Modified AAVRH74 VP1 capsid protein and its use, recombinant viral particle, polynucleotide, recombinant expression system, host cell, method for modifying polynucleotide and / or protein in cell and kit.

Description

1 / 59 “MODIFIED AAVRH74 VP1 CAPSID PROTEIN AND USE THEREOF, RECOMBINANT VIRAL PARTICLE, POLYNUCLEOTIDE, RECOMBINANT EXPRESSION SYSTEM, HOST CELL, METHOD FOR MODIFYING POLYNUCLEOTIDE AND / OR PROTEIN IN CELL AND KIT” SPLIT APPLICATION OF PATENT APPLICATION BR1120200183547, dated 03 / 14 / 2019 DESCRIPTIVE REPORT REFERENCE TO RELATED REQUESTS

[001] This Patent Application claims priority to the Application of US Provisional Patent Serial No. 62 / 644,317, filed March 16, 2018, the contents of which are incorporated herein by reference. STATEMENT REGARDING GOVERNMENT SUPPORT

[002] This invention was made with the support of the Government of United States under Grant No. TR001068 issued by the National Institutes of Health. The Government has certain rights in the invention. SEQUENCE LISTING [002.1] This patent application contains a Listing of The sequence was submitted electronically in ASCII format and is incorporated here by reference in its entirety. The so-called ASCII copy, created on February 26, 2019, was named 1068877170_SL.txt and has a size of 18,492 bytes. Petition 870260076337, dated 07 / 30 / 2026, p. 86 / 177 2 / 59 BACKGROUND

[003] Efforts have been made to modify AAV capsids for enhanced gene delivery. For example, WO 2017 / 165859A1 describes a viral capsid modification in which Cas9 is fused or conjugated to the viral capsid protein to promote customizable gene editing. Additional references have described the use of DNA family shuffling (Grimm to Kay), lysine substitutions in AAVrh74 (US 2015 / 006556), mutations at positions 195, 199, 201, or 202 of AAVrh74 (US 9,840,719), and modifications in AAVrh48 (EP 2359866). Further references detail libraries of random peptides that are displayed on the surface of an AAV (e.g., Muller (2003) Nat. Biotechnol., Perabo (2003) Mol. Ther. and 7,588,722 (Grimm and Kay)).

[004] Gene therapy treatments for Dystrophies Muscle cells will require systemic delivery of genes to muscle cells throughout the body. The most efficient delivery option is to use the body's circulatory system to distribute the virus to peripheral muscle cells. One problem with systemic delivery is that most AAV serotypes have a natural propensity to infect liver cells [1]. There are approximately 20 times more liver cells than muscle cells in the body, through which the virus must pass during systemic delivery [2]. It is estimated that more than 50% of AAV vectors become trapped in the liver after systemic intravenous injection.

[005] Currently, more than 50% of the systemically injected virus is lost in the liver where little therapeutic effect is generally produced [4]. By altering the targeting of vector delivery to the liver and increasing specific binding and transduction to the muscle, specific gene expression for the muscle and the therapeutic benefit for the patient can be significantly increased while reducing potential side effects. Currently, the Petition 870260076337, dated 07 / 30 / 2026, page 87 / 177 3 / 59 Clinical trials for the treatment of Duchenne Muscular Dystrophy require the delivery of high vector levels (>5E+12 vector genomes per kilogram) to attempt to achieve therapeutic levels of gene expression in the muscle. Muscle-specific promoters are used to reduce “off-target” gene expression, but have no effect on increasing total gene expression levels. By increasing the efficiency of muscle-specific transduction, the total dose required to achieve a therapeutic benefit can be significantly reduced.

[006] This disclosure addresses the limitations of the prior art and also provides related advantages. SUMMARY

[007] The Applicant proposes an approach to increase the systemic delivery of the vector to muscle cells that would reduce or eliminate infection of liver cells as the virus circulates. The Applicant hypothesized that modified AAVrh74 capsids target myoblasts and muscle satellite cells more efficiently than unmodified AAVrh74 vectors.

[008] This disclosure relates to a modified viral capsid protein comprising or alternatively consisting essentially of, or further consisting in addition to, a viral capsid protein modified by amino acid substitution or insertion between 1 and 7 amino acids. The Applicant has generated three mutants. One of the mutants (AAVmut4, asparagine to isoleucine at amino acid 502 of the VP1 capsid) increases gene delivery globally to all tissues tested up to 56 times (increase between 3 and 56 times depending on the tissue) greater transduction efficiency. Another mutant (AAVmut5, tryptophan to arginine at amino acid 505 of the VP1 capsid) increases gene delivery to the heart almost 50 times more than AAVrh74. Petition 870260076337, dated 07 / 30 / 2026, page 88 / 177 4 / 59 A third mutant (AAVYIG or AAVYIGSR591) targets a receptor found primarily on satellite cells, which are considered to be muscle stem cells, although satellite cell tropism has not yet been confirmed by IHC staining. Notably, based on the Applicant's knowledge of the AAV crystal structure and the alpha 7 beta 1 integrin, AAVYIG or AAVYIGSR591 is believed to have a higher affinity for skeletal muscle and a lower affinity for the liver.

[009] To avoid being limited to theory, the Applicant provides methods for achieving therapeutic benefits for a patient by increasing the effective dose that reaches the target tissue such as the heart or muscle without increasing the total dose to the patient. By reducing the total dose required to achieve a therapeutic benefit, fewer viral antigens are delivered to the patient, ideally resulting in reduced immune responses to the vector and greater safety. The manufacture of a gene therapy pharmaceutical product sufficient to conduct late-stage clinical trials is the main obstacle to further development. Reducing the dose requirements to achieve therapeutic benefit will result in reduced manufacturing requirements, reduced manufacturing costs, faster development of clinical trials, and a greater capacity to treat more patients.

[0010] Consequently, this disclosure relates to modified capsid proteins, isolated polynucleotides, methods for preparing modified capsid proteins, recombinant viral particles, and recombinant expression systems for producing modified viral particles. One aspect of the disclosure relates to a modified viral capsid protein comprising or alternatively consisting essentially of, or further consisting of, a viral capsid protein modified by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, Petition 870260076337, dated 07 / 30 / 2026, page 89 / 177 5 / 59 optionally of AAVrh74. In additional embodiments, the modification comprises the substitution of isoleucine with asparagine at amino acid position 502 in VP1 of AAVrh74 or an equivalent modification. In some embodiments, the modification comprises the substitution of tryptophan with arginine at amino acid 505 in VP1 of AAVrh74. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74 or an equivalent thereof. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter normal rh74 receptor binding.

[0011] Also disclosed here is an isolated polynucleotide encoding a modified viral capsid protein comprising or alternatively consisting essentially of or further consisting of a modified viral capsid protein altered by amino acid modification or insertion between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally from AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in the VP1 of AAVrh74. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells.In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74 or an equivalent thereof. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter normal rh74 receptor binding. Petition 870260076337, dated 07 / 30 / 2026, page 90 / 177 6 / 59

[0012] Disclosed herein is a recombinant viral particle comprising or alternatively consisting essentially of, or further consisting of, a modified capsid protein comprising or alternatively consisting essentially of, or further consisting of, a modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally from AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in the VP1 of AAVrh74. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells.In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some embodiments, this peptide has a high affinity for the Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter the binding of the normal rh74 receptor. In particular aspects, a recombinant viral particle comprises or alternatively consists essentially of 5 or more modified capsid proteins per viral particle (and / or per modified viral capsid).

[0013] In other aspects, a recombinant viral particle comprises or alternatively consists essentially of between 1 and 5 modified capsid proteins per viral particle (and / or per modified viral capsid). Additional aspects consider a polynucleotide encoding the modified viral capsid protein through the modification or insertion of between 1 and 7 amino acids disclosed herein. In some embodiments, the viral capsid protein is a VP1, optionally of AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine for asparagine in Petition 870260076337, dated 07 / 30 / 2026, page 91 / 177 7 / 59 amino acid position 502 in VP1 of AAVrh74 or an equivalent modification. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in VP1 of AAVrh74. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter normal rh74 receptor binding.

[0014] Also provided are the modified capsids disclosed herein which optionally comprise a transgene or a CRISPR system for gene modification. Additionally provided are polynucleotides encoding the modified capsids and vectors encoding said polynucleotides, as well as complements and equivalents of each other. Further aspects relate to a host cell that produces the viral particle and / or comprises the vector disclosed herein. Further aspects relate to an expression system for the production of the viral particle disclosed herein.

[0015] This disclosure also provides compositions comprising a carrier and one or more modified capsids, a polynucleotide, a vector, a plasmid, a host cell or an expression system. Additionally, a kit is provided comprising one or more modified capsid proteins, a polynucleotide, a vector, a plasmid, a host cell or an expression system and instructions for use.

