Methods and compositions for treating skeletal muscle disorders

Abstract

Described herein are regulatory promoters that provide for an expression level and expression pattern that is required for treatment of LGMD2A by delivery and expression of CAPN3. Described herein are nucleic acids comprising a regulatory promoter, wherein the regulatory promoter comprises a nucleic acid sequence with at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:7, 10, 12 or 13.

Description

METHODS AND COMPOSITIONS FOR TREATING SKELETAL MUSCLEDISORDERSBACKGROUND OF THE INVENTION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 694,720, filed on September 13, 2024, the entirety of which is incorporated herein by reference.

[0002] The application contains a Sequence Listing in compliance with ST.26 format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on September 12, 2025 is named UCLAP0215WO.xml and is 50,849 bytes in size.

[0003] This invention was made with government support under NS117912 awarded by the National Institutes of Health. The government has certain rights in the invention.I. Field of the Invention

[0004] This invention relates to the field of gene therapy and methods of treating skeletal disorders.II. Background

[0005] Limb girdle muscular dystrophy type 2A (LGMD2A AKA, LGMD1R) is an autosomal recessive (AR) muscle wasting disorder due to mutations in the CAPN3 gene encoding calpain 3 protease (Ono et al. (2016) Biochimie. 122: 169-187). LGMD2A is the most prevalent of the AR LGMDs and yet there is no treatment for patients, who are usually wheelchair dependent a decade after diagnosis. It has been shown that shown that overexpression of Capn3 in skeletal muscle is non-toxic (Spencer et al. (2002) Proc. Natl. Acad. Sci. USA, 99(13): 8874-8879), and that a Capn3 cDNA fits without modification in AAV vectors. These properties make gene therapy for LGMD2A a realistic goal.

[0006] LGMD2A, however, is unique with respect to gene therapy, because Capn3 expression in the heart is toxic (Roudaut et al. (2013) Circulation, 128(10): 1094-1104), which is not the case with other gene therapies for neuromuscular disorders such as Duchenne muscular dystrophy (DMD) and other LGMDs (Crudele & Chamberlain (2019) Hum. Mol. Genet. 28(R1): R102-R107. doi: 10.1093 / hmg / ddzl28). While pre-clinical, proof of concept studies have successfully accomplished AAV-Capn3 overexpression in mice (Roudaut et al.UCLA.P0215WO Specification.docx - 1 -(2013) Circulation, 128(10): 1094-1104), gene therapy for humans with LGMD2A has not been successfully developed as a therapeutic.. Nor has any study been conducted to examine the minimal amount of Capn3 needed for a therapeutic effect. Furthermore, because LGMD2A preferentially impacts slow fibers (Kramerova et al. (2012) Hum. Mol. Genet. 21(14): 3193- 3204), it is desirable that the therapeutic construct that is ultimately used in clinical trials is optimized for slow fiber expression. Furthermore calpain 3 is not expressed in the heart and it’s expression there appears to be toxic in mice (Roudaut et al. (2013) Circulation, 128(10): 1094-1104). Thus, expression in slow and fast skeletal muscle but not the heart will provide the optimal gene expression profile for CAPN3 delivered by AAV vectors.SUMMARY OF THE INVENTION

[0007] Described herein are regulatory promoters that provide for an expression level and expression pattern that is required for treatment of LGMD2A by delivery and expression of CAPN3. Described herein are nucleic acids comprising a regulatory promoter, wherein the regulatory promoter comprises a nucleic acid sequence with at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 7, 10, 12 or 13. The regulatory promoter may comprise a nucleic acid sequence that has the nucleic acid sequence of SEQ ID NO:7, 10, 12 or 13. The regulatory promoter may comprise or exclude a nucleic acid sequence that has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of SEQ IDN0:7, 10, 12 or 13. Also provided is anucleic acid comprising a regulatory promoter, wherein the regulatory promoter comprises regulatory element A that has a nucleic acid sequence with at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 and regulatory element B that has a nucleic acid sequence with at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:2, 12, or 13. The regulatory promoter may comprise regulatory element A that has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 and regulatory element B that has a nucleic acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:2, 12, or 13. Also described is a nucleic acid comprising the sequence of one of SEQ ID NOS: 17-21, or an amino acid sequence that has at least 80% sequence identity to oneUCLA.P0215WO Specification. docx - 2 -of SEQ ID NOS: 17-21. The nucleic acid may comprise the sequence of one on SEQ ID NOS: 17-21. The nucleic acid may comprise a nucleic acid sequence that has or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of one of SEQ ID NOS: 17-21.

[0008] Included in the disclosure are proteins encoded by the nucleic acid, viral particles and virus comprising the nucleic acid, and host cells comprise the nucleic acid.

[0009] Also provided is a method of making an engineered host cell comprising transferring a nucleic acid of the disclosure into the host cell. The disclosure also describes a method of treating a subject with a skeletal muscle disorder, said method comprising administering a nucleic acid, viral particle, host cell, or virus of the disclosure to the subject. Further disclosed is a method of expressing a transgene in a muscle cell of a subject, the method comprising administering the nucleic acid, viral particle, host cell, or virus of the disclosure to the subject. Also described is a method comprising incubating a host cell of the disclosure under conditions suitable for the production of viral particles and isolating viral particles.

[0010] The regulatory element A may have the nucleic acid sequence of SEQ ID NO: 1 and regulatory element B may have the nucleic acid sequence of SEQ ID NO:2, 12, or 13. Element A and element B may be separated by less than 100 nucleotides. Element A and Element B may be separated by at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides, or any derivable range therein. Element A may be upstream of element B. Element B may be upstream of element A. The term upstream with reference to Element A being upstream of Element B, means that element A is closer to the 5’ end of the nucleic acid than Element B. Intervening nucleotides may be between element A and B unless stated otherwise. Similarly, an element or region is downstream if it is closer to the 3’ end of the nucleic acid. The nucleic acid may be an isolated nucleic acid. The nucleic acid may be integrated into the genome of a cell or virus.

[0011] The nucleic acid may further comprise a nucleic acid sequence encoding a transgene. The transgene may be downstream of the regulatory promoter. The transgene may be downstream of element A. The transgene may be downstream of element B. Element A may be downstream of the transgene. Element B may be downstream from the transgene. Element A and the transgene may be separated by at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,UCLA.P0215WO Specification. docx - 3 -10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides, or any derivable range therein. Element B and the transgene may be separated by at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides, or any derivable range therein. The regulatory promoter may exclude a wild-type promoter nucleic acid sequence.

[0012] The transgene may comprise or exclude the full-length cDNA of the gene. The transgene may comprise or exclude a UTR-deleted cDNA. The transgene may comprise or exclude a Kozak-optimized cDNA. The transgene may comprise or exclude Calpain 3 (CAPN3). The transgene may comprise or exclude a human protein. The transgene may comprise the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that has at least 75% sequence identity to the amino acid sequence of SEQ ID NO:6. The transgene may comprise an amino acid sequence that has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of SEQ ID NO:6. The nucleic acid sequence encoding the transgene may be codon optimized. The nucleic acid encoding the transgene may be one that is not codon optimized. The nucleic acid sequence encoding the transgene is CpG depleted. The nucleic acid encoding the transgene may comprise the nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence that has at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. The nucleic acid encoding the transgene may comprise a nucleic acid sequence that has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0013] The nucleic acid may be one that does not comprise a miRNA binding site. The nucleic acid may be one that does not comprise a miR208 binding site. The nucleic acid may be further defined as an expression cassette. The expression cassette may be present in a gene therapy vector. The cassette may be present in a gene therapy vector selected from the group consisting of a lentiviral vector (LV), an adenovirus vector (AV), and an adeno-associated viral vector (AAV). The gene therapy vector may exclude a lentiviral vector (LV), an adenovirusUCLA.P0215WO Specification. docx - 4 -vector (AV), or an adeno-associated viral vector (AAV). The gene therapy vector may comprise or exclude an adeno-associated viral vector (AAV). The AAV vector may comprise or exclude a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9. The AAV vector may comprise an AAV2 backbone. The AAV vector may comprise an AAV2 vector pseudotyped with an AAV6 capsid (AAV2 / 6). The gene therapy vector may be a lentiviral vector. The vector may comprise or exclude an HIV-1 lentiviral vector.

[0014] The nucleic acid may further comprise or exclude a nucleic acid sequence comprising inverted terminal repeats (ITR) and wherein a first ITR is upstream of the regulatory promoter and a second ITR is downstream of the transgene. The ITR(s) may comprise the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:3. The ITR(s) may comprise a nucleic acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:3.

[0015] The nucleic acid may comprise an intron between the nucleic acid sequence encoding the transgene and the regulatory promoter. The intron may comprise the nucleic acid sequence of SEQ ID NO:4 or 8 or a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:4 or 8. The intron may comprise a nucleic acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:4 or 8.

