectors and / or a composition thereof to a cell. Upon delivery of said vectors and / or compositions to the tissue, and thereupon into the cells of the mammal, the transfected cells will express the therapeutic composition. In some embodiments, said vectors may be administered to a mammal to alter gene expression and / or to re-engineer or alter the genome. For example, the said vectors or compositions may be administered to a mammal to correct the dystrophin gene in a mammal. The mammal may be human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, or chicken, and preferably human, cow, pig, or chicken.

[0094] In some embodiments, said vectors and / or compositions are administered to a subject by any route selected from oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular administration or combinations thereof. In certain embodiments, said vectors and / or compositions are administered to a subject (e.g., a subject suffering from DMD) intramuscularly, intravenously or a combination thereof. For veterinary use, said vectors and / or compositions may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian may readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. In some embodiments, said vectors and / or compositions are administered by any one of traditional syringes, needleless injection devices, (e.g., microprojectile bombardment gene guns), or other physical methods such as electroporation ("EP"), "hydrodynamic method", or ultrasound.

[0095] Any delivery method and / or route of administration described above may be utilized with a myriad of cell types. Cell types may include, but are not limited to, immortalized myoblast cells, such as wild-type and DMD patient derivedlines (e.g., 6.48-50 DMD, DMD 6594 (del48-50), DMD 8036 (del48-50), C25C14 and DMD-7796 cell lines), primal DMD dermal fibroblasts, induced pluripotent stem cells, bone marrow-derived progenitors, skeletal muscle progenitors, human skeletal myoblasts from DMD patients, CD133+ cells, mesoangioblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and MyoD- or Pax7 -transduced cells, or other myogenic progenitor cells. Immortalization of human myogenic cells can be used for clonal derivation of genetically corrected myogenic cells.4. Examples

[0096] Suitable modifications and adaptations of the compositions and methods of the present disclosure, as described herein, are readily apparent, appreciable, and applicable to one of skill in the art. Such modifications and / or adaptations may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein.

[0097] Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended to illustrate some aspects and embodiments of the disclosure without limiting, in any way, the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties for all purposes. The present disclosure details multiple embodiments and aspects, illustrated by the following non-limiting examples. a. Example 1: Deamidation Analysis of model peptides using the AccuMAP kit

[0098] Two peptides: 1) YHLNGR (SEQ ID NO: 9) and 2) YLGPFNGLDK (SEQ ID NO: 10) that were generated by the trypsin / Lys-C digestion of the AAV8 capsid proteins were selected as model peptides to evaluate the level of deamidation artifacts generated during analysis. These peptides were selected as model peptides because they both have the NG motif that is most susceptible to deamidation. In addition, in the absence of the protein higher-order structure, peptides are more prone to intakehigher level of deamidation due to the accessibility of reaction sites. To represent the sample preparation conditions of the AAV8 capsid proteins, both peptides were analyzed via three different workflows, including the low pH AccuMAP workflow (pH 5.0), and two conventional peptide mapping workflows that utilize slightly basic conditions, with Tris (pH 7.5) and ammonium bicarbonate (pH 7.8) buffers. The treated peptides were then analyzed by LC-MS / MS and the level of deamidation on each individual peptide generated from all three workflows were compared. Untreated peptides that have not gone through any sample preparation workflow were analyzed as control to evaluate the endogenous level of deamidation present in each peptide.

[0099] The untreated YHLNGR (SEQ ID. NO: 9) peptide analyzed as a control showed no detectable level of deamidation. As shown in Figure 3, the same peptide when analyzed using the pH 5.0 workflow showed only 0.1% (s = 0) deamidation, confirming that very low level of artifactual deamidation occurred under these conditions. However, when the same peptide was analyzed using the Tris (pH 7.5) workflow, 4.6% (s = 0.6) deamidation was detected while the ammonium bicarbonate (pH 7.8) workflow showed 93.9% (s = 0.1) deamidation, confirming significantly higher levels of artifactual deamidation as compared to the low pH workflow. Moreover, the results confirm the effects of various salts and buffers as previously reported, even at the same or similar pH (38). Similarly, the untreated peptide YLGPFNGLDK (SEQ ID NO: 10) showed 0.2% deamidation when analyzed by LC-MS / MS. The same peptide when analyzed by the pH 5.0 workflow showed 0.3% (s = 0.1) deamidation, as compared to 5.6% (s = 0.7) deamidation using the Tris (pH 7.5) workflow and 77.7% (s = 0.4) deamidation using the ammonium bicarbonate (pH 7.8) workflow. These data demonstrated that the workflow developed by using the low pH method significantly minimized the introduction of deamidation artifacts during sample preparation as compared to conventional peptide mapping workflows that utilize neutral to slightly basic pH for sample preparation.Example 2: Comparative deamidation analysis between the low pH workflow and conventional peptide mapping workflows (pH 7 - 8)