[0016] A method of treating a target disease or dysfunctional tissue in an individual in need thereof is further disclosed herein, comprising or alternatively essentially consisting or further consisting in administering to the individual an effective quantity of a viral particle. Petition 870260076337, dated 07 / 30 / 2026, page 92 / 177 8 / 59 recombinant comprising or alternatively consisting essentially of, or additionally consisting of, a modified capsid protein comprising or alternatively consisting essentially of, or additionally consisting of, a viral capsid protein modified by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally from AAVrh74. In additional embodiments, the modification comprises the substitution of isoleucine with asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In additional embodiments, this viral particle increases the treatment delivery efficacy by approximately 3 to 56 times to diseased or dysfunctional tissue compared to AAVrh74. In some embodiments, the modification comprises the substitution of tryptophan with arginine at amino acid 505 in the VP1 of AAVrh74.In additional modalities, the diseased or dysfunctional tissue is cardiac tissue. Also in additional modalities, this viral particle increases the effectiveness of treatment delivery by approximately 50 times or more to cardiac tissue compared to AAVrh74. In some modalities, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some modalities, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some modalities, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that is likely to alter the binding of the normal rh74 receptor. In some modalities, the viral particle comprises an efficient amount of treatment suitable for the diseased or dysfunctional tissue. In some modalities, the treatment is CRISPR / Cas9-based gene editing. BRIEF DESCRIPTION OF THE FIGURES Petition 870260076337, dated 07 / 30 / 2026, page 93 / 177 9 / 59

[0017] FIGS. 1A-1E show the structural analysis of the capsid library of adeno-associated virus serotype 9 (AAV9). (a) (a) Graphical representation of the AAV9 VP3 subunit monomer obtained using SWISS-MODEL with the AAV8 crystal structure serving as a template (pdb id: 2QA0). The GH loop containing amino acids 390-627 (VP1 numbering) is in gray. (b) Surface rendering of an AAV9 capsid model with 60 VP3 subunits generated using T = 1 icosahedral symmetry coordinates in VIPERdb. The GH loop regions of different VP3 subunits, surrounding the five-fold icosahedral pore and interdigitating on the three-fold symmetry axis, are highlighted in gray. (c) Graph of the AAV9 VP3 subunit trimer generated in VIPERdb with point mutations from 43 representative clones of the AAV9 library represented by gray spheres. (d) Side view of the capsid trimer (90° rotation) showing most of the point mutations (gray spheres) in clusters on the outer loops.(e) Spherical roadmap projection of surface residues within the capsid trimer region. Residues highlighted in red represent a subset of ten AAV9 variants containing altered residues prominently located on the capsid surface. Figure reproduced from Pulicherla et al. [7].

[0018] FIG. 2 shows markers that can be separated by FACS for muscle stem cell isolation. Figure reproduced from Wang et al.,

[13] .

[0019] FIG. 3 shows the biodistribution of the mutant vector of AAVrh74. Male CD-1 mice received intravenous injection of 1E+11 Vg of AAVrh74 or two AAVrh74 capsid mutants / report viruses with lucEYFP and 3 weeks later tissues were collected and analyzed for vector genome copies by qPCR.

[0020] FIG. 4 shows the acquisition of images in live animals of AAVmut4 and mut5 luminescence. Adult CD-1 mice Petition 870260076337, dated 07 / 30 / 2026, page 94 / 177 10 / 59 received caudal vein injection of 1E+11 Vg of AAVrh74 or AAVmut4 virus containing the same luciferase vector. Images of the mice were obtained 3 weeks after injection regarding luciferase expression. Two separate experiments were performed under the same conditions. Exp. 1, same animals in groups A and B with photon quantification in the head (A) or photon quantification in the legs (B). Exp. 2 was a repetition of Exp. 1 with separate animals performed 4 weeks later. The leftmost animal in each group of 4 received the AAVrh74 control virus injection, then 3 animals from each group received the AAVmut4 virus injection.

[0021] FIG. 5 shows live animal luminescence imaging of AAVYIG or AAVYIGSR591. Adult CD-1 mice received caudal vein injection of 1E+11 Vg of AAVYIG or AAVYIGSR591 virus containing the luciferase vector. Images were obtained from mice 3 weeks post-injection regarding luciferase expression with whole-body photon counts above each mouse in the ventral or dorsal position. DETAILED DESCRIPTION

[0022] Embodiments according to this disclosure will be described more fully later herein. Aspects of the disclosure may, however, be incorporated in different forms and should not be interpreted as being limited by the embodiments presented herein. Rather, these embodiments are provided so that this disclosure is thorough and complete and fully conveys the scope of the invention to those skilled in the art. The terminology used in the description herein is intended to describe only particular embodiments and is not intended to be limiting.

[0023] Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as is commonly understood by someone skilled in the art. Petition 870260076337, dated 07 / 30 / 2026, p. 95 / 177 11 / 59 to which the invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and the relevant art and should not be interpreted in an idealized or overly formal sense as defined herein. Although not explicitly defined below, these terms should be interpreted according to their common meaning.

[0024] 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 herein by reference in their entirety.

[0025] Unless otherwise indicated, the practice of the present technology will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the expertise of the technique.

[0026] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination. Furthermore, the disclosure also considers that, in some embodiments, any feature or combination of features presented herein may be excluded or omitted. For illustration, if the Descriptive Report states that a complex comprises components A, B, and C, it is specifically intended that any one of A, B, or C, or a combination thereof, may be omitted and disregarded individually or in any combination.

[0027] Unless explicitly stated otherwise, all specified modalities, features and terms are intended to include both the modality, feature or term mentioned and its biological equivalents.

[0028] All numerical designations, for example, pH, Petition 870260076337, dated 07 / 30 / 2026, p. 96 / 177 12 / 59 Temperature, time, concentration, and molecular weight, including ranges, are approximations that 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%. It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term “approximately.” It should also be understood, although not always explicitly stated, that the reagents described herein are merely examples and that their equivalents are known in the art.

[0029] Throughout this disclosure, various published publications, patents, and Patent Description Reports are referred to by an identifying citation or by an Arabic numeral. The full citation for publications identified by an Arabic numeral is found immediately preceding the Claims.Disclosures of these publications, patents, and patent descriptions are incorporated herein by reference in the present disclosure in their entirety to describe more fully the state of the art to which the invention belongs. Definitions

[0030] 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 expertise of the technique. See, for example, Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (1987)); the Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, a Laboratory and Animal Cell Culture Handbook (RI Freshney, ed. (1987)). Petition 870260076337, dated 07 / 30 / 2026, p. 97 / 177 13 / 59

[0031] As used in the description of the invention and in Claims attached, it is intended that the singular forms “um”, “uma”, “o” and “a” also include the plural forms, unless the context clearly indicates otherwise.

[0032] As used herein, the term “comprising” is intended to mean that compositions and methods include the cited elements but do not exclude others. As used herein, the transitional expression consisting essentially of (and grammatical variants) should be interpreted as encompassing the cited materials or steps and that which does not materially affect the basic and novel feature(s) of the cited embodiment. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising”. “Consisting of” should mean the exclusion of more than trace elements of other ingredients and steps of substantial methods for the administration of the compositions disclosed herein. The aspects defined by each of these transitional terms are within the scope of the present disclosure.

[0033] It is understood that the term “approximately”, as used herein when referring to a measurable value such as a quantity or a concentration and the like, covers variations of 20%, 10%, 5%, 1%, 0.5% or even 0.1% of the specified quantity.

[0034] The terms “acceptable”, “efficient” or “sufficient” when used to describe the selection of any components, ranges, dosage forms, etc. disclosed herein mean that said component, range, dosage form, etc. is appropriate for the disclosed purpose.

[0035] Furthermore, as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0036] The term “cell” as used herein may refer to a prokaryotic or eukaryotic cell, optionally obtained from an individual Petition 870260076337, dated 07 / 30 / 2026, p. 98 / 177 14 / 59 or a commercially available source.

[0037] “Eukaryotic cells” comprise all kingdoms of life except Monera. They can be easily distinguished by a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes, or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically cited, the term “host” includes a eukaryotic host, including, for example, yeast cells, higher plants, insects, and mammals. Non-limiting examples of eukaryotic cells or hosts include apes, cattle, pigs, mice, rats, birds, reptiles, and humans, for example, HEK293 cells and 293T cells.

[0038] “Prokaryotic cells” generally lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and Archaea. In addition to chromosomal DNA, these cells may also contain genetic information in a circular loop called an episome. Bacterial cells are very small, approximately the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells exhibit three main shapes: rod-shaped, spherical, and spiral. Instead of undergoing elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus bacteria, E. coli bacteria, and Salmonella bacteria.

[0039] The term “encodes” as applied to nucleic acid sequences refers to a polynucleotide that is said to “encode” a polypeptide if, in its native state or when manipulated through methods well known to those skilled in the art, it can be transcribed and / or translated to produce mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of this nucleic acid and the coding sequence can be deduced from it. Petition 870260076337, dated 07 / 30 / 2026, p. 99 / 177 15 / 59

[0040] The terms “equivalent” or “biological equivalent” are used interchangeably when referring to a particular mode, biological or cellular material and mean those that have minimal homology while still maintaining the desired structure or functionality. Non-limiting examples of equivalent polypeptides include a polypeptide that has at least 60% or alternatively at least 65% or alternatively at least 70% or alternatively at least 75% or alternatively 80% or alternatively at least 85% or alternatively at least 90% or alternatively at least 95% identity with respect to polypeptide sequences or a polypeptide that is encoded by a polynucleotide or its complement that hybridizes under high stringency conditions to a polynucleotide encoding such polypeptide sequences. High stringency conditions are described herein and incorporated herein by reference.Alternatively, an equivalent thereof is a polypeptide encoded by a polynucleotide or a complement thereof, which has at least 70% or alternatively at least 75% or alternatively 80% or alternatively at least 85% or alternatively at least 90% or alternatively at least 95% identity or at least 97% sequence identity with the reference polynucleotide, for example, the wild-type polynucleotide.