[0016] The nucleic acid may comprise the nucleic acid sequence of SEQ ID NOV or a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NOV. The nucleic acid may comprise a nucleic acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid sequence of SEQ ID NOV.

[0017] The nucleic acid may further comprise a promoter that directs the expression of the nucleic acid. The promoter may be a regulatory promoter described herein. .

[0018] The host cell may be a viral packaging cell or may exclude a viral packaging cell. The virus, nucleic acid, and / or viral particles may include the capsid variants described in WO2024168266; WO2023196967; WO2021077000; W02022020616; WO2021050974; WO2021222831; US20220228173; WO2023039476; WO2023015297; or W02023060142.UCLA.P0215WO Specification. docx - 5 -The host cell may comprise the nucleic acid extra-chromosomally. The host cell may comprise a nucleic acid of the disclosure that has been integrated the genome of the host cell.

[0019] The subject may be one that has a skeletal muscle disorder. The methods may include methods in which there is substantially no expression of the transgene in cardiac muscle cells. The methods may include methods in which there is no detectable expression of the transgene in cardiac muscle cells. The transgene may be one that is expressed in slow fiber muscle cells in the methods of the disclosure. The compositions may be administered systemically, parenterally, orally, nasally, intramuscularly or by a route of administration described herein.

[0020] The transgene may be expressed at higher levels in slow fiber muscle cells as compared to cardiac muscle cells. The transgene may expressed in the subject and the expression level of the transgene may be at levels at least 2-fold, or at least 3 -fold, or at least 4-fold, or at least 5-fold, or at least 6-fold , or at least 7-fold , or at least 8-fold , or at least 9- fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 500-fold, or at least 1000-fold higher levels (or any derivable range therein) in skeletal muscle cells than in cardiac muscle cells. The skeletal muscle disorder may comprise Limb girdle muscular dystrophy (LGMD). The skeletal muscle disorder may comprise or exclude . The skeletal muscle disorder may comprise or exclude X-linked myotubular myopathy. The skeletal muscle disorder may comprise or exclude Facioscapulohumeral muscular dystrophy (FSHD). The skeletal muscle disorder may comprise or exclude FKRP-related muscular dystrophy (LGMD2I). The skeletal muscle disorder may comprise or exclude Nemaline myopathy. The subject may be a human subject. The subject may be a laboratory test animal such as a rat, mouse, rabbit, pig, goat, or horse. The subject may be a mammal. The subject may be a guinea pig, a horse, or a nonhuman primate. The muscle cells described herein may be further defined as a myocyte. The cells may be defined as cardiac muscle cells, skeletal muscle cells, or smooth muscle cells.

[0021] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0022] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0023] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can referUCLA.P0215WO Specification. docx - 6 -to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z ” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment or aspect.

[0024] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0025] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0026] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0027] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0028] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.UCLA.P0215WO Specification. docx - 7 -

[0029] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments and aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0031] FIG. 1A-1B A: Construct to drive slow and fast skeletal muscle expression without expression in heart named “3 SURE”. A slow enhancer named SURE was added to a CK8e promoter variant called RC#3. RC3 alone did not express highly in fast muscles like gastric or TA. The addition of the SURE enhancer boosted expression in fast and slow. B shows additional constructs tested in Example 1.

[0032] FIG. 2. AAVMyola delivery of cassettes with SM Mut (#3), RC#7, SM Mut + SURE (3SURE) or 3 +7. After systemic delivery of a high dose, there is >100% calpain 3 expression in both fast and slow twitch muscle and no expression in the heart.

[0033] FIG. 3. Addition of the SURE enhancer (SEQ ID NO: 1) impacts calpain 3 expression in both fast and slow twitch muscle. Shown are tibialis anterior and plantaris. This data further shows that RC3 alone did not express well in fast muscles tibialis anterior (TA) and plantaris. Addition of the slow SURE enhancer boosts expression. It is not obvious because it is a slow enhancer and yet expression in fast is boosted.

[0034] FIG. 4. Testing RC# 8, generated based on the Tnni promoter with addition of a fast enhancer and packaged in MyoAAV with FLAG. Vectors were r.o. injected to C3KO (2E14 vg / kg). The data show a low amount of protein in the gastrocnemius, but good levels in the soleus for RCs# 8 and no expression in the heart. This data shows that simply adding a fast enhancer to a slow promoter does not increase expression in gastric.

[0035] FIG. 5. Testing RC# 11 packaged in AAV Myo and r.o. injected to C3KO (2E14 vg / kg). Construct #11 showed low expression in soleus and gastric while there was moreUCLA.P0215WO Specification. docx - 8 -protein in diaphragm. RC11, a computer generated promoter, has low expression in slow and still expresses in heart (neither is desirable).

[0036] FIG. 6 Dose response and histology. RC#3SURE was packaged in MyoAAV and injected systemically by RO injection to C3KO at lE13vg / kg, 5E13vg / kg and 2E14 vg / kg. Even the highest levels of calpain 3 did not induce toxicity.

[0037] FIG. 7A-7B shows embodiments from prior publications. A. Expression of Calpain3 protein under the control of the Desmin promoter caused expression in heart and skeletal muscle. miRNA208 binding site was used to block expression in heart, but is likely not effective enough to prevent toxicity. B. The Calpain 3 levels were 5-15% relative to WT. The promoter was not expressed in slow fibers.

[0038] FIG. 8 : The CK8e promoter is commonly used in AAV-gene therapy vectors for skeletal muscle. This promoter is not highly active in slow muscle. It is not sufficient for gene therapies that need to detarget the heart and to express in slow fibers.

[0039] FIG. 9: Testing of Capn3 expression with different regulatory cassettes in slow (soleus) muscle fibers, fast (gastric) muscle fibers, and the heart.

[0040] FIG. 10: RC3 and SURE-RC3 both express in fast and slow and detarget the heart, but RC1 does not express at therapeutically significant levels in slow fibers.

[0041] FIG. 11. Testing of Capn3 expression with different regulatory cassettes and combinations thereof in the diaphragm, which has a significant number of slow fibers.DETAILED DESCRIPTION OF THE INVENTION

[0042] The current disclosure describes gene therapy methods and vectors for treating muscular dystrophies in which expression of the transgene is high in both fast and slow type skeletal muscle and low in the heart and liver. One such disease is type 2A (LGMD2A AKA, LGMD1R), an autosomal recessive (AR) muscle wasting disorder due to mutations in the CAPN3 gene encoding calpain 3 protease. LGMD2A is the most prevalent of the AR LGMDs and yet there is currently no treatment for patients, who are usually wheelchair dependent a decade after diagnosis. The inventors have shown that overexpression of Capn3 in skeletal muscle is non-toxic, and that a Capn3 cDNA fits without modification in AAV. These properties make gene therapy for LGMD2A a realistic goal. LGMD2A is unique with respect to gene therapy, because Capn3 expression in the heart is toxic (Roudaut et al. (2013) Circulation, 128(10): 1094-1104), which is not the case with other gene therapies for neuromuscular disorders such as Duchenne muscular dystrophy (DMD) and other LGMDs.UCLA.P0215WO Specification. docx - 9 -While pre-clinical, proof of concept studies have been successfully accomplished showing that AAVCapn3 can induce calpain 3 protein expression in muscle, human gene therapy constructs having the appropriate expression patterns of the Capn3 gene have not been developed. Furthermore, the amount of Capn3 required for a therapeutic effect is unknown. Because LGMD2A preferentially impacts slow fibers, it is critical that the therapeutic construct that is ultimately used in clinical trials is optimized for slow fiber expression.

[0043] To accomplish the goal of generating efficacious regulatory cassettes to drive calpain 3 expression for use in gene therapy vectors, the inventors designed and tested structural different regulatory promoters to determine whether the appropriate expression level and expression pattern can be achieved to treat LGMD2A.I. Nucleic Acids and Proteins

[0044] As used herein, a “protein” “peptide” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wildtype versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.

[0045] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specificUCLA.P0215WO Specification. docx - 10 -polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0046] In certain aspects, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein. Nucleic acids that encode the epitope to which certain of the antibodies provided herein are also provided. Nucleic acids encoding fusion proteins that include these peptides are also provided. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0047] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.

[0048] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, nucleic acids, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.UCLA.P0215WO Specification. docx - 11 -

[0049] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0050] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, at least, or at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 (or any derivable range therein) or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0051] In certain aspects the size of a protein, polypeptide (wild-type or modified), or nucleic acid may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1400, 1600, 1800, or 2000UCLA.P0215WO Specification. docx - 12 -amino acid residues or nucleic acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).

[0052] The polypeptides, proteins, or nucleic acids encoding such polypeptides or proteins of the disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to at least, or at most 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOS: 1-21. In specific aspects, the peptide or polypeptide is or is based on a human sequence. In certain aspects, the peptide or polypeptide is not naturally occurring and / or is in a combination of peptides or polypeptides.

