Gene therapy for treating limb-girdle muscular dystrophy R9 and congenital muscular dystrophy 1C

Abstract

This invention relates to a method and materials for treating limb-girdle muscular dystrophy R9 (LGMDR9) and congenital muscular dystrophy 1C (MDC1C) using a bicistronic recombinant adeno-associated virus encoding fukutin-related protein (FKRP) and follistatin (FST). This therapy is unique in that it can reconstruct lost muscle strength while simultaneously preventing the subsequent development of muscle disease.

Description

Technical Field

[0001] This application claims the priority of U.S. Provisional Patent Application No. 63 / 460,124, filed on April 18, 2023, which is hereby incorporated by reference in its entirety. Incorporation by Reference of Sequence Listing This application includes a sequence listing in computer-readable form, which is incorporated by reference in its entirety as a separate part of this disclosure and is specified as follows: 59037_SeqListing.xml, size: 26,083 bytes, creation date: April 17, 2024.

[0002] The present invention relates to methods and materials for treating limb-girdle muscular dystrophy R9 (LGMDR9) and congenital muscular dystrophy 1C (MDC1C) using a bicistronic recombinant adeno-associated virus encoding fukutin-related protein (FKRP) and follistatin (FST).

Background Art

[0003] Gene replacement therapies using adeno-associated virus (AAV) have now been shown to significantly improve patient outcomes in several genetic diseases, including hemophilia A, Leber congenital amaurosis, Duchenne muscular dystrophy, and spinal muscular atrophy type 1. In all of these examples, normal or miniaturized normal copies of the disease-causing mutant gene have been introduced to replace the lost gene function. Such an approach can have a major impact on disease progression, but gene replacement typically does not reverse an already existing disease. Such drawbacks can be very important in degenerative diseases such as muscular dystrophy, where muscle weakness is most frequently caused by the progressive loss of muscle tissue. In theory, gene replacement could completely prevent muscular dystrophy if given at the pre-disease stage, but most gene therapies will likely need to be given after patients have already become debilitated by the disease, especially for adult-onset disorders. In the case of muscular dystrophy, this will manifest as impairment of motor function due to loss of muscle mass and strength.

[0004] Limb-girdle muscular dystrophy R9 (LGMDR9; formerly known as LGMD2I) is caused by homozygous recessive loss of functional mutations in the FKRP gene (Non-Patent Literature 1), and FKRP mutations cause LGMDR (Non-Patent Literature 2, Non-Patent Literature 3), and the more severe congenital muscular dystrophy 1C (MDC1C) (Non-Patent Literature 1, Non-Patent Literature 3, Non-Patent Literature 4) by reducing the functional glycosylation of alpha-dystroglycan (aDG). Dystroglycans are members of the dystrophin-associated glycoprotein (DAG) complex, which is a complex that links ECM proteins to the F-actin cytoskeleton in muscle cells across the membrane (Non-Patent Literature 5). DG is post-translationally cleaved by autoproteolytic mechanisms to produce aDG, an extracellular membrane-associated glycoprotein, and beta-dystroglycan (bDG (also known as J3 dystroglycan (J3DG))), a transmembrane glycoprotein (Non-Patent Literature 6). Extracellular matrix (ECM) proteins, including laminin that covers the muscle fiber membrane (Non-Patent Documents 7, 8, and 9), bind to aDG via essential repeat glycosaminoglycan disaccharides produced by glycans present in their mucin domains, particularly LARGE (Xylalpha1,3-GlcAbeta1,3) (Non-Patent Document 10). The production of these glycans requires at least 18 genes, including FKRP, which help construct the glycan substrate that acts to enable LARGE to complete the glycosylation pathway. Without proper glycosylation, ECM proteins cannot bind to aDG within the muscle membrane, leading to muscular dystrophy. These hereditary disorders are collectively known as dystroglycanopathy.