[0041] Non-limiting examples of equivalent polypeptides include a polynucleotide that has at least 60% or alternatively at least 65% or alternatively at least 70% or alternatively at least 75% or alternatively 80% or alternatively at least 85% or alternatively at least 90% or alternatively at least 95% or alternatively at least 97% identity with a reference polynucleotide. An equivalent also means a polynucleotide or its complement that hybridizes under conditions of high stringency with a reference polynucleotide. Petition 870260076337, dated 07 / 30 / 2026, pp. 100 / 177 16 / 59

[0042] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (e.g., 80%, 85%, 90%, or 95%) of “sequence identity” with another sequence means that, when aligned, the percentage of bases (or amino acids) is the same when comparing the two sequences. Alignment and percentage of homology or sequence identity can be determined using software programs known in the art, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, pre-established parameters are used for alignment. A non-limiting example of an alignment program is BLAST, which uses pre-established parameters. In particular, examples of programs include BLASTN and BLASTP, which use the following pre-established parameters: Genetic code=default; filter=none; filament=both; Limit=60; Expectation=10; Matrix=BLOSUM62; Descriptions=50 sequences; Separate by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percentage of identity can be determined by incorporating them into clustalW (available at the web address:genome.jp / tools / clustalw / , last accessed on January 13, 2017).

[0043] “Homology” or “identity” or “similarity” refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for the purpose of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between the Petition 870260076337, dated 07 / 30 / 2026, pp. 101 / 177 17 / 59 sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences in this disclosure.

[0044] “Homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0045] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized through hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein bonding, or any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a larger process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0046] Examples of stringent hybridization conditions include: Examples of moderate hybridization conditions include: incubation temperatures of approximately 25°C to approximately 37°C; hybridization buffer concentrations of approximately 6*SSC to approximately 10*SSC; formamide concentrations of approximately 0% to approximately 25%; and washing solutions of approximately 4*SSC to approximately 8*SSC. Examples of moderate hybridization conditions include: incubation temperatures of approximately 40°C to approximately 50°C; buffer concentrations of approximately 9*SSC to approximately 2*SSC; formamide concentrations of approximately 30% to approximately 50%; and washing solutions of approximately 5*SSC to approximately 2*SSC. Petition 870260076337, dated 07 / 30 / 2026, page 102 / 177 High stringency conditions include: incubation temperatures of approximately 55°C to approximately 68°C; buffer concentrations of approximately 1*SSC to approximately 1.1*SSC; formamide concentrations of approximately 55% to approximately 75%; and washing solutions of approximately 1*SSC, 1.1*SSC, or deionized water. In general, hybridization incubation times are 5 minutes to 24 hours, with 1, 2, or more washing steps, and washing incubation times are approximately 1, 2, or 15 minutes. SSC is 1.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems may also be employed.

[0047] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0048] The term “isolate” as used here refers to molecules or biological agents or cellular materials that are substantially free of other materials.

[0049] As used herein, the term “functional” can be used to modify any molecule, biological agent, or cellular material to signify that it performs a particular specified effect.

[0050] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. Petition 870260076337, dated 07 / 30 / 2026, pp. 103 / 177 19 / 59

[0051] The terms “protein”, “peptide”, and “polypeptide” are used interchangeably and in their broadest sense refer to a compound of two or more amino acid subunits, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. Alternatively, the subunit may be linked by other bonds, for example, ester, ether, etc. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise the sequence of a protein or peptide. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0052] As used herein, the term “recombinant expression system” refers to a genetic construct or constructs for the expression of certain genetic material formed through recombination.

[0053] A “gene delivery vehicle” is defined as any molecule that can carry polynucleotides inserted into a host cell. Examples of gene delivery vehicles are liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metallic particles; and bacteria or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the technique that have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as for simple protein expression.

[0054] A polynucleotide disclosed herein may be delivered to a cell or tissue using a gene delivery vehicle. “Gene delivery,” “gene transfer,” “transduction,” and the like, as used herein, are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”). Petition 870260076337, dated 07 / 30 / 2026, pp. 104 / 177 20 / 59 in a host cell, regardless of the method used for introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., through viral infection / transfection or various other protein- or lipid-based gene delivery complexes) as well as techniques that facilitate the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery, and various other techniques used for polynucleotide introduction). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contain a host cell-compatible origin of replication or integrate into a host cell replicon such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.A number of vectors are known that are capable of mediating the transfer of genes into mammalian cells, as is known in the art and described herein.

[0055] A “plasmid” is an extrachromosomal DNA molecule separate from chromosomal DNA that is capable of replicating independently of chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically provide a selective advantage under certain environmental conditions. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar circumstances.

[0056] The “plasmids” used in genetic engineering are called “plasmid vectors.” Many plasmids are commercially available for these uses. The gene to be replicated is inserted into copies of a plasmid that contain genes that make cells resistant to particular antibiotics and a multiple cloning site. Petition 870260076337, dated 07 / 30 / 2026, pp. 105 / 177 21 / 59 (MCS or polylinker), which is a short region containing several commonly used restriction sites that allow for the easy insertion of DNA fragments at this location. Another important use of plasmids is to produce large quantities of proteins. In this case, researchers cultivate bacteria containing a plasmid carrying the gene of interest. Just as the bacteria produce proteins to confer antibiotic resistance, they can also be induced to produce large quantities of proteins from the inserted gene.

[0057] A “yeast artificial chromosome” or “YAC” refers to a vector used to clone large fragments of DNA (greater than 100 kb and up to 3000 kb). It is an artificially constructed chromosome containing the telomeric, centromeric, and origin of replication sequences necessary for replication and preservation in yeast cells. Constructed using an initial circular plasmid, these are linearized through the use of restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule through the use of cohesive ends.Yeast expression vectors, such as YACs, YIps (yeast integration plasmid), and YEps (yeast episomal plasmid), are extremely useful since eukaryotic protein products with post-translational modifications can be obtained, as yeasts themselves are eukaryotic cells. However, it has been observed that YACs are more unstable than BACs, producing chimeric effects.

[0058] A “viral vector” is defined as a recombinantly produced virus or viral particle comprising a polynucleotide that will be delivered to a host cell, in vivo, ex vivo or in vitro.

[0059] Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors, alphaviral vectors, and the like. Vectors based on tobacco mosaic virus (TMV) Petition 870260076337, dated 07 / 30 / 2026, pp. 106 / 177 Infectious 22 / 59 vectors can be used to produce proteins and have been reported to express Griffithsin and tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphaviral vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof and a therapeutic gene. Further details for modern vector methods for use in gene transfer can be found, for example, in Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17.

[0060] As used herein, “retrovirus-mediated gene transfer” or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred to the host cell by virtue of the virus entering the cell and integrating its genome into the host cell’s genome. The virus may enter the host cell through its normal infection mechanism or be modified so that it binds to a different host cell receptor or surface ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

[0061] Retroviruses carry their genetic information in the form of RNA; however, as soon as the virus infects a cell, the RNA is reverse transcribed into DNA that integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0062] In aspects where gene transfer is mediated by a DNA virus vector, such as an adenovirus (Ad) or a virus Petition 870260076337, dated 07 / 30 / 2026, pages 107 / 177 23 / 59 adeno-associated (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof and a transgene. Adenoviruses (Ads) are a relatively well-characterized group of homogeneous viruses, which includes more than 50 serotypes. Ads do not require integration into the host cell genome. Vectors derived from recombinant Ads, particularly those that reduce the potential for recombination and production of wild-type viruses, have also been constructed. These vectors are commercially available from sources such as Takara Bio USA (Mountain View, CA), Vector Biolabs (Philadelphia, PA), and Creative Biogene (Shirley, NY). Wild-type AAVs have high infectivity and integration specificity into the host cell genome. See, Wold and Toth (2013) Curr. Gene. Ther. 13(6):421-433, Hermonat & Muzyczka (1984) Proc. Natl. Academic. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0063] Vectors containing both a promoter and a cloning site to which a polynucleotide can be operationally bound are well known in the art. These vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and / or transcription in vitro, it may be necessary to remove, add, or alter the 5' and / or 3' untranslated portions of the clones to eliminate potentially inappropriate alternative translation start codons or other sequences that may interfere with or reduce expression at the transcriptional or translational level. Alternatively, consensus ribosome binding sites can be inserted immediately 5' from the start codon to increase expression.

[0064] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or a fragment thereof may be used in the methods disclosed herein. In addition to polynucleotide delivery Petition 870260076337, dated 07 / 30 / 2026, pp. 108 / 177 24 / 59 for a cell or a population of cells, the direct introduction of the proteins described here into the cell or population of cells can be performed using the non-limiting technique of protein transfection; alternatively, culture conditions that can increase the expression and / or promote the activity of the proteins disclosed here are other non-limiting techniques.

[0065] As used herein, the term “viral capsid” or “capsid” refers to the protein coat or outer covering of a viral particle. Capsids function to encapsidize, protect, transport, and release a viral genome within the host cell. Capsids are generally comprised of oligomeric structural protein subunits (“capsid proteins”). As used herein, the term “encapsulated” means embedded within a viral capsid.

[0066] As used herein, the term “helper” in reference to a virus or plasmid refers to a virus or plasmid used to provide the additional components needed for replication and packaging of a viral particle or recombinant viral particle, such as the modified AAV disclosed herein. The components encoded by a helper virus may include any genes needed for virion assembly, capsidation, genome replication, and / or packaging. For example, the helper virus may encode enzymes needed for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0067] As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus. An adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells where certain functions are supplied by a co-infecting helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is perfectly Petition 870260076337, dated 07 / 30 / 2026, pp. 109 / 177 25 / 59 It is expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized after the publication dates of the reviews because it is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, apparently all AAV serotypes exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins such as those expressed in AAV2.The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annulating segments at the terminals that correspond to inverted terminal repeat sequences (ITRs). Similar infectivity patterns also suggest that replication functions in each serotype are under similar regulatory control.

[0068] An “AAV vector” as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by terminal repeat sequences (ITRs) of the AAV. These AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing products of the rep and cap genes.