[0053] The substitution at the amino acid or nucleic acid at position 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,UCLA.P0215WO Specification. docx - 13 -85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,UCLA.P0215WO Specification. docx - 14 -735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962963. 964. 965. 966. 967. 968. 969. 970. 971. 972. 973. 974. 975. 976. 977. 978. 979. 980. 981982, 983, 984, 985, 986, 987, 9: 58, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300,UCLA.P0215WO Specification. docx - 15 -1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315,1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330,1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345,1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360,1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375,1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390,1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405,1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420,1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435,1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450,1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465,1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480,1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495,1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510,1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525,1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540,1541, 1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555,1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570,1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585,1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600,1601, 1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615,1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630,1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645,1646, 1647, 1648, 1649, 1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660,1661, 1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675,1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690,1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705,1706, 1707, 1708, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720,1721, 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735,1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750,1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765,1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780,1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795,1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810,UCLA.P0215WO Specification. docx - 16 -1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825,1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840,1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855,1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870,1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885,1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900,1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915,1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930,1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945,1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960,1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975,1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990,1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005,2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020,2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035,2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050,2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065,2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080,2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095,2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 2110,2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125,2126, 2127, 2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140,2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155,2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 2170,2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185,2186, 2187, 2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200,2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215,2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227, 2228, 2229, 2230,2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245,2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260,2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275,2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290,2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305,2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320,UCLA.P0215WO Specification. docx - 17 -2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335,2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347, 2348, 2349, 2350,2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365,2366, 2367, 2368, 2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380,2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395,2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410,2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425,2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440,2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455,2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470,2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485,2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500,2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515,2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530,2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545,2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560,2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575,2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590,2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605,2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620,2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635,2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650,2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665,2666, 2667, 2668, 2669, 2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680,2681, 2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693, 2694, 2695,2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705, 2706, 2707, 2708, 2709, 2710,2711, 2712, 2713, 2714, 2715, 2716, 2717, 2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725,2726, 2727, 2728, 2729, 2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740,2741, 2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753, 2754, 2755,2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765, 2766, 2767, 2768, 2769, 2770,2771, 2772, 2773, 2774, 2775, 2776, 2777, 2778, 2779, 2780, 2781, 2782, 2783, 2784, 2785,2786, 2787, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800,2801, 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813, 2814, 2815,2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825, 2826, 2827, 2828, 2829, 2830,UCLA.P0215WO Specification. docx - 18 -2831, 2832, 2833, 2834, 2835, 2836, 2837, 2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845,2846, 2847, 2848, 2849, 2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860,2861, 2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872, 2873, 2874, 2875,2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2885, 2886, 2887, 2888, 2889, 2890,2891, 2892, 2893, 2894, 2895, 2896, 2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905,2906, 2907, 2908, 2909, 2910, 2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920,2921, 2922, 2923, 2924, 2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932, 2933, 2934, 2935,2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944, 2945, 2946, 2947, 2948, 2949, 2950,2951, 2952, 2953, 2954, 2955, 2956, 2957, 2958, 2959, 2960, 2961, 2962, 2963, 2964, 2965,2966, 2967, 2968, 2969, 2970, 2971, 2972, 2973, 2974, 2975, 2976, 2977, 2978, 2979, 2980,2981, 2982, 2983, 2984, 2985, 2986, 2987, 2988, 2989, 2990, 2991, 2992, 2993, 2994, 2995,2996, 2997, 2998, 2999, or 3000 of SEQ ID NOS: 1-21 may be with alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, in the case of polypeptides or adenine, cytosine, guanine, or thymine, in the case of nucleic acids.

[0054] In some aspects, the protein, polypeptide, or nucleic acid may comprise amino acids or nucleotides 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any derivable range therein) of SEQ ID NOS: 1-21.UCLA.P0215WO Specification. docx - 19 -

[0055] In some aspects, the protein, polypeptide, or nucleic acid may comprise amino acids or nucleotides 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any derivable range therein) of SEQ ID NOS: 1-21 and have or have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOS: 1-21.

[0056] In some aspects, the protein, polypeptide, or nucleic acid may comprise, comprise at least, or comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,UCLA.P0215WO Specification. docx - 20 -249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any derivable range therein) contiguous amino acids or nucleic acids of SEQ ID NOS: 1-21.

[0057] In some aspects, the polypeptide, protein, or nucleic acid may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 (or any derivable range therein) contiguous amino acids of SEQ ID NOS: 1-21 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to one of SEQ ID NOS: 1-21.

[0058] In some aspects there is a nucleic acid molecule or polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,UCLA.P0215WO Specification. docx - 21 -141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 , 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176 , 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195 , 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 , 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233 , 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 , 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290 , 291, 292,293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309 , 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328 , 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347 , 348, 349,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366 , 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385 , 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404 , 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423 , 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442 , 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463,464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480 , 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 , 500, 501,502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518 , 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537 , 538, 539,540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556 , 557, 558,559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575 , 576, 577,578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594 , 595, 596,597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613 , 614, 615,616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632 , 633, 634,635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651 , 652, 653,654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670 , 671, 672,673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689 , 690, 691,692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708 , 709, 710,711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727 , 728, 729,730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746 , 747, 748,749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765 , 766, 767,768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784 , 785, 786,UCLA.P0215WO Specification. docx - 22 -787, 788, 789, 790, 791, 792, 793, 794, 795, 796 , 797, 798, 799, 800, 801, 802, 803, 804, 805,806, 807, 808, 809, 810, 811, 812, 813, 814, 815 , 816, 817, 818, 819, 820, 821, 822, 823, 824,825, 826, 827, 828, 829, 830, 831, 832, 833, 834 , 835, 836, 837, 838, 839, 840, 841, 842, 843,844, 845, 846, 847, 848, 849, 850, 851, 852, 853 , 854, 855, 856, 857, 858, 859, 860, 861, 862,863, 864, 865, 866, 867, 868, 869, 870, 871, 872 , 873, 874, 875, 876, 877, 878, 879, 880, 881,882, 883, 884, 885, 886, 887, 888, 889, 890, 891 , 892, 893, 894, 895, 896, 897, 898, 899, 900,901, 902, 903, 904, 905, 906, 907, 908, 909, 910 , 911, 912, 913, 914, 915, 916, 917, 918, 919,920, 921, 922, 923, 924, 925, 926, 927, 928, 929 , 930, 931, 932, 933, 934, 935, 936, 937, 938,939, 940, 941, 942, 943, 944, 945, 946, 947, 948 949, or 950 of any of SEQ ID NOS: 1-21 and comprising at least, at most, or exactly 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123:, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 , 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 16L , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180:, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 , 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 , 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 231 , 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256^ , 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 215 , 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294^ , 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313 , 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332^ , 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 35L , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370^ , 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389^ , 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408^ , 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427^ , 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446^ , 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465^ , 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484 , 485, 486, 487, 488, 489, 490, 491, 492, 493,UCLA.P0215WO Specification. docx - 23 -494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512,513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550,551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588,589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607,608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645,646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721,722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740,741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759,760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778,779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797,798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816,817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854,855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873,874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892,893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911,912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930,931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, or 950 (or any derivable range therein) contiguous amino acids or nucleotides of any of SEQ ID NOS: 1-21.

[0059] The nucleotide as well as the protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.UCLA.P0215WO Specification. docx - 24 -

[0060] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0061] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0062] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0063] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type (or any range derivable therein). A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0064] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminalUCLA.P0215WO Specification. docx - 25 -sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0065] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0066] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0067] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0068] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.UCLA.P0215WO Specification. docx - 26 -

[0069] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. In further aspects, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0070] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (—0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain aspects, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the invention, those that are within ±1 are included, and in other aspects of the invention, those within ±0.5 are included.

[0071] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity valuesUCLA.P0215WO Specification. docx - 27 -have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (_0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain aspects, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other aspects, those which are within ±1 are included, and in still other aspects, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0072] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0073] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy.org / proteomics / protein structure.

[0074] In some aspects of the invention, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5) confer orUCLA.P0215WO Specification. docx - 28 -modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such aspects, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).A. Vectors

[0075] Polypeptides of the disclosure may be encoded by a nucleic acid molecule comprised in a vector. The term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. A nucleic acid sequence can be “heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference).

[0076] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.B. Promoters and Enhancers

[0077] A “promoter” is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase andUCLA.P0215WO Specification. docx - 29 -other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0078] Naturally, it may be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al., 2001, incorporated herein by reference). The promoters employed may be constitutive, tissuespecific, or inducible and in certain aspects may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.