[0005] The introduction of the AAV.FKRP gene substitution has been shown to restore functional glycosylation of aDG and prevent disease pathology in mouse models of LGMDR9 (Non-Patent Literature 11, 12, 13, 14). However, such therapeutic effects require careful administration because overexpression of FKRP can cause dose-dependent pathology (Non-Patent Literature 14). Ribitol and NAD+glycan therapy also showed therapeutic effects in FKRP mutant mice (Non-Patent Literature 15, 16). Additional ribitol may increase the concentration of CDP-ribitol, a substrate required for FKRP glycosylation of aDG by ribitol-6-phosphate (Non-Patent Literature 15). Gene substitution studies in LGMDR9 mouse models clearly show that single FKRP gene therapy can prevent loss of gait and muscle pathology in young mice, but the therapeutic effect is much lower when administered to older mice where the disease is already pronounced (Non-Patent Literature 17, 18).

[0006] While these proof-of-concept results for gene replacement are encouraging, LGMDR9 is a very slow-progressing, genetically heterogeneous disease, making it extremely difficult to test treatments that only halt disease progression. An MRI study of LGMDR9 patients by Volker Straub et al. suggested 0–2% annual fat replacement of muscle mass in 14 different leg muscles (Non-Patent Literature 19). While the MRI study over one year clearly showed muscle mass loss, measurements of hip, knee, or ankle flexion / extension or adduction / abduction, or the time to walk or stand up, differed significantly when comparing 32 subjects over the same period (Non-Patent Literature 19). Therefore, while gene replacement for these diseases may halt subsequent muscle damage and loss, the slow and variable clinical progression of LGMDR9 makes it extremely difficult, perhaps impossible, to demonstrate clinically significant improvement in motor function over a one-year clinical trial period.

[0007] Therefore, the need in the art for novel and effective methods for treating LGMDR9 and MDC1C remains. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Brockington et al.,Hum Mol Genet 10:2851-9,2001 [Non-Patent Document 2] Brockington et al., Am J Hum Genet 69(6):1198-209,2001 [Non-Patent Document 3] Brown et al., Am.J. Pathol.164(2):727-37,2004 [Non-Patent Document 4] Longman et al.,Hum Mol Genet.12(21):2853-61,2003 [Non-Patent Document 5] Ervasti et al.,Cell 66(6):1121-31,1992 [Non-Patent Document 6] Akhaven et al., FASEB 22(2):612-21,2008 [Non-Patent Document 7] Ervasti et al., J.Cell Biol 122(4):809-23,1993 [Non-Patent Document 8] Michele et al.J.Biol.Chem.278(18):15457-60,2003 [Non-Patent Document 9] Michele et al. Nature 418 [Non-Patent Document 10] Inamori et al.Science 335(6064):93-6,2012 [Non-Patent Document 11] Xu et al.Mol Ther,21(10):p.1832-40,2013 [Non-Patent Document 12] Qiao et al.Mol Ther,2014.22(11):p.1890-9,2014 [Non-Patent Document 13] Awano et al.Am J Pathol,2015.185(7):p.2025-37,2015 [Non-Patent Document 14] Gicquel et al.,Hum Mol Genet.26(10):p.1952-1965,2017 [Non-Patent Document 15] Kanagawa et al.J.Neuromuscul Dis.4(4):259-267,2017 [Non-Patent Document 16] Baily et al.,Skelet Muscle 9(1):21,2019 [Non-Patent Document 17] Vannoy et al.Mol Ther Methods Clin Dev 5:31-42,2017 [Non-Patent Document 18] Vannoy et al.Mice.Mol Ther Methods Clin Dev 11:106-120,2018 [Non-Patent Document 19] Willis et al.,PLoS One 8(8):e70993,2013 [Overview of the project]

[0009] A bicistronic gene therapy approach that adds muscle-building components to gene replacement is disclosed herein. Such therapies have the potential to reconstruct muscle loss while simultaneously halting subsequent disease progression. For example, the bicistronic gene therapy disclosed herein is used for FKRP in dystrophilia-girdle muscular dystrophy R9 (LGMDR9), an adult-onset muscular dystrophy. P448LThe study was conducted using a mouse model, a knock-in mouse model containing the P448L human disease mutation (Chan et al., Hum Mol Genet, 2010.19(20):p.3995-4006, 2010).