[0069] An “AAV virion” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an AAV vector with an encapsulated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene that will be supplied to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector”. Thus, the production of the vector particle Petition 870260076337, dated 07 / 30 / 2026, pp. 110 / 177 26 / 59 of AAV necessarily includes the production of the AAV vector, such that a vector is contained within an AAV vector particle.

[0070] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long, including two 145-nucleotide inverted terminal repeats (ITRs). There are several AAV serotypes. The nucleotide sequences of the AAV serotype genomes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 {1983}; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; The AAV-5 genome is provided in GenBank Accession No. AF085716; the complete AAV-6 genome is provided in GenBank Accession No. NC_00 1862; at least parts of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos.AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The AAV rh.74 genome sequence is provided in US Patent 9,434,928, incorporated herein by reference. The in cis sequences that direct viral DNA replication (rep), encapsulation / packaging, and integration into the host cell chromosome are contained within the AAV ITRs. Three AAV promoters (named p5, p9, and p40 due to their relative locations on the map) control the expression of the two internal open reading frames of AAV that encode the rep and cap genes. The two rep promoters (p5 and p9), coupled with differential splicing of the individual AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) originating from the rep gene.Rep proteins possess various enzymatic activities that are ultimately... Petition 870260076337, dated 07 / 30 / 2026, pp. 111 / 177 27 / 59 responsible for viral genome replication. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at position 95 of the AAV genome map. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0071] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. Infection of cultured cells with AAV is non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, allowing for targeting to many different tissues in vivo. In addition, AAV slowly transduces cells that are dividing or not and can persist essentially for the entire lifespan of these cells in the form of a transcriptionally active nuclear episome (extrachromosomal element). The AAV viral progenome is inserted in the form of cloned DNA into plasmids, which makes the construction of recombinant genomes possible.Furthermore, since the signals that direct AAV replication and genome encapsulation are contained within the ITRs of the AAV genome, some or all of the approximately 4.3 kb internal genome (which encodes the replication and capsid structural proteins, rep-cap) can be replaced by foreign DNA. To generate AAV vectors, the rep and cap proteins can be provided in trans. Another significant characteristic of AAV is that it is an extremely stable and friendly virus. It readily withstands the conditions used to inactivate adenoviruses (56° to 65°C for several hours), making refrigerated preservation of AAV less critical. AAV can even be lyophilized. Finally, cells infected with AAV are not resistant to superinfection.

[0072] Several studies have demonstrated protein expression Petition 870260076337, dated 07 / 30 / 2026, page 112 / 177 28 / 59 mediated by recombinant AAV over a long period (> 1.5 years) in muscle. See, Clark et al., Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 8098-8108 (1996). See also, Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, since muscle is highly vascularized, transduction of recombinant AAV resulted in the appearance of transgene products in the systemic circulation after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94: 13921-13926 (1997). In addition, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for the correct glycosylation, folding, and secretion of antibodies, indicating that muscle is capable of stably expressing secreted protein therapeutic agents.The recombinant AAV (rAAV) genomes of the invention comprise a nucleic acid molecule encoding γsarcoglycan (e.g., SEQ ID NO: 1) and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genomes can be from any AAV serotype for which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also considered. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of several AAV serotypes are known in the art.

[0073] The term “Cas9” refers to an endonuclease associated with CRISPR referred to by this name. Non-limiting examples of Cas9s are provided here, for example, Cas9 provided in UniProtKB G3ECR1 (CAS9_STRTR) or Cas9 from Staphylococcus aureus, as well as Cas9 from Petition 870260076337, dated 07 / 30 / 2026, pp. 113 / 177 29 / 59 “dead” nuclease, orthologs, and biological equivalents of each of these. Orthologs include, but are not limited to, Cas9 from Streptococcus pyogenes (“spCas9”); Cas9 from Streptococcus thermophiles, Legionella pneumophilia, Neisseria lactamica, Neisseria meningitides, Francisella novicida; and Cpf1 (which performs cutting functions analogous to Cas9) from various bacterial species including Acidaminococcus spp. and Francisella novicida U112.

[0074] As used herein, the term “CRISPR” refers to a sequence-specific gene manipulation technique that relies on the regularly interspaced short palindromic repeats pathway. CRISPR can be used to perform gene editing and / or gene regulation, as well as to simply target proteins to a specific genomic location. Gene editing refers to a type of genetic engineering in which the nucleotide sequence of a target polynucleotide is altered by introducing deletions, insertions, or base substitutions into the polynucleotide sequence. In some respects, CRISPR-mediated gene editing utilizes non-homologous end linkage (NHEJ) or homologous recombination pathways to perform the edits. Gene regulation refers to increasing or decreasing the production of specific gene products such as protein or RNA.

[0075] The term “gRNA” or “guide RNA” as used herein refers to guide RNA sequences used to target specific genes for correction using the CRISPR technique. Techniques for designing therapeutic donor gRNAs and polynucleotides for target specificity are well known in the art. For example, Doench, J., et al. Nature Biotechnology 2014; 32(12):1262-7, Mohr, S. et al. (2016) FEBS Journal 283: 3232-38 and Graham, D., et al. Genome Biol. 2015; 16: 260. The gRNA comprises or alternatively consists essentially or further consists additionally of a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA); or a polynucleotide comprising CRISPR RNA Petition 870260076337, dated 07 / 30 / 2026, pp. 114 / 177 30 / 59 (crRNA) and in trans activating CRIPSPR RNA (tracrRNA). In some respects, a gRNA is synthetic (Kelley, M. et al. (2016) J of Biotechnology 233 (2016) 74-83). As used herein, a biological equivalent of a gRNA includes, but is not limited to, polynucleotides or targeting molecules that can guide a Cas9 or its equivalent to a specific nucleotide sequence such as a specific region of a cell's genome.

[0076] A “composition” is intended to mean a combination of an active polypeptide, polynucleotide or antibody and another compound or composition, either inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0077] A “pharmaceutical composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with an inert or active carrier such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0078] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, and emulsions, such as an oil-in-water or water-in-oil emulsion, and various types of wetting agents. Compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0079] A “subject” for diagnosis or treatment is a cell or an animal such as a mammal or a human being. A subject is not limited to a specific species and includes non-human animals subjected to diagnosis and treatment and are those subjected to infections or animal models, for example, apes, murines such as rats, mice, chinchillas, canines such as dogs, leporids such as rabbits, livestock, sport animals and companion animals. Human patients are also included within the term. Petition 870260076337, dated 07 / 30 / 2026, pp. 115 / 177 31 / 59

[0080] The term “tissue” is used here to refer to the tissue of a living or dead organism or any tissue derived from or designed to mimic a living or dead organism. Tissue may be healthy, dead, and / or have genetic mutations. Biological tissue may include any individual tissue (e.g., a collection of cells that may be interconnected) or a group of tissues that constitutes an organ or a part or region of an organism’s body. Tissue may comprise a homogeneous cellular material or may be a composite structure such as that found in body regions including the thorax, which, for example, may include lung tissue, skeletal tissue, and / or muscle tissue.Examples of tissues include, but are not limited to, those derived from the liver, lungs, thyroid, skin, pancreas, blood vessels, bladder, kidneys, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart, and intestines, including any combination thereof.

[0081] As used herein, “treating” or “treatment” of a disease in an individual refers to (1) preventing the occurrence of symptoms or disease in an individual who is predisposed or does not yet exhibit symptoms of the disease; (2) inhibiting the disease or halting its development; or (3) improving or causing regression of the disease or symptoms of the disease. As understood in the art, “treatment” is an approach to achieving beneficial or desired results, including clinical results.For the purposes of this technology, beneficial or desired results may include, but are not limited to, relief or improvement of one or more symptoms, reduction in the extent of a condition (including a disease), stabilization (i.e., non-aggravation) of the state of a condition (including a disease), slowing or reduction in the rate of the condition (including a disease), progression, improvement or palliation of the condition (including a disease), states and remission (whether partial or total), whether detectable or undetectable. Petition 870260076337, dated 07 / 30 / 2026, pp. 116 / 177 32 / 59 Methods of Conducting Dissemination Modified Viral Capsids and Preparation Methods

[0082] AAV vector delivery currently relies on serotype selection for tissue targeting based on the virus's natural tropism or through direct injection into target tissues. If systemic delivery is required to achieve maximum therapeutic benefit, then serotype selection is the only available option for tissue targeting combined with tissue-specific promoters. As described herein, the Applicant has approximately identified the specific amino acids responsible for receptor binding and entry, thus reducing the need for elaborate mutagenesis studies to identify critical amino acids in AAVrh74 [7-11] (FIG. 1). With the critical regions for non-targeting to the liver identified, the Applicant has developed modified capsids that enhance muscle-specific vector transduction.The positive impact of combining reduced vector loss in the liver with specific delivery of the gene of interest to muscle cells will greatly increase the chances of finding the threshold for clinical efficacy in the systemic treatment of neuromuscular diseases.

[0083] This disclosure relates to modified capsid proteins, isolated polynucleotides, methods for preparing modified capsid proteins, recombinant viral particles, and recombinant expression systems for producing modified viral particles. One aspect of the disclosure relates to a modified viral capsid protein comprising or alternatively consisting essentially of, or further consisting of, a viral capsid protein modified by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally of Petition 870260076337, dated 07 / 30 / 2026, pp. 117 / 177 33 / 59 AAVrh74. In additional embodiments, the modification comprises the substitution of isoleucine with asparagine at amino acid position 502 in VP1 of AAVrh74 or an equivalent modification. In some embodiments, the modification comprises the substitution of tryptophan with arginine at amino acid position 505 in VP1 of AAVrh74. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that is likely to alter normal rh74 receptor binding. The modified capsids can be incorporated into other viral delivery systems.

[0084] In a further aspect, the modified viral particles also comprise a transgene for gene modification and / or a CRISPR system for gene modification.