[0079] Various elements / promoters may be employed in the context of the present invention to regulate the expression of a gene. Examples of such inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include but are not limited to Immunoglobulin Heavy Chain (Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.; 1990), Immunoglobulin Light Chain (Queen et al., 1983; Picard et al., 1984), T Cell Receptor (Luria et al., 1987; Winoto et al., 1989; Redondo et al.; 1990), HLA DQ and / or DQ; Sullivan et al., 1987), Interferon (Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988), Interleukin-2 (Greene et al., 1989), Interleukin-2 Receptor (Greene et al., 1989; Lin et al., 1990), MHC Class II 5 (Koch et al., 1989), MHC Class II HL A-DR; Sherman et al., 1989), Actin (Kawamoto et al., 1988; Ng et al.; 1989), Muscle Creatine Kinase (MCK) (Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989), Prealbumin (Transthyretin) (Costa et al., 1988), Elastase I (Ornitz et al., 1987), Metallothionein (MTII) (Karin et al., 1987; Culotta et al., 1989), Collagenase (Pinkert et al., 1987; Angel et al., 1987), Albumin (Pinkert et al., 1987; Tronche et al., 1989, 1990), Fetoprotein (Godbout et al., 1988; Campere et al., 1989), y-Globin (Bodine et al., 1987; Perez-Stable et al., 1990), -Globin (Trudel et al., 1987), c-fos (Cohen et al., 1987), c-Ha-Ras (Triesman, 1986; Deschamps et al., 1985), Insulin (Edlund et al., 1985), Neural Cell Adhesion Molecule (NCAM) (Hirsh et al., 1990), 1-Antitrypain (Latimer et al., 1990), H2B (TH2B) Histone (Hwang et al., 1990), Mouse and / or Type I Collagen (Ripe et al., 1989), Glucose-Regulated Proteins (GRP94 and GRP78)UCLA.P0215WO Specification. docx - 30 -(Chang et al., 1989), Rat Growth Hormone (Larsen et al., 1986), Human Serum Amyloid A (SAA) (Edbrooke et al., 1989), Troponin I (TN I) (Yutzey et al., 1989), Platelet-Derived Growth Factor (PDGF) (Pech et al., 1989), Duchenne Muscular Dystrophy (Klamut et al., 1990), SV40 (Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988), Polyoma (Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell et al., 1988), Retroviruses (Kriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989), Papilloma Virus (Campo et al., 1983; Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987), Hepatitis B Virus (Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988), Human Immunodeficiency Virus (Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989), Cytomegalovirus (CMV) IE (Weber et al., 1984; Boshart et al., 1985; Foecking et al., 1986), Gibbon Ape Leukemia Virus (Holbrook et al., 1987; Quinn et al., 1989).

[0080] Inducible elements include, but are not limited to MT II - Phorbol Ester (TFA) / Heavy metals (Palmiter et al., 1982; Haslinger et al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989); MMTV (mouse mammary tumor virus) - Glucocorticoids (Huang et al., 1981; Lee et al., 1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988); Interferon - poly(rl)x / poly(rc) (Tavernier et al., 1983); Adenovirus 5 E2 - E1A (Imperiale et al., 1984); Collagenase - Phorbol Ester (TP A) (Angel et al., 1987a); Stromelysin- Phorbol Ester (TP A) (Angel et al., 1987b); SV40 - Phorbol Ester (TP A) (Angel et al., 1987b); Murine MX Gene - Interferon, Newcastle Disease Virus (Hug et al., 1988); GRP78 Gene - A23187 (Resendez et al., 1988); B-2-Macroglobulin - IL-6 (Kunz et al., 1989); Vimentin - Serum (Rittling et al., 1989); MHC Class I Gene H-2b - Interferon (Blanar et al., 1989); HSP70- E1A / SV40 Large T Antigen (Taylor et al., 1989, 1990a, 1990b); Proliferin - Phorbol Ester / TPA (Mordacq et al., 1989); Tumor Necrosis Factor - PMA (Hensel et al., 1989); and Thyroid Stimulating Hormone Gene - Thyroid Hormone (Chatteijee et al., 1989).UCLA.P0215WO Specification. docx - 31 -

[0081] The particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell, preferably a bacterial cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter.C. Initiation Signals and Internal Ribosome Binding Sites (IRES)

[0082] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.

[0083] In certain aspects of the disclosure, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5’ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). In some aspects, a 2A peptide is used to create multigene or polyscistronic messages.D. Selectable and Screenable Markers

[0084] In certain aspects of the invention, cells containing a nucleic acid construct of the current disclosure may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker. As an alternative, 2AUCLA.P0215WO Specification. docx - 32 -peptides could be used to introduce ribosomal skips to enable expression of multiple polypeptidic or protein sequences.II. Host Cells

[0085] As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

[0086] Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).

[0087] Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available. In some aspects, the expression system comprises a second generation lentiviral packaging system in 293T cells. In some aspects, the gag, pol, and rev are on the same plasmid.

[0088] The insect cell / baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.

[0089] In addition to the disclosed expression systems of the invention, other examples of expression systems include STRATAGENE®’s COMPLETE CONTROL™ InducibleUCLA.P0215WO Specification. docx - 33 -Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.III. Viral Particles and Methods of Producing Viral Particles

[0090] The engineered viruses of the disclosure may comprise viruses that do not normally cause disease or other negative effects in the infected host or cell. Alternatively, the virus may be an attenuated virus, i.e. a virus which has mutated or which has been engineered or otherwise treated such that it does not support pathogenic infection of healthy or nontarget cells.

[0091] In some aspects, the virus can be derived from the Lenti virus genus. Non-limiting examples of lentiviral vectors include those derived from a lentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, an Simian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1 (HTLV-1), HTLV-2 or equine infection anemia virus (E1AV). The nucleic acid may comprise an epHIV7 vector backbone. Other suitable viral vectors include, for example, pRSV-Rev, pMDLg / pRRE, psPAX2, pCMV delta R8.2, pMD2.G, pCMV-VSV- G, pCMV-dR8.2 dvpr, pCI-VSVG, pCPRDEnv, pLTR-RD114A, pLenti-III (Applied Biological Materials; cat # LV587);87 pLentiCRISPR v.l (Addgene; cat #52963);88 pl56RRLsinppt (Addgene; cat #42795);89 pFUGW (Addgene; cat #14883);90 pFUG (Addgene; cat #14882);90 pHAGE (Addgene; cat #46793);91 pHRsin (Addgene; cat #12265);92 pLenti (AMP) (Addgene; cat #61422);93 pLKO. l (Addgene; cat #10878);94 pLL3.7 m (Addgene; cat #89362);95 Puro.cre (Addgene; cat #17408);96 pRSIEG (Cellecta; SVSHU6EG-L); pLenti7.3 (Thermo Fisher Scientific; cat #V53406); pLenti (OriGene; cat #PS100109); pSF_Lenti (Sigma; cat #OGS269); pLV-GFPSpark (Sinobiological; cat #LVCV- 01); pLVX (Takara; cat #632164); pALD-Lenti (Aldevron), pLenti (Vigene; cat #P100020), pLenti CMV (Addgene), and pLV. In some aspects, the vector backbone does not contain an antibiotic resistance gene. In some aspects, the vector backbone does not contain a beta-lactam resistance gene.UCLA.P0215WO Specification. docx - 34 -

[0092] In some aspects, the recombinant retrovirus can be derived from HIV, SIV, or FIV. In further aspects, the recombinant retrovirus can be derived from the human immunodeficiency virus (HIV) in the Lentivirus genus. Lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The higher complexity enables the lentivirus to modulate the life cycle thereof, as in the course of latent infection. A typical lentivirus is the human immunodeficiency virus (HIV), the etiologic agent of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages.

[0093] In one aspect, provided herein are packaging cells or systems comprising the nucleic acids as described herein. The packaging cells may be any cell suitable for virus production. In some aspects, the packaging cells is a mammalian cell that is used to make virus. Any of a wide variety of cells can be selected for in vitro production of a virus. Eukaryotic cells are typically used, particularly mammalian cells including human, simian, canine, feline, equine and rodent cells. In illustrative examples, the cells are human cells. In further illustrative aspects, the cells reproduce indefinitely, and are therefore immortal. Examples of cells that can be used in the present invention include NIH 3T3 cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293T cells and any cells derived from such cells. Highly transfectable cells, such as human embryonic kidney 293T cells, can be used. By "highly transfectable" it is meant that at least about 50%, more preferably at least about 70% and most preferably at least about 80% of the cells can express the genes of the introduced DNA.

[0094] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCLIO), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0095] In any of the aspects disclosed herein, the methods of making virus can include growing a mammalian packaging cells to 50%, 60%, 70%, 80%, 90% or 95% confluence or confluence to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% peak cell density and then splitting or diluting the cells. In some aspects, a stirred tank reactor can be used to grow theUCLA.P0215WO Specification. docx - 35 -cells. In some aspects, the cells can be split at least about 1:2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 12, 1 : 15, or 1 :20 using methods a skilled artisan will understand. In some aspects, the cells can be diluted to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% peak cell density.IV. Treatment of Disease

[0096] In various embodiments nucleic acids are provided that preferentially express a gene (or cDNA) in skeletal muscle as compared to expression in cardiac muscle. In certain embodiments gene therapy vectors are provided that contain these nucleic acids and uses thereof are provided.