[0010] The data provided herein demonstrate that the inclusion of two transcriptional regulatory sequences, e.g., a promoter with an enhancer, can synergistically amplify dual transgene expression in skeletal muscle with muscular dystrophy. Such a gene therapy approach has restored lost walking ability, essentially reversing the disease, something that monogenetic replacement therapy has not been shown to do. In addition, the bicistronic (two-gene) vector described herein amplifies the expression levels of both therapeutic transgenes compared to the use of a single promoter / enhancer alone. This disclosure provides a polynucleotide sequence comprising, in 5' to 3' order, AAV ITR, a first transcriptional regulatory sequence, an intron, a first transgene sequence, a polyadenylation signal sequence, a second transcriptional regulatory sequence (having a promoter, enhancer, and intron), a second (but different) transgene sequence, a second polyadenylation signal sequence, and AAV ITR.

[0011] Transcriptional regulatory sequences include, but are not limited to, promoters, enhancers, and / or polyadenylation signal sequences. Examples of transcriptional regulatory sequences include the cytomegalovirus (CMV promoter), CMV enhancer, miniCMV promoter, MHCK7, CK8 promoter, chicken β-actin promoter, P546 promoter, monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Rous sarcoma virus promoter, and human gene promoters, including, but are not limited to, actin promoters, myosin promoters, elongation factor-1a promoters, hemoglobin promoters, and creatine kinase promoters.

[0012] In exemplary embodiments, the first transcriptional regulatory sequence is a muscle-specific regulatory element. The term “muscle-specific regulatory element” refers to a nucleotide sequence that regulates the expression of a coding sequence that is specific to expression in muscle tissue. These regulatory elements include enhancers and promoters. This disclosure provides a construct comprising a muscle-specific regulatory element MHCK7 promoter (a muscle creatine kinase promoter having a hybrid intron (7), an MCK promoter, and an MCK enhancer / or an alpha-myosin heavy chain (MHC) complex enhancer).

[0013] Examples of muscle-specific promoters include one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer-binding factor (MEF) binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MHC enhancer-promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast-twitch muscle troponin C gene element, slow-twitch muscle cardiac troponin c gene element, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factor (HIF)-response element (HRE), steroid-inducible element, and glucocorticoid-response element (GRE).

[0014] In some embodiments, the first transgene is a gene encoding a protein that is reduced, deleted, or eliminated in subjects suffering from degenerative muscle disorders such as muscular dystrophy. Exemplary transgenes include calpain gene 3 (CAPN3), alpha sarcoglycan gene (SGCA), beta sarcoglycan gene (SGCB), gamma sarcoglycan gene (SGCG), detra sarcoglycan gene (SGCD), epsilon sarcoglycan gene (SGCE), terethionine gene (TCAP), tripaltate motif-containing protein 32 (TRIM32), fukutin-related protein gene (FKRP), fukutin gene (FKTN), protein O-mannosyltransferase 1 gene (POMT2), protein O-mannosyltransferase 2 gene (POMT2), anoctamin 5 gene (ANO5), and protein O-binding mannose N- Actetylglucosyltransferase 1 gene (POMGnT1), dystroglycan gene (DAG1), decine gene (DES), GDP-mannose pyrophosphorylase B gene (GMPPB), isoprenoid synthase domain-containing gene (ISPD), alpha-glucosidase gene (GAA), LIM dyssin finger domain-containing 2 gene (LIMS2), vascular epicardial structure gene (BVES), tosin 1A interaction protein gene (TORIA1P1), protein O-glucosyltransferase 1 gene (POGLUT1), dysferin gene (DYSF), dystrophin gene (DMD), UDP-GlcNAc epimerase / ManNAc These include the 6 kinase gene (GNE), UDP-N-acetyl-galactosaminyltransferase 2 gene (GALGT2 or B4GALNT2), myotirin gene (MYOT), lamin A / C gene (LMNA), and caveolin gene (CAV1).

[0015] In an exemplary embodiment, the first transgene is a fukutin-related protein (FKRP) gene. The FKRP gene sequence contains nucleotide sequence 3024-4511 of SEQ ID NO: 1, or contains a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotide sequence 3024-4511 of SEQ ID NO: 1, and encodes a functional FKRP protein. The FKRP gene encodes a protein containing the amino acid sequence of SEQ ID NO: 3.

[0016] In some embodiments, the second transgene is a gene encoding a muscle-building protein. Muscle-building proteins are proteins that build new muscle mass and induce muscle growth, and include proteins that stimulate muscle growth signals or inhibit inhibitory muscle growth signals. Exemplary transgenes include follistatin genes (FST), e.g., follistatin 344 (FS344) for follistatin 317 (FS317), insulin-like growth factor 1 gene (IGF1), heparin-binding epidermal growth factor-like growth factor gene (HB-EGF), or maternal (SMAD7) for the decapentaplesic homolog 7 gene.