[0085] Also disclosed here is an isolated polynucleotide encoding a modified viral capsid protein comprising or alternatively consisting essentially of or further consisting of a modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally of AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in the VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification is directed towards a Petition 870260076337, dated 07 / 30 / 2026, pp. 118 / 177 34 / 59 receptor found primarily in satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter normal rh74 receptor binding.

[0086] A recombinant viral particle is disclosed herein comprising or alternatively consisting essentially of, or further consisting of, a modified capsid protein comprising or alternatively consisting essentially of, or further consisting of, a modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally from AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In further embodiments, this viral particle increases the effectiveness of treatment delivery by approximately 3 to 56 times to diseased or dysfunctional tissue compared to AAVrh74.In some embodiments, the modification involves replacing tryptophan with arginine at amino acid 505 in the VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In additional embodiments, the diseased or dysfunctional tissue is cardiac tissue. Also in additional embodiments, this viral particle increases the efficacy of treatment delivery by approximately 50 times or more for cardiac tissue compared to AAVrh74. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally. Petition 870260076337, dated 07 / 30 / 2026, pp. 119 / 177 35 / 59 muscle stem cells. In some modalities, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some modalities, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter the binding of the normal rh74 receptor. In some modalities, the viral particle comprises an efficient amount of a suitable treatment for diseased or dysfunctional tissue. In some modalities, the treatment is gene editing based on CRISPR / Cas9.

[0087] The modified virus, for example, AAV, can be packaged using a viral packaging system such as a retrovirus, adenovirus, herpesvirus, or baculovirus packaging system. In some embodiments, packaging is achieved through the use of a helper virus or a helper plasmid and a cell line. The helper virus or helper plasmid contains sequence elements that facilitate the delivery of genetic material into cells. Alternatively, the helper plasmid or a polynucleotide comprising the helper plasmid is stably incorporated into the genome of a packaging cell line, so that the packaging cell line does not require further transfection with a helper plasmid.

[0088] A helper plasmid may comprise, for example, at least one viral helper DNA sequence derived from a replication-incompetent viral genome that encodes in trans all virion proteins necessary for packaging a replication-incompetent AAV and for producing virion proteins capable of packaging the replication-incompetent AAV at a titer algo, without producing replication-competent AAV. The viral DNA sequence lacks the region encoding the enhancer and / or the native 5' LTR promoter of the virus and does not Petition 870260076337, dated 07 / 30 / 2026, pp. 120 / 177 36 / 59 possesses both the psi function sequence responsible for helper genome packaging and the 3' LTR, but encodes a foreign polyadenylation site, for example, the SV40 polyadenylation site, and a foreign enhancer and / or promoter that directs efficient transcription in a cell type where virus production is desired. The virus is a leukemia virus such as Moloney Murine Leukemia Virus (MMLV), Human Immunodeficiency Virus (HIV), or Gibbon Monkey Leukemia Virus (GALV). The foreign enhancer and promoter may be the immediate initial enhancer and promoter (IE) of human cytomegalovirus (HCMV), the enhancer and promoter (U3 region) of Moloney Murine Sarcoma Virus (MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3 region of Spleen Focus-Forming Virus (SFFV), or the HCMV IE enhancer linked to the native Moloney Murine Leukemia Virus (MMLV) promoter.The helper plasmid may consist of two retroviral helper DNA sequences encoded by the plasmid based on expression vectors, for example, when a first helper sequence contains the cDNA encoding the gag and pol proteins of MMLV or ecotropic GALV and a second helper sequence contains the cDNA encoding the env protein.The Env gene, which determines the host range, can be derived from genes encoding the xenotropic, amphoteric, ecotropic, polytropic (focus-forming in minks) or 10A1 murine leukemia virus env proteins, or the Gibbon Monkey Leukemia Virus (GALV) env protein, the Human Immunodeficiency Virus (IHV) env protein (gp160), the Vesicular Stomatitis Virus (VSV) G protein, the type I and type II env gene products of Human T-cell Leukemia Virus (HTLV), the chimeric envelope gene derived from combinations of one or more of the aforementioned env genes, or chimeric envelope genes encoding the cytoplasm and transmembrane of the aforementioned env gene products and a monoclonal antibody directed against a specific surface molecule on a desired target cell. Petition 870260076337, dated 07 / 30 / 2026, pp. 121 / 177 37 / 59

[0089] In the packaging process, helper plasmids and plasmids encoding viral proteins are transiently co-transfected into a first population of mammalian cells capable of producing viruses, such as human embryonic kidney cells, for example, 293 cells (ATCC No. CRL1573, ATCC, Rockville, Md.) to produce supernatants containing recombinant retroviruses at high titers. In another method of the invention, this first population of transiently transfected cells is then co-cultured with mammalian target cells, for example, human lymphocytes, to transduce the target cells with the foreign gene with high efficiency. Compositions

[0090] The invention also provides a composition or kit comprising any one or more of the viral vectors, isolated cells, packaging system, viral particles described herein and a carrier. In one aspect, the carrier is a pharmaceutically acceptable carrier. These compositions can be used therapeutically as described herein and can be used in combination with other known therapies. Methods of Administering Modified Viral Particles

[0091] A non-human transgenic animal comprising a modified viral capsid protein altered by amino acid modification or insertion between 1 and 7 amino acids is provided herein. In some embodiments, the viral capsid protein is a VP1, optionally from AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification Petition 870260076337, dated 07 / 30 / 2026, pp. 122 / 177 38 / 59 involves the substitution of tryptophan for arginine at amino acid 505 in VP1 of AAVrh74. In some ways, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some ways, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter normal rh74 receptor binding.

[0092] A gene editing method is disclosed herein comprising, essentially consisting of, or further consisting of, contacting a cell with a recombinant viral particle comprising or alternatively essentially consisting of a modified capsid wherein the modified capsid comprises or essentially consists of a modified viral capsid protein comprising or alternatively essentially consisting of, or further consisting of, the modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally from AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In further embodiments, the modification comprises the substitution of isoleucine by asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification.In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, Petition 870260076337, dated 07 / 30 / 2026, pp. 123 / 177 39 / 59 The modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter the binding of the normal rh74 receptor. In some embodiments, contact occurs in vivo or in vitro. In some embodiments, the viral particle comprises one or more polynucleotides, and at least one of the polynucleotides comprises or essentially consists of, or additionally consists of, a polynucleotide encoding a guide RNA (gRNA).In some embodiments, at least one of the polynucleotides comprises, or alternatively consists essentially of, or additionally consists of, a therapeutic polypeptide. In some embodiments, the viral particle comprises a Cas9 or an equivalent thereof.

[0093] A gene editing method is further disclosed herein for an individual in need thereof, comprising administering to the individual an efficient amount of recombinant viral particle comprising or alternatively consisting essentially of a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or further consisting further of, a modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally of AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine by asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In some aspects, other amino acids in the peptide are Petition 870260076337, dated 07 / 30 / 2026, pp. 124 / 177 40 / 59 modified, but this substitution is maintained. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter normal rh74 receptor binding.In some embodiments, the viral particle comprises one or more polynucleotides, and at least one of the polynucleotides comprises, essentially consists of, or additionally consists of a polynucleotide encoding a guide RNA (gRNA). In some embodiments, at least one of the polynucleotides comprises, essentially consists of, or additionally consists of a therapeutic polypeptide. In some embodiments, the viral particle comprises a Cas9 or an equivalent thereof.

[0094] A non-human transgenic animal is provided herein comprising a recombinant viral capsid particle comprising or alternatively consisting essentially of, or further consisting additionally of, a recombinant viral particle comprising or alternatively consisting essentially of, a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or further consisting additionally of, a modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, Petition 870260076337, dated 07 / 30 / 2026, pp. 125 / 177 41 / 59 optionally of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In additional embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in VP1 of AAVrh74 or an equivalent modification. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74.In some respects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter the binding of the normal rh74 receptor.

[0095] In some respects, the polynucleotide encoding gRNA comprises or alternatively consists essentially of or also consists additionally of a fusion polynucleotide comprising CRISPR RNA (crRNA) and CRISPR RNA with in trans activation (tracrRNA); or a polynucleotide comprising CRISPR RNA (crRNA) and CRISPR RNA with in trans activation (tracrRNA). In some respects, gRNA is specific to a region of DNA that requires gene editing in the individual or in the cell that requires it.

[0096] In some respects, the recombinant viral particle also comprises a therapeutic polynucleotide. A therapeutic polynucleotide is any polypeptide that can be used to target a DNA sequence that needs to be edited, provide a repair template for a DNA sequence that needs to be edited, or Petition 870260076337, dated 07 / 30 / 2026, pages 126 / 177 42 / 59 provide a replacement for a DNA sequence that needs to be edited. In additional aspects, the therapeutic polypeptide comprises a wild-type sequence of a gene that needs to be edited in the individual or cell that requires it.