[0100] As with analysis of the model peptide above, deamidation of AAV8 generated from the pH 5.0 method was compared to those generated from traditional tryptic peptide mapping workflows utilizing slightly basic buffer systems for sample preparation — Tris (pH 7.5) and ammonium bicarbonate (pH 7.8).

[0101] The results for AAV8 deamidation were significantly different using the three workflows. As expected, the deamidation hotspots, especially the NG motifs that are most susceptible to deamidation showed the highest level of differences between the three workflows. Extracted ion chromatograms (EICs) of one such peptide, YHLNGR that contains the N513G motif are shown in Figure 4. N513(G) residue showed 22% deamidation under the Tris (pH 7.5) workflow and 93% deamidation under the ammonium bicarbonate (pH 7.8) workflow (Table 1). On the contrary, the same peptide showed only 0.2% deamidation under pH 5.0 conditions. Similarly, N56(G), N262(G), N384(G) and N539(G) also showed significantly lower level of deamidation under pH 5.0 conditions as compared to the Tris (pH 7.5) and ammonium bicarbonate (pH 7.8) workflows. Amongst the other remaining hotspots, N254(H), N499(S), N516(S) also showed a similar trend with lowest level of deamidation observed under the AccuMAP workflow (pH 5.0). No deamidation was observed at the three remaining hotspots, N93(H), N669(S) and N692(S), under any of the workflows. A comparison of the levels of deamidation observed for AAV8 under the three workflows is provided in Table 1. This data confirmed that the AccuMAP workflow significantly reduced the levels of artifactual deamidation for AAV8 as compared to conventional peptide mapping workflows employing slight basic buffer systems and therefore provided a more accurate picture of endogenous deamidation in AAV8. b. Example 3: Deamidation analysis of AAV8 capsid proteins using the low pH workflow

[0102] The AAV8 capsid proteins have a total of 58 asparagine residues. Out of the 58 total asparagine residues, there are 5 NG motifs: N56(G), N262(G), N384(G),N513(G) and N539(G) that are most susceptible to deamidation. In addition, there are 2 NH: N93(H), N254(H) and 4 NS: N499(S), N516(S), N598(S) and N692(S) motifs that are also potential hotspots for deamidation. Analysis of deamidation in AAV8 capsid proteins using the pH 5.0 workflow provided sequence coverage on 56 of the 58 asparagine residues. Total coverage was obtained on all the deamidation hotspots present in the AAV8 capsid proteins using this method. As shown in Table 2, low levels of deamidation (<1%) were observed at three asparagine residues: N254(H), N478(W) and N516(S). A second lot of AAV8 analyzed under this workflow also showed very low levels of deamidation (<1%). However, there were some differences in the levels of deamidation between the two lots with the second lot showing a slightly higher level of deamidation. N56(G), N93(H) and N539(G) showed low level of deamidation in the second lot but no deamidation was detected for these asparagine residues in the first lot. Deamidation results from both lots of AAV8 are summarized in Table 2. c. Example 4: Sequence coverage and characterizing additional post-translations modifications