[0017] In an exemplary embodiment, the second transgene is a follistatin (FST) gene encoding the protein morphology FS344. The FST(FS344) gene sequence contains nucleotides 5393-6427 of SEQ ID NO: 1, or a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 5393-6427 of SEQ ID NO: 1, and encodes a functional FS344 protein. The FST(FS344) gene encoded a protein containing the amino acid sequence of SEQ ID NO: 5.

[0018] A polynucleotide sequence comprising AAV ITR, a first transcriptional regulatory sequence operably ligated to a nucleotide sequence encoding an FKRP protein such as a protein containing the amino acid sequence of SEQ ID NO: 3, in the order of 5' to 3', a second transcriptional regulatory sequence operably ligated to a nucleotide sequence encoding an FS344 protein such as a protein containing the amino acid sequence of SEQ ID NO: 5, and AAV ITR.

[0019] The FKRP gene sequence contains nucleotide sequences 3024-4511 of SEQ ID NO: 1, or contains nucleotide sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 3024-4511 of SEQ ID NO: 1, and encodes a functional FKRP protein. The FKRP gene encodes a protein containing the amino acid sequence of SEQ ID NO: 3.

[0020] The FS344 gene sequence contains nucleotides 5393-6427 of SEQ ID NO: 1, or a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 5393-6427 of SEQ ID NO: 1, and encodes a functional FS344 protein. The FS344 gene encoded a protein containing the amino acid sequence of SEQ ID NO: 5.

[0021] In any of the polynucleotides disclosed herein, the first transcriptional regulatory sequence is the MHCK7 promoter. For example, the MKCK promoter sequence includes nucleotides 2056-2846 of SEQ ID NO: 1. Furthermore, in any of the polynucleotides disclosed herein, the second transcriptional regulatory sequence is the CMV promoter and / or the CMV promoter. For example, the CMV promoter includes nucleotides 4591-5117 of SEQ ID NO: 1.

[0022] In some embodiments, any of the polynucleotides disclosed herein further comprises an SV40 enhancer and / or an intron such as an SV40 intron or a chimeric intron. For example, the SV40 enhancer comprises nucleotides 2858-3004 of SEQ ID NO: 1 or nucleotides 5188-5382 of SEQ ID NO: 1. Furthermore, any of the polynucleotides disclosed herein further comprises a polyadenylation signal sequence, which is optionally a synthetic polyadenylation signal sequence. The polynucleotide sequences disclosed herein include inverted end repeats (ITRs), such as mutant ITRs or wild-type ITRs.

[0023] This disclosure also provides polynucleotide sequences that constitute an AAV genome. For example, this disclosure provides an AAV genome or polynucleotide sequence comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 1847-6672 of SEQ ID NO: 1. In addition, this disclosure provides an AAV genome or polynucleotide sequence comprising nucleotides 1847-6672 of SEQ ID NO: 1.

[0024] In the context of nucleic acid sequences or amino acid sequences, the terms “sequence identity,” “percent sequence identity,” or “percent identity” refer to residues in two sequences that are identical when aligned to the greatest extent possible. The length of the sequence identity comparison can be the full length of the genome, the full length of the gene coding sequence, or, if desired, a fragment of at least about 500–5000 nucleotides. However, identity between smaller fragments, e.g., at least about 9 nucleotides, typically at least about 20–24 nucleotides, at least about 28–32 nucleotides, at least about 36 nucleotides, or more, may also be desired. The percentage of sequence identity can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, ClustalW2, and BLAST. In one embodiment, when BLAST is used as an alignment tool, the following default parameters are used: Genetic code = standard; filter = none; strand = both; cutoff = 60; prediction = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swissprotein+Spupdate+PIR.

[0025] In addition, the rAAV genome provided herein hybridizes under stringent conditions to the polynucleotide sequence of nucleotides 1847-2013 of SEQ ID NO: 1 or its complement.