[0097] The nucleic acids and gene therapy vectors, such as the AAV vectors, for example, are provided that are suitable for the treatment of limb girdle muscular dystrophy type R1 / 2A (LGMDR1 / 2A). There are 26 genetically defined autosomal recessive LGMDs and LGMDR1 is the most prevalent. LGMDR1 is caused by loss of function mutations in the CAPN3 gene. At this time, there are no therapies available for patients.

[0098] The nucleic acids of the disclosure are not expressed or are expressed at a low, nontoxic level in the heart and expressed highly in both fast and slow skeletal muscle fibers. The nucleic acids comprising the regulatory promoters of the disclosure can be used in combination with CAPN3 (the gene), both of which can be cloned between the inverted terminal repeats of AAV in a plasmid and used to generate viral vectors.

[0099] Mechanisms of pathogenesis in LGMDR1 are unique and, at least the following distinctive features need to be considered when designing gene therapy vectors (e.g., AAV- vectors) for LGMDR1 : 1) CAPN3 expression in the heart can be fatal; and 2) LGMDR1 involves loss of the slow oxidative phenotype, so slow fibers must be targeted.

[0100] Successful gene therapy for LGMD2A involves producing beneficial Cap3 levels in fast and slow fibers without producing toxic Capn3 levels in cardiac and non-muscle tissues. This is facilitated by the regulatory cassettes (RCs) that exhibit fiber type-specific transcription. Importantly, because the Capn3 cDNA is only ~2.6 kb, RC miniaturization is not necessary, and the additional >1 kb of vector packaging space provides opportunities for designing larger RCs with greater skeletal muscle fiber type specificity.

[0101] Due to cardiotoxicity issues, optimal LGMD2A gene therapy requires very low to no Capn3 production in cardiac muscle. Many current RCs thus have the deleterious side effect of causing Capn3 -mediated cardiotoxicity. Gene therapy strategies for LGMD2A include producing optimal Capn3 levels in both slow and fast skeletal muscle fibers whileUCLA.P0215WO Specification. docx - 36 -simultaneously limiting Capn3 levels in cardiac muscle to prevent toxicity. Designing regulatory cassettes (RCs) that function in this fashion is complex because Type I fibers and cardiomyocytes share numerous transcription factors (TFs). RCs with high activities in slow fibers contain CEs that bind these TFs, so are active in cardiac muscle as well.

[0102] In view of these considerations, a nucleic acid that expresses a gene at higher levels in skeletal muscle than in cardiac muscle is provided. The nucleic acid may express the gene (transgene) at levels at least 2-fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold , or at least 7-fold , or at least 8-fold , or at least 9-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 500-fold, or at least 1000-fold higher levels in skeletal muscle than in cardiac muscle. The nucleic acid may provide substantially no expression of the transgene in cardiac muscle. When the gene is a Capn3 gene, the nucleic acid may express the transgene at levels in cardiac muscle that produce substantially no adverse effects in the cardiac muscle. The nucleic acid may express the gene in slow fiber muscle fibers. The nucleic acid may express the transgene at said higher levels in slow fiber muscle fibers as compared to cardiac muscle.V. Administration of Therapeutic Compositions

[0103] The therapeutic agents of the disclosure may be administered by intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0104] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0105] In certain instances, it will be desirable to have multiple administrations of nucleic acids or viruses of the disclosure, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.UCLA.P0215WO Specification. docx - 37 -

[0106] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0107] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0108] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0109] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0110] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.UCLA.P0215WO Specification. docx - 38 -The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.[OHl] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0112] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0113] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above..VI. SequencesUCLA.P0215WO Specification. docx - 39 -UCLA.P0215WO Specification. docx -40 -UCLA.P0215WO Specification. docx -41 -UCLA.P0215WO Specification. docx -42 -UCLA.P0215WO Specification. docx -43 -UCLA.P0215WO Specification. docx -44 -UCLA.P0215WO Specification. docx -45 -UCLA.P0215WO Specification. docx -46 -UCLA.P0215WO Specification. docx -47 -UCLA.P0215WO Specification. docx -48 -UCLA.P0215WO Specification. docx -49 -UCLA.P0215WO Specification. docx - 50 -UCLA.P0215WO Specification. docx - 51 -VII. Examples

[0114] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1: Gene therapy for Limb Girdle Muscular Dystrophy type 2A

[0115] The overarching goal of this proposal is to develop a gene therapy for limb girdle muscular dystrophy type 2A (LGMD2A AKA, LGMD1R), an autosomal recessive (AR) muscle wasting disorder due to mutations in the CAPN3 gene encoding calpain 3 proteasefl]. LGMD2A is the most prevalent of the AR LGMDs and yet there is currently no treatment for patients, who are usually wheelchair dependent a decade after diagnosis. The inventors have shown that overexpression of Capn3 in skeletal muscle is non-toxic, and that a Capn3 cDNA fits without modification in AAV. These properties make gene therapy for LGMD2A a realistic goal. LGMD2A is unique with respect to gene therapy, because Capn3 expression in the heart is toxic [3], which is not the case with other gene therapies for neuromuscular disorders such as Duchenne muscular dystrophy (DMD) and other LGMDs [4], While pre-clinical, proof of concept studies have successfully accomplished AAV-Capn3 overexpression in mice [3], gene therapy constructs for humans with LGMD2A have not been developed, especially one that considers the relative skeletal muscle vs cardiac gene expression issues. Nor has any study been conducted to examine the minimal amount of Capn3 needed for a therapeutic effect. Furthermore, because LGMD2A preferentially impacts slow fibers [5], it is critical that the therapeutic construct that is ultimately used in clinical trials is optimized for slow fiber expression.

[0116] The current project is significant because it entails development of a treatment for a patient population in which there are no therapies. This application builds on 15 years of NIH investment that led to creation of mouse models, drugs and cell lines centered on understanding the biological basis of calpainopathy.A. The effect of the FLAG epitope on the expression of CAPN3 protein.UCLA.P0215WO Specification. docx - 52 -

[0117] Adding a FLAG epitope is widely used for detection of recombinant proteins if there are no antibodies suitable for the desired application (in our case immunohistochemistry to follow CAPN3 expression in slow and fast muscles). However, the inventors showed that adding the FLAG epitope had a negative impact on calpain 3 protein. Since the 2 vector preps (AAV#1 and AAV# 14) were packaged one year apart, and had different levels of empty to full capsids they chose to repeat this experiment using vectors produced at the same time. They made new constructs of RC#3 (SEQ ID NO:2) that were different only by the presence of the FLAG tag. These two constructs were packaged to MyoAAV and 1E1 Ivg were injected into the gastrocnemius of C3KO mice. As shown in FIG. 4, expression of CAPN3 was significantly higher without FLAG. These data strongly indicate that adding the FLAG epitope at the C terminus of CAPN3 significantly reduced CAPN3 levels, possibly making CAPN3 more unstable.B. Packaging AAV-CAPN3 constructs with MyoAAV and AAVMyo capsids

[0118] New myotropic AAV variants have been identified through screening peptide display libraries. The inventors have incorporated two such variants into the program (MyoAAV1or AAV-Myo2) and these were used to package the new constructs. Because these studies were underway before the discovery of the negative effect of the FLAG, all constructs had FLAG tags. High titer vectors using RC # 3, 7, 8, 9 in MyoAAV were produced, and RCs # 3, 4, 7, 8, 9, 10, 11, 12 and 13 in AAVMyo were produced. All these AAVs were generated using a codon optimized / CpG depleted (COD) CAPN3 transgene with FLAG.

[0119] Even though the inventors now have established that the FLAG is not optimal for CAPN3 expression, they continued to test all of them to see if differences in regulatory cassette activity could be discerned. The data revealed that very little calpain 3 protein could be detected after dosing due to the FLAG. The inventors plan to re-clone most of these RCs without FLAG, except for RCs 12 and 13 which showed no activity. All novel AAVs have been injected intramuscularly and retro-orbitally into C3KO mice (n=4 for each AAV). Due to high number of mice required for these injections, the injections were done in groups over a 2-month period. The data showed that RC#3 performed best in gastrocnemius but RC#7 performed best in soleus (see FIGS. 1-2), although RC7 did not show expression in soleus (FIG. 2) in this experiment. Since RC#3 looked promising and since they had already generated and packaged this construct in MyoAAV without the FLAG, the inventors carried out systemic administration and evaluation (Fig. 3). RC#3 is detargeted from the heart but expressed in gastroc and quad.UCLA.P0215WO Specification. docx - 53 -The inventors did not see expression in soleus (not shown). Expression is estimated to be approximately 15% of WT, but more quantitative westerns will need to be carried out. Furthermore, the dose was based on qPCR which underestimates the titer, so the doses are most likely much lower than indicated. These preps will be re-titered by ddPCR. The inventors are currently dosing this vector at higher doses by systemic administration and will carry out functional testing. They have now generated a new RC that includes RC#3 but with a slow enhancer to boost expression in slow muscles.1. Development and optimization of laboratory protocols for packaging MyoAAV (Spencer lab) and production and purification of AAVMyo (Chamberlain lab).2. Packaging and testing in vivo of 14 different AAVs in two different myotropic vectors.C. Testing the effect of codon optimization on the expression of CAPN3 protein.