[0097] Further aspects relate to methods of treatment of an individual who has a disease, disorder or condition which includes the administration of the modified AAV disclosed herein to the individual. In some respects, the disease, disorder, or condition is selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's disease, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler-Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesterolemia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysaccharidosis (I-IX), hepatitis B, and hepatitis C. In one respect, hemophilia is characterized by a deficiency of one or more of factor VIII or factor IX.In some respects, muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0098] In some respects, the guide RNA and / or therapeutic polynucleotide is designed and / or selected to treat a disease, disorder or condition selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's disease, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler-Najjar syndrome, Petition 870260076337, dated 07 / 30 / 2026, pages 127 / 177 43 / 59 Pompe disease, X-linked retinoschisis, homozygous familial hypercholesterolemia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysaccharidosis (I-IX), hepatitis B, and hepatitis C. In one aspect, hemophilia is characterized by a deficiency of one or more factors VIII or IX. In some aspects, muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0099] In some respects, the guide RNA and / or therapeutic polynucleotide is designed and / or selected to treat or repair a selected gene from the Factor VIII (F8, NM_000132, NM_019863), Factor IX (F9, NM_000133, NM_001313913), dystrophin (DMD, NM_000109, NM_004006, NM_004007, NM_004009, NM_004010), dysferlin (DYSF, NM_001130455, NM_001130976, NM_001130977, NM_001130978, NM_001130979), emerin (EMD, NM_000117), lamina A / C (LMNA, NM_001257374, NM_001282624, NM_001282625, NM_001282626, NM_005572), double homeobox 4 (DUX4, NM_001205218, NM_001278056, NM_001293798, NM_001306068), myotonin-protein kinase (MDPK, NM_001081560, NM_001081562, NM_001081563, NM_001288764, NM_001288765), cellular nucleic acid binding protein (CNBP, NM_003418, NM_001127192, NM_001127193, NM_001127194, NM_001127195), polyadenylate binding protein-2 (PABP-2, NM_004643), Alpha-1-antitrypsin, superoxide dismutase (SOD1, NM_000454), alsin (ALS2, NM_001135745, NM_020919), senataxin helicase (SETX, NM_015046), spatacsin (SPG11, NM_001160227, NM_025137), RNA-binding FUS / TLS protein (FUS, NM_001010850, NM_001170634, NM_001170937, NM_004960), vesicle-associated membrane protein B / C (VAPB, NM_001195677, NM_004738), angiogenin (ANG, NM_001145, NM_001097577), TAR DNA-binding protein 43 (TARDBP, Petition 870260076337, dated 07 / 30 / 2026, pp. 128 / 177 44 / 59 NM_007375), Polyphosphoinositide phosphatase (FIG4, NM_014845), optineurin (OPTN, NM_001008211, NM_001008212, NM_001008213, NM_021980), ataxin-2 (ATXN2, NP_001297050, NP_001297052, NP_002964), valosin-containing protein (VCP, NM_007126), ubiquilin-2 (UBQLN2, NM_013444), sigma-1 receptor (SIGMAR1, NM_001282205, NM_001282206, NM_001282207, NM_001282208, NM_001282209), Charged multivesicular body protein 2b (CHMP2B, NM_001244644, NM_014043), profilin-1 (PFN1, NM_005022), erbB-4 receptor tyrosine protein kinase (ERBB4, NM_001042599, NM_005235), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1, NM_002136, NM_031157), matrin-3 (MATR3, NM_199189, NM_001194954, NM_001194955, NM_001194956, NM_001282278), tubulin alpha-4A chain (TUBA4A, NM_001278552, NM_006000), chromosome 9 open reading frame 72 (C9orf72, NM_145005, NM_001256054, NM_018325), CHCD10, SQSTM1 (NM_001142298), TBK1, apolipoprotein E (NM_001302691, NM_000041, NM_001302688, NM_001302689, NM_001302690), SMN1 (NM_000344), SMN2 (NM_017411, NM_022875, NM_022876, NM_022877), CTFR (NM_000492), beta globin HBB PDB, CHM, alpha-synuclein (SNCA, NM_000345), parkin (PRKN, NM_004562), leucine-rich repeat kinase 2 (LRRK2 or dardarin, NM_198578), PTEN-induced putative kinase 1 (PINK1, NM_032409), DJ-1 (NM_001123377), acid maltase (NM_000152), UDP-glucuronosyltransferase 1 (NM_000463), PPT-1 (NM_000310) or ATP13A2 (NM_001141973).

[00100] Additional aspects of the invention relate to compositions comprising a carrier and the modified virus described herein. Briefly, the pharmaceutical compositions of the present invention may comprise the modified viral particle described herein, in combination with one or more pharmaceutically acceptable carriers, diluents or excipients. These compositions may comprise buffers such as neutral buffered saline solution, saline solution Petition 870260076337, dated 07 / 30 / 2026, pp. 129 / 177 45 / 59 phosphate-buffered and similar; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure may be formulated for oral, intravenous, topical, enteral and / or parenteral administration. In certain embodiments, the compositions of this disclosure are formulated for intravenous administration.

[00101] Experts in the art consider that gRNAs can be generated for targeted specificity to treat a specific gene, optionally a gene associated with a disease, disorder, or condition. Thus, in combination with Cas9, guide RNAs facilitate the targeted specificity of the CRISPR / Cas9 system. Additional aspects such as promoter selection, as discussed earlier, can provide further mechanisms to achieve targeted specificity – for example, selecting a promoter for the guide RNA that encodes a polynucleotide that facilitates expression in a particular organ or tissue. Consequently, the selection of gRNAs suitable for the particular disease, disorder, or condition is considered here.

[00102] The administration of the modified AAV or compositions may be carried out in a single dose, continuously or intermittently throughout the course of treatment. Administration may occur via any suitable route of administration, including, but not limited to: intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, subretinal, intravitreal, intra-articular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal and inhalation.

[00103] The methods for determining the most efficient means and dosage of administration are known to experts in the technique and vary with the composition used for therapy, the purpose of the therapy, and the individual to be treated. Single or multiple administrations. Petition 870260076337, dated 07 / 30 / 2026, pp. 130 / 177 46 / 59 can be performed with the dose level and pattern being selected by the attending physician. It is noted that the dosage may be impacted by the route of administration. Dosage formulations and methods of administration of suitable agents are known in the art. Non-limiting examples of such suitable dosages may range from 1E+9 vector genomes to 1E+17 vector genomes per administration.

[00104] In a further aspect, the modified viral particle and compositions of the invention may be administered in combination with other treatments, for example, approved treatments suitable for the particular disease, disorder or condition. A non-limiting example includes the treatment of muscular dystrophy with a combination of the modified viral particle and one or more steroids. Treatment success can be determined by monitoring clinical or subclinical evidence of gene modification, which is determined by the attending physician.

[00105] This administration of the modified viral particle or compositions of the invention can be performed to generate an animal model of the desired disease, disorder or condition for experimental and verification assays. Modified AAV Capsids and Particles

[00106] This disclosure also provides a specific embodiment, for example, a modified adeno-associated virus (AAV) comprising a recombinant viral particle comprising or alternatively consisting essentially of a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or further consisting of, a modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally from AAVrh74. In Petition 870260076337, dated 07 / 30 / 2026, pp. 131 / 177 In some embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in VP1 of AAVrh74 or an equivalent modification. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid position 505 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells. In some embodiments, the modification is an insertion of the YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in VP1 of AAVrh74. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained.In some embodiments, this peptide has a high affinity for Alpha 7 beta 1 integrin and / or is positioned in a region that will likely alter the binding of the normal rh74 receptor.

[00107] Adeno-associated virus (AAV) vectors are replication-defective vectors that are engineered to deliver genetic material to cells. These are non-enveloped viruses that, in their vector form, possess only the inverted terminal repeats (ITRs) of the original virus. The structural and enzymatic proteins of the AAV are delivered “in trans” by additional plasmids and are transfected together into a cell to generate engineered particles for gene delivery. AAVs have been widely used for gene therapy and, more specifically, with CRISPR / Cas9 systems – due to their safety and efficiency. The AAV efficiently infects a variety of cells, and during the infection process, the capsid binds and enters the nucleus where the vector genome is delivered.

[00108] The AAV structural particle is composed of 60 protein molecules consisting of VP1, VP2, and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins, and 50 VP3 proteins. Petition 870260076337, dated 07 / 30 / 2026, pp. 132 / 177 48 / 59 arranged in an icosahedral structure. AAV2 particles have been shown to support the insertion of peptides and proteins at various sites within the capsid structure. The ability to introduce single peptides into the capsid has led to the development of AAV particles with altered tropism, allowing the virus to bind to and infect cells and tissues that are normally refractory to infection. In addition, large peptides and even functional proteins have been introduced into the capsid of AAV2 vectors with varying degrees of success. A functional green fluorescent protein (GFP, 30 kD MW) containing the AAV capsid was generated and produced infectious virus that was used to track infections in cells.

[00109] One of the limitations of AAV vectors for gene delivery is the limited size of the genetic insert that can be efficiently packaged within the particles. For example, the wild-type AAV2 genome size is 4679 single-stranded DNA bases. Packaging even one of the new smaller Cas9 variants (Staphylococcus aureus Cas9, SaCas9, 130 kD PM) requires approximately 3255 bp for the coding region alone. Adding a ubiquitous or tissue-specific promoter to the construct can add another 500-800 bp. Including another 500 bp for a poly-A addition sequence and the ITRs brings the vector close to the packaging capacity of an AAV particle. To achieve functional CRISPR / Cas9 gene correction, a guide RNA (gRNA) with the target sequence must also be included. To have this RNA expressed, a minimal polIII promoter and termination sequence are additionally required.Together, these elements are too large to be combined within an efficiently packaged AAV vector. One could choose to package the Cas9 construct and guide RNA expression cassettes into separate vectors, but for these to be functional, both viruses must infect the same target cells.

[00110] Additional aspects of disclosure relate to a system Petition 870260076337, dated 07 / 30 / 2026, pp. 133 / 177 49 / 59 recombinant expression for the production of such modified AAV. In some embodiments, the recombinant expression system comprises a large number of plasmids; the large number encoding all the AAV virus proteins - VP1, VP2, and VP3. In some embodiments, each viral protein is encoded on a different plasmid. In some embodiments, one or more viral proteins are encoded on the same plasmid. In some embodiments, at least one viral protein is encoded in the form of a Cas9 fusion protein.