[0103] Digestion of the AAV8 capsid proteins using the Promega AccuMAP kit yielded a very high sequence coverage of 95% (Figure 5) and therefore allowed the characterization of 56 / 58 total asparagine residues in AAV8. The high sequence coverage provided by this method has also allowed the characterization of the other PTMs present in AAV8. MS / MS spectrum of the triply charged ion, m / z: 726.0104 corresponding to the mass of 2175.0107 Da confirmed the presence of the VP1 N- terminal peptide Ac-AADGYLPDWLEDNLSEGIR (SEQ ID NO: 11) with methionine loss and N-terminal acetylation. Similarly, multiple b-series fragment ions from the MS / MS spectrum of the triply charged ion, m / z: 1140.4693 corresponding to the VP3 N-terminal peptide Ac- AAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDR (SEQ ID NO: 12) confirmed the N-terminal methionine loss and acetylation for VP3. Fragment ions b9, bl 2, bl3 and bl4 with a mass shift of +80 Da in the MS / MS spectrum of the triply charged ion, m / z of 738.6592 corresponding to the mass of the VP1 N-terminal peptide Ac-AADGYLPDWLEDNLSEGIR (SEQ ID NO: 11) confirmed phosphorylation at Y6 residue. Phosphorylation was also confirmed at S149 and SI 53 from the MS / MS spectrum of the quadruply charged ion with m / z: 551.5262, corresponding to the mass of the peptide KRPVEPSPQRSPDSSTGIGK (SEQ ID NO: 13). A +80 Da mass shift for the ylO fragment ion and presence of the unmodified y9 ion confirmed the phosphorylation at SI 53. Whereas a mass shift of +80 Da on the bl2 ion while the presence of the unmodified yl3 fragment ion confirmed the phosphorylation at S149. Low level oxidation was observed (< 1%) at multiple methionine and tryptophan residues.

[0104] A separate Asp-N digestion was performed (pH 7.5) to characterize the deamidation on the two asparagine residues that were missing from the AccuMAP digestion process, namely N35Q and N737L and gain 100% sequence coverage for the AAV8 capsid proteins. The AspN digestion data showed no detectable level of deamidation for N35Q and N737L residues. As these two asparagine residues are not deamidation hotspots the chances of introduction artifactual deamidation during proteolysis were low. Therefore, the AspN digestion was carried out following a conventional peptide mapping workflow (pH 7.5). d. Example 5: Conservation analysis and extension to additional AAV serotypes

[0105] Having established a robust low pH peptide mapping method for deamidation characterization in AAV capsid proteins that can effectively eliminate deamidation artifacts with high sequence coverage, this prophetic example extends this analysis to various other AAV capsid proteins. Based upon the sequence homology between the AAV serotypes, it is likely that deamidation is conserved — at least at critical residues. Indeed, there are numerous conserved asparagine residues between AAV8 and at least AAV9, AAVrh74, MyoAAV9-4E, and AAV5 (Figures 6A-6D).

[0106] Using the methods previously described above, comparative deamidation analysis is performed for each of these viral capsid proteins. Such analyses further demonstrate the use of this method as a platform approach for structural analysis of AAV capsid proteins.

[0107] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.TABLE 1. Percent Deamidation obtained for AAV8 when analyzed under the low pH AccuMAP workflow, pH 5.0 and the conventional peptide mapping workflows (Tris, pH 7.5 and ammonium bicarbonate, pH 7.8) employing neutral to slightly alkaline conditions for proteolysis.TABLE 2. Listing of select vector components and other nucleotide sequences related to the disclosure.

Claims

CLAIMS1. A method for using a pH buffer to detect deamidated amino acid residues in a viral capsid, the method comprising:(a) denaturation and reduction of the capsid;(b) proteolytic digestion of the capsid with one or more proteases to generate several polypeptide fragments; and(c) mass spectrometry analysis of the polypeptide fragments.

2. The method of claim 1, wherein the pH buffer is an acidic buffer.

3. The method of claim 2, wherein the pH buffer is at or less than pH 5.

4. The method of claim 1, wherein the pH buffer is a basic buffer.

5. The method of claim 4, wherein the pH buffer is at or less than pH 7.5.

6. The method of claim 4, wherein the pH buffer is pH 7.8.

7. The method of any one of claims 1-6, wherein the capsid is denatured and reduced in step(a) with 6M guanidine HC1 and lOmM TCEP.

8. The method of claim 7, further comprising an optional buffer exchange step to reduce the concentration of guanidine HC1 to IM.

9. The method of any one of claims 1-3 or 7-8, wherein the one or more proteases in step(b) comprises recombinant Lys-C.

10. The method of any one of claims 1-9, wherein the one or more proteases in step (b) comprises trypsin.

11. The method of any one of claims 1-10, wherein the mass spectrometry analysis in step (c) is Quadropole Time-of-Flight (QTOF) mass spectrometry.