[0120] In performing these experiments, The inventors noticed that the codon optimized transgene seemed to express more poorly than the WT transgene. Quantitative analysis of WBs compared the level of calpain 3 with calpains ’A (lower band on the blots). CK8e and SM mut promoters produced very similar level of CAPN3 expression in gastrocnemius. Nevertheless, WT CAPN3 expression with AAV6#1 was on average 2 times higher after injection of COD CAPN3 AAV#3. This comparison suggests that using codon optimized CAPN3 had a negative effect on CAPN3 levels (Fig.4). Taken together, our data indicate that in addition to the FLAG destabilizing calpain 3, and they believe the codon optimization negatively affected CAPN3 expression levels. The inventors further investigated codon optimization and engineered transcripts that had therapeutic levels of CAPN3 expression in fast and slow muscle fibers. These sequences include the DNA sequences of SEQ ID NOS: 17-21.UCLA.P0215WO Specification. docx - 54 -D. References

[0121] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. Ono, Y., et al., An eccentric calpain, CAPN3 / p94 / calpain-3. Biochimie, 2016. 122: p. 169- 87.2. Spencer, M.J., et al., Stable expression of calpain 3 from a muscle transgene in vivo: immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation. Proc Natl Acad Sci U S A, 2002. 99(13): p. 8874-9.3. Roudaut, C., et al., Restriction of calpain3 expression to the skeletal muscle prevents cardiac toxicity and corrects pathology in a murine model of limb-girdle muscular dystrophy. Circulation, 2013. 128(10): p. 1094-104.4. Crudele, J.M. and J.S. Chamberlain, AAV-based gene therapies for the muscular dystrophies. Hum Mol Genet, 2019.5. Kramerova, I., et al., Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3. Hum Mol Genet, 2012. 21(14): p. 3193-204.6. Kramerova, I., et al., Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro. Hum Mol Genet, 2004. 13(13): p. 1373-88.7. Kramerova, I., et al., Calpain 3 participates in sarcomere remodeling by acting upstream of the ubiquitin-proteasome pathway. Hum Mol Genet, 2005. 14(15): p. 2125-34.8. Spencer, M.J. and R.L. Mellgren, Overexpression of a calpastatin transgene in mdx muscle reduces dystrophic pathology. Hum Mol Genet, 2002. 11(21): p. 2645-55.9. Guyon, J.R., et al., Calpain 3 cleaves filamin C and regulates its ability to interact with gamma- and delta-sarcoglycans. Muscle Nerve, 2003. 28(4): p. 472-83.10. Kramerova, I., et al., Calpain 3 participates in sarcomere remodeling by acting upstream of the ubiquitin-proteasome pathway. Hum Mol Genet, 2007. 16(8): p. 1006.11. Ermolova, N., I. Kramerova, and M.J. Spencer, Autolytic activation of calpain 3 proteinase is facilitated by calmodulin protein. J Biol Chem, 2015. 290(2): p. 996-1004.12. Ermolova, N., et al., Pathogenity of some limb girdle muscular dystrophy mutations can result from reduced anchorage to myofibrils and altered stability of calpain 3. Hum Mol Genet, 2011. 20(17): p. 3331-45.13. Kramerova, I., et al., Novel role of calpain-3 in the triad-associated protein complex regulating calcium release in skeletal muscle. Hum Mol Genet, 2008. 17(21): p. 3271-80.UCLA.P0215WO Specification. docx - 55 -14. Kramerova, I., et al., Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy). Hum Mol Genet, 2016. 25(11): p. 2194-2207.15. Kramerova, I., et al., Regulation of the M-cadherin-beta-catenin complex by calpain 3 during terminal stages of myogenic differentiation. Mol Cell Biol, 2006. 26(22): p. 8437-47.16. Kramerova, I., et al., Mitochondrial abnormalities, energy deficit and oxidative stress are features of calpain 3 deficiency in skeletal muscle. Hum Mol Genet, 2009. 18(17): p. 3194- 205.17. Ramos, J.N., et al., Development of Novel Micro-dystrophins with Enhanced Functionality. Mol Ther, 2019. 27(3): p. 623-635.18. Chamberlain, J.R. and J.S. Chamberlain, Progress toward Gene Therapy for Duchenne Muscular Dystrophy. Mol Ther, 2017. 25(5): p. 1125-1131.19. Salva, M.Z., et al., Design of tissue-specific regulatory cassettes for high-level rAAV- mediated expression in skeletal and cardiac muscle. Mol Ther, 2007. 15(2): p. 320-9.20. Amacher, S.L., J.N. Buskin, and S.D. Hauschka, Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle. Mol Cell Biol, 1993. 13(5): p. 2753-64.21. Nguyen, Q.G., et al., Differences in the function of three conserved E-boxes of the muscle creatine kinase gene in cultured myocytes and in transgenic mouse skeletal and cardiac muscle. J Biol Chem, 2003. 278(47): p. 46494-505.22. Chistiakov, D.A., A.N. Orekhov, and Y.V. Bobryshev, Cardiac-specific miRNA in cardiogenesis, heart function, and cardiac pathology (with focus on myocardial infarction). J Mol Cell Cardiol, 2016. 94: p. 107-121.23. van Rooij, E., et al., A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance. Dev Cell, 2009. 17(5): p. 662-73.24. Young, C.S., et al., Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne Muscular Dystrophy and Application of a CRISPR / Cas9 Gene Editing Therapy. J Neuromuscul Dis, 2017. 4(2): p. 139-145.25. Young, C.S., et al., A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells. Cell Stem Cell, 2016. 18(4): p. 533-40.26. Baneijee-Basu, S. and A. Buonanno, cis-acting sequences of the rat troponin I slow gene confer tissue-and development-specific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice. Mol Cell Biol, 1993. 13(11): p. 7019-28.UCLA.P0215WO Specification. docx - 56 -27. Mullen, A. J. and P. J. Barton, Structural characterization of the human fast skeletal muscle troponin I gene (TNNI2). Gene, 2000. 242(1-2): p. 313-20.28. Corin, S.J., et al., Structure and expression of the human slow twitch skeletal muscle troponin I gene. J Biol Chem, 1994. 269(14): p. 10651-9.29. Corin, S.J., et al., Delineation of a slow-twitch-myofiber-specific transcriptional element by using in vivo somatic gene transfer. Proc Natl Acad Sci U S A, 1995. 92(13): p. 6185-9.30. Nelson, D.M., et al., Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins. Hum Mol Genet, 2018. 27(12): p. 2090- 2100.31. Halbert, C.L., J.M. Allen, and J.S. Chamberlain, AAV6 VectorProduction and Purification for Muscle Gene Therapy. Methods Mol Biol, 2018. 1687: p. 257-266.32. Nelson, D.M., et al., Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins. Hum Mol Genet, 2018.33. Thomson, K.S., et al., Translation of Cardiac Myosin Activation with 2-deoxy-ATP to Treat Heart Failure via an Experimental Ribonucleotide Reductase-Based Gene Therapy. JACC Basic Transl Sci, 2016. 1(7): p. 666-679.34. Foster, H., et al., Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2 / 8 gene transfer. Mol Ther, 2008. 16(11): p. 1825-32.35. Blankinship, M.J., et al., Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6. Mol Ther, 2004. 10(4): p. 671-8.36. Halbert, C.L., et al., Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration. J Virol, 1997. 71(8): p. 5932-41.37. Gregorevic, P., M.J. Blankinship, and J.S. Chamberlain, Viral vectors for gene transfer to striated muscle. Curr Opin Mol Ther, 2004. 6(5): p. 491-8.38. Gregorevic, P., et al., Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat Med, 2004. 10(8): p. 828-34.39. Gregorevic, P., et al., rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nat Med, 2006. 12(7): p. 787-9.40. El-Khoury, V., et al., Disruption of autophagy by the histone deacetylase inhibitor MGCD0103 and its therapeutic implication in B-cell chronic lymphocytic leukemia. Leukemia, 2014. 28(8): p. 1636-46.UCLA.P0215WO Specification. docx - 57 -41. Kawai, H., et al., Clinical, pathological, and genetic features of limb-girdle muscular dystrophy type 2A with new calpain 3 gene mutations in seven patients from three Japanese families. Muscle Nerve, 1998. 21(11): p. 1493-501.42. Cox, G.A., et al., Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity. Nature, 1993. 364(6439): p. 725-9.43. Ermolova, N.V., et al., Long-term administration of the TNF blocking drug Remicade (cVlq) to mdx mice reduces skeletal and cardiac muscle fibrosis, but negatively impacts cardiac function. Neuromuscul Disord, 2014. 24(7): p. 583-95.44. Tabebordbar, M. et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell 184, 4919-4938 e4922, doi: 10.1016 / j.cell.2021.08.028 (2021).45. Weinmann, J. et al. Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nat Commun 11, 5432, doi: 10.1038 / s41467-020- 19230-w (2020).UCLA.P0215WO Specification. docx - 58 -Example 2: RC3, an AAV Regulatory Cassette for Skeletal Muscle-Restricted Expression