[0026] This disclosure also provides recombinant adeno-associated viruses (rAAVs) comprising any of the polynucleotide sequences described herein. For example, rAAVs include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAVrh.74, AAVrh.10, Anc80, AAV7m8, AAVMyo, MYOAAV capsid protein, or variants thereof. In some embodiments, rAAV is the AAVrh.74 serotype. In some embodiments, rAAV is the AAVMYO3a serotype.

[0027] This disclosure also provides recombinant AAV particles comprising any of the polynucleotide sequences disclosed herein or any of the rAAVs disclosed herein. In another embodiment, the Disclosure provides a method for producing rAAV vector particles, which comprises culturing cells transfected with any rAAV vector of the Disclosure and recovering rAAV particles from the supernatant of the transfected cells. The Disclosure also provides viral particles comprising any of the recombinant AAV vectors of the Disclosure.

[0028] This disclosure also provides compositions comprising any of the rAAVs disclosed herein or any of the rAAV particles described herein. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier. The compositions may also comprise other components such as diluents and adjuvants. The acceptable carriers, diluents and adjuvants are nontoxic to the recipient, preferably inert at the dosage and concentration employed, and comprise buffers and surfactants such as Pluronic®.

[0029] This disclosure also provides a method for treating muscular dystrophy, which comprises administering to a subject in need of treatment any of the rAAVs disclosed herein, or any rAAV particles disclosed herein, or any of the compositions disclosed herein. For example, the subject is suffering from limb-girdle muscular dystrophy (LGMD), such as LGMDR9, or congenital muscular dystrophy 1C (CMD1C). In any of the disclosed methods, the rAAV, rAAV particles, or composition is administered by systemic administration, intramuscular injection, or intravenous injection.

[0030] This disclosure also provides compositions for treating muscular dystrophy in subjects requiring treatment, the compositions comprising any of the rAAVs disclosed herein, or any rAAV particles disclosed herein, or any of the compositions disclosed herein. For example, the subject suffers from limb-girdle muscular dystrophy (LGMD), such as LGMDR9, or congenital muscular dystrophy 1C (CMD1C). In any of the disclosed compositions, the rAAV, rAAV particles, or compositions are administered by systemic administration, intramuscular injection, or intravenous injection.

[0031] This disclosure also provides the use of any of the rAAVs disclosed herein, or any rAAV particles disclosed herein, or any of the compositions disclosed herein, for the preparation of agents for treating muscular dystrophy in subjects requiring such treatment. For example, subjects suffering from limb-girdle muscular dystrophy (LGMD), such as LGMDR9, or congenital muscular dystrophy 1C (CMD1C). In any of the disclosed uses, the rAAV, rAAV particles, or compositions are administered by systemic administration, intramuscular injection, or intravenous injection.

[0032] As used herein, “subject” can be any animal and may be referred to as “patient.” Preferably, the subject is a vertebrate, and more preferably, the subject is a mammal such as livestock (e.g., cattle, horses, pigs) or pets (e.g., dogs, cats). In some embodiments, the subject is a human. [Brief explanation of the drawing]