12. A method for using a pH 5.0 buffer to detect deamidated amino acid residues in one or more viral capsid proteins, the method comprising:(a) denaturing the one or more viral capsid proteins in 6M guanidine HC1;(b) reducing the one or more viral capsid proteins in lOmM TCEP;(c) performing a first proteolytic digestion of the one or more viral capsid proteins to generate several polypeptide fragments with about 1: 10 (protease: capsid) recombinant Lys-C for about 18 hours at 37 °C;(d) performing a second proteolytic digestion of the several polypeptide fragments with about 1:5 trypsin for about 4 hours at 37 °C;(e) quenching the first and second proteolytic digestions with about 2% formic acid;(f) performing a chromatographic separation of the several polypeptide fragments with a 2.1 x 150 mm BEH Cl 8 column with a 1.7 micron pore size, wherein a first mobile phase comprises 0.1% formic acid and a second mobile phase comprises 0.1% formic acid in 90% acetonitrile, wherein equilibrium of the second mobile phase is increased from about 2% to about 45% over about 80 minutes, to about 70% over about 5 minutes and about 100% over about 2 minutes; and(g) introducing the separated peptides into a mass spectrometer.

13. A modified adeno-associated virus (AAV) capsid protein comprising one or more deamidated asparagine residues.

14. The AAV capsid protein of claim 13, wherein the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, and MyoAAV15. The AAV capsid protein of claim 14, wherein said deamidated asparagine residues in an AAV8 capsid protein or its equivalent conserved residue are selected from N56, N254,N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N516, N539, N598, N629, N652, N664, and N717.

16. The AAV capsid protein of claim 15, wherein said deamidated asparagine residues are selected from N254, N478, N513, N516, and N539.

17. The AAV capsid protein of claim 14, wherein said deamidated asparagine residues in an AAVrh74 capsid protein or its equivalent conserved residue are selected from N56, N254, N262, N304, N305, N384, N409, N471, N478, N497, N499, N501, N513, N598, N629, N652, N664, and N717.

18. The AAV capsid protein of claim 14, wherein said deamidated asparagine residues in an AAV9 capsid protein or its equivalent conserved residue are selected from N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N627, N650, N662, and N715.

19. The AAV capsid protein of claim 14, wherein said deamidated asparagine residues in an MyoAAV9-4E capsid protein or its equivalent conserved residue are selected from N56, N253, N261, N303, N304, N408, N476, N495, N497, N511, N513, N637, N660, N672, and N725.

20. The AAV capsid protein of claim 14, wherein said deamidated asparagine residues in an AAV5 capsid protein or its equivalent conserved residue are selected from N55, N243, N292, N374, N399, N462, N481, N585, and N639.

21. A modified AAV capsid protein, wherein said capsid protein is 95% identical or greater to the capsid protein of any one of claims 13-20.

22. A modified AAV capsid protein, wherein said capsid protein is 99% identical or greater to the capsid protein of any one of claims 13-20.

23. A vector expressing the capsid protein of any one of claims 13-22.

24. The vector of claim 23, wherein the vector comprises at least one bidirectional promoter.

25. The vector of any one of claims 23 or 24, wherein the vector comprises a ubiquitous promoter or a tissue-specific promoter operably linked to one or more encoded therapeutic polynucleotides.

26. The vector of claim 25, wherein the tissue-specific promoter is a muscle specific promoter.

27. The vector of claim 25, wherein the tissue-specific promoter is a neuron specific promoter.

28. The vector of claim 25, wherein the one or more encoded therapeutic polynucleotide is selected from a group consisting of a microdystrophin, alpha-sarcoglycan, beta- sarcoglycan, gamma-sarcoglycan, FKRP, SMN1, SMN2, a guide RNA (gRNA), a CRISPR Cas nuclease, dysferlin, calpain, and acid alpha-glucosidase (GAA).

29. A cell comprising:(i) the capsid protein of any one of claims 13 -22; or(ii) the vector of claims 23-28.

30. The cell of claim 29, wherein the cell is a eukaryotic cell or a prokaryotic cell.

31. The cell of claim 29, wherein the cell is a yeast cell, an insect cell, a mammalian cell, or a bacterial cell.

32. A kit comprising:(i) the capsid protein of any one of claims 13 -22; or(ii) the vector of claims 23-28.

33. A method of treating a disease in a patient in need thereof, the method comprising administering to the patient the capsid protein of any one of claims 13-22 or the cell of claim 29.

34. The method of claim 33, wherein the disease is Duchenne muscular dystrophy (DMD).

35. The method of claim 33, wherein the disease is Limb Girdle muscular dystrophy (LGMD).

36. The method of claim 33, wherein the disease is Pompe disease.

37. The method of any one of claims 33-36, wherein the protein or the cell is administered to the subject intramuscularly, intravenously, or a combination thereof.

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