[0122] LGMD2A, also known as calpainopathy, is the most common autosomal recessive form of limb-girdle muscular dystrophy. It is caused by mutations in the CAPN3 gene, which encodes calpain-3, a calcium-dependent protease critical for muscle function. There is currently no cure for LGMD2A, and current treatments are largely supportive, such as physical therapy to maintain mobility, orthopedic interventions for contractures, respiratory support in advanced cases, anti-fibrotic agents, and muscle regeneration enhancers. Researchers have explored gene therapies that deliver CAPN3 gene. The challenges with gene therapies that deliver the CAPN3 gene include the toxicity of CAPN3 expression in the heart and the challenge of delivering therapeutically high expression levels of CAPN3 in fast and slow muscle fibers. The Ck8e promoter is commonly used for neuromuscular disease indications. However, it expresses in the heart and exhibits much higher expression in fast fibers compared to slow fibers (FIG. 8). Sahenk et al (Mol Ther Methods Clin Dev. 2021 Jun 24;22:401-414) used a mutant Ck8e promoter (tMCK promoter) to express human CAPN3 in mouse models. The results showed improved muscle structure and function, with no observed toxicity. However, mouse muscle fibers are almost entirely comprised of fast fibers, while human muscles are about half fast and half slow muscle fibers. In fact, Wang et al. (Gene Ther 75, 1489-1499) studied the same tMCK promoter and found that, while tMCK is detargeted from the heart, it attains only about 15% of wild type calpain 3 levels and it does not express in slow fibers. Therefore, the therapeutic efficacy of this promoter is not sufficient, and it is critical that any gene therapy strategy that delivers a functional CAPN3 gene detarget expression of the transgene in the heart and overexpress the CAPN3 protein in both fast and slow muscle fibers.

[0123] The inventors found that simply combining slow and fast enhancers in different combinations does not provide the desired expression pattern (heart detargeting and expression in both slow and fast muscles). Shown in FIG. 9 are examples of calpain 3 expression from different regulatory cassettes after in vivo dosing of AAV-RC-Capn3. In this experiment, mice were dosed with AAVMyo-packaged with the RC and the wild type calpain 3 cDNA. Mice were dosed at 2el4 vg / kg RO in vivo and analyzed 5 weeks post dosing. The sequences of RC#7, 8, and 11 are below:UCLA.P0215WO Specification. docx - 59 -UCLA.P0215WO Specification. docx -60-UCLA.P0215WO Specification. docx -61 -

[0124] The inventors developed adeno-associated virus (AAV) regulatory cassettes designed for gene therapy of neuromuscular disorders. The cassette enables transgene expression in both fast- and slow-twitch skeletal muscle fibers while preventing expression in non-target tissues such as the heart and liver. This expression pattern is critical for the treatment of several neuromuscular conditions, including Limb-girdle muscular dystrophy type 2A (LGMD2A, also known as LGMD1R), an autosomal recessive muscle-wasting disorder caused by mutations in the CAPN3 gene, X-linked myotubular myopathy, FacioscapulohumeralUCLA.P0215WO Specification. docx - 62 -muscular dystrophy (FSHD), FKRP -related muscular dystrophy (LGMD2I), and Nemaline myopathy, among others.

[0125] The inventors engineered a synthetic regulatory cassette termed SURE-RC3, which drives robust expression in both slow and fast skeletal muscle fibers while excluding expression in cardiac and hepatic tissues. SURE-RC3 incorporates: i) a modified version of the muscle creatine kinase (MCK) promoter, which includes specific mutations to eliminate cardiac expression without compromising skeletal muscle activity; and ii) a human enhancer element, SURE (7), to augment transcriptional output in skeletal muscle. A humanized version of RC3, incorporating the human mutant MCK promoter, has also been developed. As shown in FIG. 10, the incorporation of the SURE enhancer boosted the Calpain 3 levels in the slow (soleus) and fast (gastrocnemius) fibers. This increase was unpredictable, since we had previously found that dosing with a combination of vectors that express well in one fiber type or the other is not sufficient to produce therapeutic levels of calpain 3 (FIG. 11).

[0126] The SURE-RC3 was tested in vivo in Q / / w3-knockout mice (13), a well-established model of LGMD2A. After 5 weeks post dosing, AAV-mediated delivery at doses of 5 * 1013and 2 x io14vg / kg resulted in:• >100% restoration of calpain 3 protein levels across multiple skeletal muscles (including soleus, diaphragm, tibialis anterior, and gastrocnemius)• No detectable expression in heart or liver using an antibody that can detect 3% calpain 3• Functional correction of fatty acid metabolism abnormalities in treated Capn3-nvX\ mice• Confirmed activity in primary human myotubes

[0127] These findings support the utility of RC3 as a versatile and clinically relevant regulatory cassette for skeletal muscle-targeted gene therapy in multiple neuromuscular disorders.UCLA.P0215WO Specification. docx - 63 -REFERENCES1. Roudaut, C., Le Roy, F., Suel, L., Poupiot, J., Charton, K., Bartoli, M., and Richard, I. (2013). Restriction of calpain3 expression to the skeletal muscle prevents cardiac toxicity and corrects pathology in a murine model of limb-girdle muscular dystrophy. Circulation 128, 1094-1104. 10.1161 / CIRCULATIONAHA.113.001340.2. Sahenk, Z., Ozes, B., Murrey, D., Myers, M., Moss, K., Yalvac, M.E., Ridgley, A., Chen, L., and Mendell, J.R. (2021). Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A / R1. Mol Ther Methods Clin Dev 22, 401-414. 10.1016 / j.omtm.2021.06.010.3. Wang, B., Li, J., Fu, F.H., Chen, C., Zhu, X., Zhou, L., Jiang, X., and Xiao, X. (2008). Construction and analysis of compact muscle-specific promoters for AAV vectors. Gene Ther15, 1489-1499. 10.1038 / gt.2008.104.4. Ono, Y., Ojima, K., Shinkai-Ouchi, F., Hata, S., and Sorimachi, H. (2016). An eccentric calpain, CAPN3 / p94 / calpain-3. Biochimie 122, 169-187. 10.1016 / j biochi.2015.09.010.5. Martin, C., and Servais, L. (2025). X-linked myotubular myopathy: an untreated treatable disease. Expert Opin Biol Ther 25, 379-394. 10.1080 / 14712598.2025.2473430.6. Preston, M.K., and Wang, L.H. (1993). Facioscapulohumeral Muscular Dystrophy. In GeneReviews((R)), M.P. Adam, J. Feldman, G.M. Mirzaa, R.A. Pagon, S.E. Wallace, and A. Amemiya, eds.7. Calvo, S., Vullhorst, D., Venepally, P., Cheng, J., Karavanova, I., and Buonanno, A. (2001). Molecular dissection of DNA sequences and factors involved in slow muscle-specific transcription. Mol Cell Biol 21, 8490-8503. 10.1128 / MCB.21.24.8490-8503.2001.8. Nguyen, Q.G., Buskin, J.N., Himeda, C.L., Fabre-Suver, C., and Hauschka, S.D. (2003). Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types. Transgenic Res 12, 337-349. 10.1023 / a: 1023369225799.9. Nguyen, Q.G., Buskin, J.N., Himeda, C.L., Shield, M.A., and Hauschka, S.D. (2003). Differences in the function of three conserved E-boxes of the muscle creatine kinase gene in cultured myocytes and in transgenic mouse skeletal and cardiac muscle. J Biol Chem 278, 46494-46505. 10.1074 / jbc.M308194200.10. Shield, M.A., Haugen, H.S., Clegg, C.H., and Hauschka, S.D. (1996). E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice. Mol Cell Biol16, 5058-5068. 10.1128 / MCB.16.9.5058.UCLA.P0215WO Specification. docx - 64 -11. Amacher, S.L., Buskin, J.N., and Hauschka, S.D. (1993). Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle. Mol Cell Biol 13, 2753-2764. 10.1128 / mcb.13.5.2753.12. Apone, S., and Hauschka, S.D. (1995). Muscle gene E-box control elements. Evidence for quantitatively different transcriptional activities and the binding of distinct regulatory factors. J Biol Chem 270, 21420-21427. 10.1074 / jbc.270.36.21420.13. Kramerova, I., Kudryashova, E., Tidball, J.G., and Spencer, M.J. (2004). Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro. Hum Mol Genet 13, 1373-1388. 10.1093 / hmg / ddhl53.* * *

[0128] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.UCLA.P0215WO Specification. docx - 65 -

Claims

WHAT IS CLAIMED IS:

1. A method for treating limb girdle muscular dystrophy (LGMD) in a human subject comprising administering a nucleic acid comprising a regulatory promoter and a transgene, wherein the regulatory promoter comprises SEQ ID NO:7 and wherein the transgene encodes for the Calpain 3 (CAPN3) protein.