[00111] Consequently, the embodiments disclosed herein refer to a recombinant expression system for the production of a modified AAV comprising a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or additionally consisting of, a modified viral capsid protein altered by the modification or insertion of amino acids between 1 and 7 amino acids. In some embodiments, the viral capsid protein is a VP1, optionally of AAVrh74. In further embodiments, the modification comprises the substitution of isoleucine for asparagine at amino acid position 502 in the VP1 of AAVrh74 or an equivalent modification. In some aspects, other amino acids in the peptide are modified, but this substitution is maintained. In some embodiments, the modification comprises the substitution of tryptophan for arginine at amino acid 505 in the VP1 of AAVrh74.In some respects, other amino acids in the peptide are modified, but this substitution is maintained. In some modalities, the modification targets a receptor found primarily on satellite cells, optionally muscle stem cells.

[00112] Some aspects relate to methods of producing modified AAVs using the recombinant expression system disclosed here.

[00113] Further aspects relate to methods of treating an individual who has an illness, a disorder or a Petition 870260076337, dated 07 / 30 / 2026, pp. 134 / 177 50 / 59 condition which includes the administration of the modified AAV disclosed herein to the individual. In some embodiments, the disease, disorder or condition is selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's disease, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler-Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesterolemia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysaccharidosis (I-IX), hepatitis B and hepatitis C. In some embodiments, hemophilia is characterized by a deficiency of one or more factors VIII or IX.In some forms, muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. Examples

[00114] The following examples are non-limiting and illustrative of procedures that may be used in various cases to effect disclosure. Additionally, all references disclosed below are incorporated herein by reference in their entirety.

[00115] AAVrh74 is an AAV serotype identified by researchers at the NCH that has been used extensively in clinical trials for the treatment of neuromuscular diseases. It efficiently infects human cardiac and skeletal muscles, and there is a low prevalence of pre-existing neutralizing antibodies in the human population. Example 1 - Determine the optimal modified serotype for maximum a Petition 870260076337, dated 07 / 30 / 2026, pp. 135 / 177 51 / 59 maximum biodistribution of myoblasts and satellite cells through vascular supply.

[00116] Muscle satellite cells are 60 times more abundant than muscle fiber cells in skeletal muscle [2]. They are a self-renewing stem cell population that facilitates the long-term regeneration capacity of skeletal muscle

[12] . Recently, several markers that can be separated by FACS have been identified for the isolation of muscle stem cells

[13] (FIG, 2). Alpha 7 beta 1 integrin has been identified as a marker of positive selection for muscle stem cells and is also the primary laminin receptor on skeletal myoblasts and adult myofibers. Expression studies have also shown that alpha 7 beta 1 integrin is abundantly expressed on cardiac and skeletal muscle tissue and only low levels are found in liver tissue. The Applicant modified the capsid of AAVrh74 to express a peptide that has been shown to bind with high affinity to the alpha 7 beta 1 integrin protein.The Applicant positioned the peptide in a region that likely alters the binding motif of the normal AAVrh74 receptor in order to utilize α7β1-integrin as the binding receptor. Two additional mutations in other AAVrh74 amino acids were also identified that are thought to be necessary for liver-specific binding and entry and have been shown to be capable of greater general and / or muscle-specific delivery of reporter genes after intravenous injection of the vector (FIGS. 3-5).

[00117] C57BL / 6J mice received intravenous injection with newly engineered modified capsid viruses or control AAVrh74 (AAVmut4, AAVmut5 and AAVYIG or YIGSR591) expressing the same luciferase-EYFP fusion protein as previously demonstrated and after 3 weeks, the mice were sacrificed by perfusion with PBS / paraformaldehyde followed by staining. Petition 870260076337, dated 07 / 30 / 2026, pages 136 / 177 52 / 59 Immunohistochemistry of muscles and sectioned organs was used to determine the specific cell populations that are transduced. Initially, mice were imaged weekly for luciferase expression using the IVIS imaging system to confirm gene expression. Three weeks after injection, the mice underwent perfusion, tissues were collected and fixed in paraformaldehyde, followed by sectioning and staining for GFP expression by immunofluorescence and counterstaining for cell type-specific antigen expression for identification. This approach enabled the Applicant to identify the specific cell populations transduced by different capsid mutant vectors to determine which is likely to have the greatest benefit for the target muscle disease.

[00118] AAVrh74 is the current standard for high levels of gene expression in skeletal and cardiac muscle and was used as the basic structure for capsid modifications. Vector biodistribution after intravenous injection into CD-1 mice was determined by qPCR three weeks after injection of control and three viruses with modified capsids (FIG. 3). The mut4 vector exhibited a higher overall vector biodistribution with significant increases in the quadriceps, diaphragm, and cardiac muscle. Whereas mut5 and YIG or YIGSR591 exhibited significantly higher vector transduction from the heart compared to AAVrh74. TABLE 1 Tissue | Amount of Mut4 change relative to AAVrh74 | Blood | 18 | Brain | 8 | Lung | 5 | Quadrilateral | 10 Petition 870260076337, dated 07 / 30 / 2026, pages 137 / 177 53 / 59 Diaphragm 17 Heart 11 Spleen 3 Kidney 8 Liver 56

[00119] FIGS. 4 and 5 show higher in vivo expression of the luciferase gene provided by mut4 and YIG or YIGSR591 compared to the parental AAVrh74. FIG. 4 shows the increased photon counts in the head or lower torso region in parentheses to show the efficiency of the mut4 virus in gene transduction and expression across a wide variety of tissues.

[00120] Intracrossed C57BL / 6J mice (6 per virus group, 3 males and 3 females) are used to confirm the previous biodistribution profile of CD-1 mice crossed with others. The 4 viruses are prepared and ready for injection into the mice upon arrival. Equivalents

[00121] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by a person skilled in the art to which this technology pertains.

[00122] The present technology described herein by way of illustration may be properly practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. should be read expansively and without limitation. Additionally, the terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of these terms and expressions to exclude any equivalents of the features. Petition 870260076337, dated 07 / 30 / 2026, pp. 138 / 177 54 / 59 shown and described or parts thereof, but it is recognized that various modifications are possible within the scope of this claimed technology.

[00123] Thus, it should be understood that the materials, methods and examples provided here are representative of preferred aspects, are examples and are not intended to be regarded as limitations of the scope of the present technology.

[00124] The present technology has been described broadly and generically herein. Each of the species and more limited subgeneric groups that fall within the generic disclosure is also part of the present technology. This includes the generic description of the present technology with a negative caveat or limitation that removes any subject matter of the genus objective, regardless of whether or not the cut material is specifically cited herein.

[00125] In addition, when the features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also described here in terms of any individual member or subgroup of members of the Markush group.

[00126] All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, this Descriptive Report, including the definitions, shall govern.