2. A nucleic acid comprising a regulatory promoter, wherein the regulatory promoter comprises SEQ ID NO:7.

3. A nucleic acid comprising a regulatory promoter, wherein the regulatory promoter comprises a nucleic acid sequence with at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 7, 10, 12 or 13.

4. The nucleic acid of claim 3, wherein the regulatory promoter comprises a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 7, 10, 12, or 13.

5. A nucleic acid comprising a regulatory promoter, wherein the regulatory promoter comprises regulatory element A that has a nucleic acid sequence with at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 and regulatory element B that has a nucleic acid sequence with at least 75% sequence identity to the nucleic acid sequence of one of SEQ ID NO:2, 12, or 13.

6. The nucleic acid sequence of claim 5, wherein regulatory element A has the nucleic acid sequence of SEQ ID NO: 1 and regulatory element B has the nucleic acid sequence of SEQ ID N0:2, 12, or 13.

7. The nucleic acid sequence of claim 6, wherein element A and element B are separated by less than 100 nucleotides.

8. The nucleic acid sequence of any one of claims 5-7, wherein element A is upstream of element B.

9. The nucleic acid sequence of any one of claims 5-7, wherein element B is upstream of element A.

10. The nucleic acid of any one of claims 3-9, wherein the nucleic acid is isolated.

11. The nucleic acid of any one of claims 3-10, wherein the nucleic acid further comprises a nucleic acid sequence encoding a transgene and wherein the regulatory promoter directs the expression of the transgene.

12. The nucleic acid of claim 11, wherein the transgene is downstream of the regulatory promoter.

13. The nucleic acid of claim 11 or 12, wherein the transgene comprises a full-length cDNA of the gene.UCLA.P0215WO Specification. docx - 66 -14. The nucleic acid of any one of claims 11-13, wherein the transgene comprises a UTR- deleted cDNA.

15. The nucleic acid of any one of claims 11-13, wherein the transgene comprises a Kozak- optimized cDNA.

16. The nucleic acid of any one of claims 11-15, wherein the transgene comprises Calpain3 (CAPN3).

17. The nucleic acid of any one of claims 11-16, wherein the transgene comprises a human protein.

18. The nucleic acid of claim 16 or 17, wherein the transgene comprises the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that has at least 75% sequence identity to the amino acid sequence of SEQ ID NO:6.

19. The nucleic acid of any one of claims 16-18, wherein the transgene is encoded on a nucleic acid having the sequence of one of SEQ ID NOs: 17-21, or a nucleic acid sequence with at least 80% sequence identity to one of SEQ ID NOs: 17-21.

20. The nucleic acid of any one of claims 11-18, wherein the nucleic acid sequence encoding the transgene is not codon optimized.

21. The nucleic acid of any one of claims 11-19, wherein the nucleic acid sequence encoding the transgene is codon optimized.

22. The nucleic acid of any one of claims 11-21, wherein the nucleic acid sequence encoding the transgene is CpG depleted.

23. The nucleic acid of any one of claims 16-22, wherein the nucleic acid encoding the transgene comprises the nucleic acid sequence of SEQ ID NO:5 or a nucleic acid sequence that has at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

24. The nucleic acid of any one of claims 3-23, wherein the nucleic acid does not comprise a miRNA binding site.

25. The nucleic acid of any one of claims 3-24, wherein the nucleic acid does not comprise a miR208 binding site.

26. The nucleic acid of any one of claims 3-25, wherein the nucleic acid is further defined as an expression cassette.

27. The nucleic acid of claim 26, wherein the expression cassette is present in a gene therapy vector.

28. The nucleic acid of claim 27, wherein said cassette is present in a gene therapy vector selected from the group consisting of a lentiviral vector (LV), an adenovirus vector (AV), and an adeno-associated viral vector (AAV).UCLA.P0215WO Specification. docx - 67 -29. The nucleic acid of claim 28, wherein said gene therapy vector comprises an adeno- associated viral vector (AAV).

30. The nucleic acid of claim 29, wherein said AAV vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9.

31. The nucleic acid of any one of claims 28-30, wherein said AAV vector comprises an AAV2 backbone.

32. The nucleic acid of claim 31, wherein said AAV vector comprises an AAV2 vector pseudotyped with an AAV6 capsid (AAV2 / 6).

33. The nucleic acid of claim 28, wherein said gene therapy vector is a lentiviral vector.

34. The nucleic acid of claim 33, wherein said vector is an HIV-1 lentiviral vector.

35. The nucleic acid of any one of claims 11-34, wherein the nucleic acid further comprises a nucleic acid sequence comprising inverted terminal repeats (ITR) and wherein a first ITR is upstream of the regulatory promoter and a second ITR is downstream of the transgene.

36. The nucleic acid of claim 35, wherein the ITR(s) comprises the nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:3.

37. The nucleic acid of any one of claims 11-34, wherein the nucleic acid comprises an intron between the nucleic acid sequence encoding the transgene and the regulatory promoter.

38. The nucleic acid of claim 37, wherein the intron comprises the nucleic acid sequence of SEQ ID NO:4 or 8 or a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 4 or 8.

39. The nucleic acid of any one of claims 3-38, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NOV or a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NOV.

40. A nucleic acid comprising the sequence of one of SEQ ID NOS: 17-21, or an amino acid sequence that has at least 80% sequence identity to one of SEQ ID NOS: 17-21.

41. The nucleic acid of claim 40, wherein the nucleic acid further comprises a promoter that directs the expression of the nucleic acid.

42. The nucleic acid of claim 41, wherein the promoter comprises the regulatory promoter of any one of claims 3-39.

43. A viral particle comprising the nucleic acid of any one of claims 3-42.

44. A host cell comprising the nucleic acid of any one of claims 3-42.

45. The host cell of claim 44, wherein the host cell is a viral packaging cell.UCLA.P0215WO Specification. docx - 68 -46. A method comprising incubating the host cell of claim 45 under conditions suitable for the production of viral particles and isolating viral particles.

47. A viral particle produced by the host cell of claim 46.

48. A virus comprising a plurality of viral particles according to claim 47 or 43.

49. A method of making an engineered host cell comprising transferring the nucleic acid of any one of claims 3-42 into the host cell.

50. A method of treating a subject with a skeletal muscle disorder, said method comprising administering the nucleic acid of any one of claims 3-42, the viral particle of claim 43 or 47, the host cell of claim 44 or 45, or the virus of claim 48 to the subject.

51. A method of expressing a transgene in a muscle cell of a subject, the method comprising administering the nucleic acid of any one of claims 3-42, the viral particle of claim 43 or 47, the host cell of claim 44 or 45, or the virus of claim 48 to the subject.

52. The method of claim 51, wherein the subject has a skeletal muscle disorder.

53. The method of any one of claims 50-52, wherein the transgene is expressed in the subject and wherein the expression level of the transgene is at levels at least 2-fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold , or at least 7-fold , or at least 8- fold , or at least 9-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 500-fold, or at least 1000-fold higher levels in skeletal muscle than in cardiac muscle.

54. The method of any one of claims 50-53, wherein there is substantially no expression of the transgene in cardiac muscle cells.

55. The method of any one of claims 50-54, wherein there is no detectable expression of the transgene in cardiac muscle.

56. The method of any one of claims 50-55, wherein the transgene is expressed in muscle cells of slow fiber muscle fibers.

57. The method of any one of claims 50-56, wherein the transgene is expressed at higher levels in slow fiber muscle cells as compared to cardiac muscle cells.

58. The method of any one of claims 50 or 52-57, wherein the skeletal muscle disorder comprises limb girdle muscular dystrophy (LGMD), Limb-girdle muscular dystrophy type 2A (LGMD2A), X-linked myotubular myopathy, Facioscapulohumeral muscular dystrophy (FSHD), FKRP-related muscular dystrophy (LGMD2I), or Nemaline myopathy.

59. The method of any one of claims 50-58, wherein the subject is a human subject.

60. A nucleic acid comprising a regulatory promoter comprising the nucleic acid sequence of SEQ ID NO: 7, 10, 12 or 13 and a transgene, wherein the regulatory promoter initiates transcription of the transgene and wherein the transgene encodes for CAPN3.UCLA.P0215WO Specification. docx - 69 -61. Viral particles comprising the nucleic acid of claim 60.

62. A composition comprising the nucleic acid of claim 60 or the viral particles of claim 61.

63. A method of treating a subject for limb girdle muscular dystrophy (LGMD) comprising administering the composition of claim 62 to a subject having LGMD.UCLA.P0215WO Specification. docx - 70 -

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