[00127] Other aspects are presented within the Claims below. References 1. Fang, H., et al., Comparison of adeno-associated virus serotypes and delivery methods for cardiac gene transfer. Hum Gene Ther Methods, Petition 870260076337, dated 07 / 30 / 2026, pp. 139 / 177 55 / 59 2012. 23(4): p. 234-41. 2. Bianconi, E., et al., An estimation of the number of cells in the human body. Ann Hum Biol, 2013. 40(6): p. 463-71. 3. Tran, T., et al., Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity. FASEB J, 2013. 27(10): p. 3991-4003. 4. Mori, S., et al., Biodistribution of a low dose of intravenously administered AAV-2, 10, and 11 vectors to cynomolgus monkeys. Jpn J Infect Dis, 2006. 59(5): p. 285-93. 5. Grimm, D., et al., In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol, 2008. 82(12): p. 5887-911. 6. Asokan, A., et al., Reengineering a receptor footprint of adeno- associated virus enables selective and systemic gene transfer to muscle. Nat Biotechnol, 2010. 28(1): p. 79-82. 7. Pulicherla, N., et al., Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer. Mol Ther, 2011. 19(6): p. 1070-8. 8. DiMattia, M.A., et al., Structural insight into the unique properties of adeno-associated virus serotype 9. J Virol, 2012. 86(12): p. 6947-58. 9. Li, C., et al., Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles. J Virol, 2012. 86(15): p. 7752-9. 10. Raupp, C., et al., The threefold protrusions of adeno-associated virus type 8 are involved in cell surface targeting as well as postattachment processing. J Virol, 2012. 86(17): p. 9396-408. Petição 870260076337, de 30 / 07 / 2026, pág. 140 / 177 56 / 59 11. Govindasamy, L., et al., Structural insights into adeno-associated virus serotype 5. J Virol, 2013. 87(20): p. 11187-99. 12. Bentzinger, C.F., et al., Cellular dynamics in the muscle satellite cell niche. EMBO Rep, 2013. 14(12): p. 1062-72. 13. Wang, Y.X., N.A. Dumont, and M.A. Rudnicki, Muscle stem cells at a glance. J Cell Sci, 2014. 127(21): p. 4543-8. 14. Loiler, S.A., et al., Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver. Gene Ther, 2003. 10(18): p. 1551-8. Sequências SEQ ID NO:1 NM_000231.2 Sarcoglicana gama (SGCG) de Homo sapiens, mRNA GAAACTCGTGAGAGCCCTTTCTCCAGGGACAGTTGCTGAAGCTTCATCC TTTGCTCTCATTCTGTAAGTCATAGAAAAGTTTGAAACATTCTGTCTGTGG TAGAGCTCGGGCCAGCTGTAGTTCATTCGCCAGTGTGCTTTTCTTAATAT CTAAGATGGTGCGTGAGCAGTACACTACAGCCACAGAAGGCATCTGCAT AGAGAGGCCAGAGAATCAGTATGTCTACAAAATTGGCATTTATGGCTGGA GAAAGCGCTGTCTCTACTTGTTTGTTCTTCTTTTACTCATCATCCTCGTTG TGAATTTAGCTCTTACAATTTGGATTCTTAAAGTGATGTGGTTTTCTCCAG CAGGAATGGGCCACTTGTGTGTAACAAAAGATGGACTGCGCTTGGAAGG GGAATCAGAATTTTTATTCCCATTGTATGCCAAAGAAATACACTCCAGAGT GGACTCATCTCTGCTTCTACAATCAACCCAGAATGTGACTGTAAATGCGC GCAACTCAGAAGGGGAGGTCACAGGCAGGTTAAAAGTCGGTCCCAAAAT GGTAGAAGTCCAGAATCAACAGTTTCAGATCAACTCCAACGACGGCAAG CCACTATTTACTGTAGATGAGAAGGAAGTTGTGGTTGGTACAGATAAACT TCGAGTAACTGGGCCTGAAGGGGCTCTTTTTGAACATTCAGTGGAGACA CCCCTTGTCAGAGCCGACCCGTTTCAAGACCTTAGATTAGAATCCCCCA CTCGGAGTCTAAGCATGGATGCCCCAAGGGGTGTGCATATTCAAGCTCA Petição 870260076337, de 30 / 07 / 2026, pág. 141 / 177 57 / 59 CGCTGGGAAAATTGAGGCGCTTTCTCAAATGGATATTCTTTTTCATAGTA GTGATGGAATGCTTGTGCTTGATGCTGAAACTGTGTGCTTACCCAAGCTG GTGCAGGGGACGTGGGGTCCCTCTGGCAGCTCACAGAGCCTCTACGAA ATCTGTGTGTGTCCAGATGGGAAGCTGTACCTGTCTGTGGCCGGTGTGA GCACCACGTGCCAGGAGCACAGCCACATCTGCCTCTGAGCTGCCTGCG TCCTCTCGGTGAGCTGTGCAGTGCCGGCCCCAGATCCTCACACCCAGG GAGCAGCTGCACATCGTGAAAGACTGAGGCAGCGTGGATGGGAAGTAA ACGCTTCCAGAGGAACTCAGAAAAAATTATGTGCCAGTGAAAGTGTTTGG ACAAAAACTACATGATCTCAAAATGCACGTGGATGTGAGACACAAAAGTT GACAAAATGGAAAAGCAATGTGTTTTTCCACTGGATTAATTTTCACCGGA ACAATTGCGAATTCTCTCTGCCTCGCCTCCCCCTATCTTGTCCGTGTGGG CACACACTGAGTGTTGAGTTGCCGTGTGGAGTTAATGTATGACGCTCCA CTGTGGATATCTAATGCCCTGTTGAGAGTAGCCTTGCTCAGTACTAAAAT GCCCCAAAGTTCTATACAGCATTTCCTTTATAGCATTCAAACCTCACATCC TCCCTTCAGTTTAATGCAAGTAAGTCAGGTTTCACAAGAAAATTTTCAAGT TTTGAAGGGAATTTGAGGTTGATCTGGTTTTCAAGATGTAGTTAAAGGAA TAAATCACTCAAAATTAAACTTTCTGTATATAGTCAATAAGCAATAAAAACC TCATTTTTCAGAGTTAAAAAA Sequência de nucleotídeos do gene do capsídeo do AAV rh74 do tipo selvagem (SEQ ID NO: 3): atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtggg acctgaaacctggagccccgaaacccaaagccaaccagcaaaagcaggacaacggccggggtctg gtgcttcctggctacaagtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggc ggacgcagcggccctcgagcacgacaaggcctacgaccagcagctccaagcgggtgacaatccgtac ctgcggtataatcacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaac ctcgggcgcgcagtcttccaggccaaaaagcgggttctcgaacctctgggcctggttgaatcgccggtt aagacggctcctggaaagaagagaccggtagagccatcaccccagcgctctccagactcctctacgg gcatcggcaagaaaggccagcagcccgcaaaaaagagactcaattttgggcagactggcgactcag agtcagtccccgaccctcaaccaatcggagaaccaccagcaggcccctctggtctgggatctggtaca atggctgcaggcggtggcgctccaatggcagacaataacgaaggcgccgacggagtgggtagttcctc Petição 870260076337, de 30 / 07 / 2026, pág. 142 / 177 58 / 59 aggaaattggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgcacctggg ccctgcccacctacaacaaccacctctacaagcaaatctccaacgggacctcgggaggaagcaccaa cgacaacacctacttcggctacagcaccccctgggggtattttgacttcaacagattccactgccacttt tcaccacgtgactggcagcgactcatcaacaacaactggggattccggcccaagaggctcaacttcaa gctcttcaacatccaagtcaaggaggtcacgcagaatgaaggcaccaagaccatcgccaataacctt accagcacgattcaggtctttacggactcggaataccagctcccgtacgtgctcggctcggcgcaccag ggctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatg gcagtcaggctgtgggccggtcgtccttctactgcctggagtactttccttctcaaatgctgagaacgggc aacaactttgaattcagctacaacttcgaggacgtgcccttccacagcagctacgcgcacagccagag cctggaccggctgatgaaccctctcatcgaccagtacttgtactacctgtcccggactcaaagcacggg cggtactgcaggaactcagcagttgctattttctcaggccgggcctaacaacatgtcggctcaggccaa gaactggctacccggtccctgctaccggcagcaacgcgtctccacgacactgtcgcagaacaacaac agcaactttgcctggacgggtgccaccaagtatcatctgaatggcagagactctctggtgaatcctggc gttgccatggctacccacaaggacgacgaagagcgattttttccatccagcggagtcttaatgtttgggaaacagggagctggaaagacaacgtggactatagcagcgtgatgctaaccagcgaggaagaaataa agaccaccaacccagtggccacagaacagcggcgtggcgataacctgcaacagcaaaacg ccgctcctattgtaggggccgtcaatagtcaaggagccttacctggcatgggcggcgcgc tgtacctgcagggtcccatctgggccaagattcctcatacggacggcaactttcatccctcgccgctgat gggaggctttggactgaagcatccgcctcgatcctgattaaaaacacacctgttcccgcggatc tccgaccaccttcaatcaggccaagctggctctctcgcgcgcgcgcgcg ggagatcgagtgggagctgcagaaggagaacagcaaacgctggaacccagagattcagtacacttc aactactacaaatctacaaatgtggactttgctgtcaatactgagggttactccgagcctcgcccca ttggcacccgttacctcaccgtaatctgtaa Amino acid sequence of wild-type rh74 AAV (SEQ ID NO: 4): MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLP GYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHAD AEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKRPKRPKRPKEPK PSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSG LGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTR TWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHFSPSP Petition 870260076337, of 30 / 07 / 2026, p. 143 / 177 59 / 59 RDWQRLINNNWGRPPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFT DSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCL EYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYLNGY RTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQN NNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGK QGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNA VNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPP PQILIKNTPVPADPPTTFNQLASFITQYSTGQVSVEIEWELQKENSKRWN PEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTTRYLTRNL* Modified Capsids - AA Sequences (SEQ ID NO: 5) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLP GYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHAD AEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVE PSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSG LGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTR TWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFT DSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCL EYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLS RTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQN NNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGK QGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNYIGSRGA VNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPP PQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWN PEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL Petição 870260076337, de 30 / 07 / 2026, pág. 144 / 177

Claims

1 / 2 CLAIMS 1. Recombinant Viral Particle, characterized in that the recombinant viral particle is a modified AAVrh74 comprising one or more modifications selected from the group of an isoleucine substitution for asparagine at amino acid position 502, an arginine substitution for tryptophan at amino acid position 505 of an AAVrh74 VP1 and an insertion of a YIG or YIGSR peptide (SEQ ID NO: 2) at amino acid position 591 in the AAVrh74 VP1 or an equivalent of each thereof.

2. Recombinant Viral Particle, according to Claim 1, characterized in that it further comprises a transgene or a CRISPR system.

3. Polynucleotide encoding the Recombinant Viral Particle, as defined in Claim 1 or 2, characterized in that it is optionally contained within a vector.

4. Recombinant Expression System, for producing the recombinant viral particle, as defined in Claim 1 or 2, characterized in that it optionally comprises a vector, as defined in Claim 3.

5. Host Cell, characterized in that it comprises the polynucleotide or vector, as defined in Claim 3.

6. Method for Modifying Polynucleotide and / or Protein in a Cell, characterized in that it comprises supplying the cell with an efficient amount of the recombinant viral particle, as defined in Claim 1 or 2.

7. Method for Modifying Polynucleotide and / or Protein in a Cell, according to Claim 6, characterized in that the cell is a mammalian cell.

8. Method for Modifying Polynucleotide and / or Protein in a Cell, according to Claim 7, characterized in that the mammalian cell is a human cell.

9. Method for Modifying Polynucleotide and / or Protein in an Individual, characterized in that it comprises administering to the individual an efficient quantity of the recombinant viral particle, as defined in Claim 1 or 2.

10. Kit, characterized in that it comprises the recombinant viral particle, as defined in Claim 2.

11. Use of Recombinant Viral Particle, as defined in Claim 1 or 2, of Polynucleotide, as defined in Claim 3, and of Recombinant Expression System, as defined in Claim 4, characterized in that it is in the manufacture of medicaments for the treatment of diseases including hemophilia, muscular dystrophy, multiple sclerosis, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's disease, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler-Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesterolemia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysaccharidosis (I-IX), hepatitis B and hepatitis C. Petition 870260076337, dated 07 / 30 / 2026, pp. 174 / 177

Images