[0033] [Figure 1-1] This provides the annotated plasmid sequence of pAAV.MHCK7.FKRP.spA.CMV.FST(FS344).spA KanR, which is sequence number 1. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above. [Figure 1-10] Same as above. [Figure 1-11] Same as above. [Figure 1-12] Same as above. [Figure 1-13] Same as above. [Figure 1-14] Same as above. [Figure 1-15] Same as above. [Figure 2] This report describes the induction of muscle mass by FST gene therapy vectors after IM injection in C57Bl / 6J mice, and the testing of bicistronic (two promoters), monocistronic, and bicistronic single-promoter-IRES-containing formulations. 1 × 10¹¹ vg was injected into the pretibialis muscle (TA), and 5 × 10¹¹ vg into the gastrocnemius muscle (Gastroc). The error is the standard deviation of muscle mass (n=4 per group). The two bicistronic promoter constructs, AAV.MHCK7.FKRP.pA.CMV.FST and AAV.MHCK7.FKRP.pA.Cbh.FST, were comparable to CMV.FST alone in terms of muscle growth and muscle mass induction, while the other formulations were less favorable. [Figure 3]This study provides the in vivo distribution of AAV after intramuscular injection. Two-month-old C57Bl / 6J mice were injected with one of several AAV vectors at a dose of 1 × 10¹¹ vg into the pretibialis muscle (TA, blue) or 5 × 10¹¹ vg into the gastrocnemius muscle (Gas). For AAVMYO3a (Myo3a), 1 × 10¹¹ vg was injected into the TA and 5 × 10¹¹ vg into the gastrocnemius muscle. At four months of age (two months after injection), the number of AAV vector genomes (vg) per nucleus was quantified in the muscles by qPCR. TA + Gas represents the mean value for both muscles. The error is the standard deviation (SD) of n=4 muscles per TA or Gastroc group, or n=8 muscles for the pooled TA + Gastroc group. [Figure 4] This report provides FKRP and FST gene expression after intramuscular (IM) injection of AAV vectors in C57Bl / 6J mice. (A, B) Human FKRP gene expression was normalized to endogenous mouse Fkrp gene expression, and (C, D) Human FST gene expression was normalized to endogenous mouse Fst gene expression in the tibialis anterior muscle (TA, A, C) and gastrocnemius muscle (Gastroc, B, D). Here, MHCK7.FKRP.pA.CMV.FST was represented by AAV serotype rh74. The error is the standard deviation (SD) for n=4 muscles per group. Figure 5 shows the gene expression measurements after intramuscular injection of AAV vectors in C57Bl / 6J mice. Human FKRP gene expression and human FST gene expression from an AAV vector normalized to wild-type mouse Fkrp gene expression, compared to endogenous wild-type mouse Fst gene expression, were measured by qRT-PCR, referencing 18S rRNA expression in the tibialis anterior (TA) and gastrocnemius (Gastroc) muscles. The error is the standard deviation of n=4 muscles per group. CMV is a stronger promoter than MHCK7. [Figure 5]This report describes gene expression measurements after intramuscular injection of AAV vectors in C57Bl / 6J mice. Human FKRP and FST gene expression from AAV vectors normalized to wild-type mouse Fkrp gene expression, compared to endogenous wild-type mouse Fst gene expression, were measured by qRT-PCR, referencing 18S rRNA expression in the tibialis anterior (TA) and gastrocnemius (Gastroc) muscles. The error is the standard deviation (SD) of n=4 muscles per group. CMV is a stronger promoter than MHCK7. [Figure 6] This study demonstrates that IV treatment with bicistronic AAV in FKRPP448L mice resulted in complete recovery of uniform normal gait in all subjects in a single gait test at 15 m / min. FKRPP448L mutant (Mut) mice were IV-administered with high-dose (HD, 5 × 10¹³ vg / kg) or low-dose (LD, 1 × 10¹³ vg / kg) monocistronic FKRP (gene substitution), follistatin (FST, muscle builder), or bicistronic (FKRP / FST, Bi) gene therapy. Mut mice showed significantly reduced gait compared to WT mice (p<0.01), while Bi HD showed significantly increased gait compared to Mut (PBS), restoring uniform WT levels (p<0.01). Errors are SD for n=5-15 / grp. Statistical analysis: Kruskal-Wallis multiple comparison test, non-parametric statistical scale. [Figure 7] This study shows the maximum tetanic force and force decline during eccentric contraction. In situ force measurements were compared in the tibialis anterior muscle of 7-month-old wild-type (WT) mice and FKRPP448L mutant mice. FKRPP448L mice were IV-treated with PBS (mock treatment), or low-dose (LD, 1 × 10¹³ vg / kg) or high-dose (HD, 5 × 10¹³ vg / kg) AAV.CMV.FST, AAV.MHCK7.FKRP, or AAV.MHCK7.FKRP.pA.CMV.FST. The study shows the maximum absolute tetanic force (A) and force decline during repetitive eccentric contraction (B). Differences in A are shown only for comparisons against WT or against PBS. The error is the standard deviation (SD) of n=7-20 (A) or 6-18 (B) muscles per group. [Figure 8]This study provides measurements of muscle mass, length, cross-sectional area, and specific force for in situ physiological studies of the tibialis anterior muscle. The pretibialis muscle was isolated for use in the physiological study, and muscle mass (A), muscle length (B), and cross-sectional area (C), as well as weight-normalized specific force (D), were measured. Errors are standard deviations (SD) of n=12–22 muscles per group (A–C) or 720 muscles per group (D). [Figure 9] This shows muscle hypertrophy after bicistronic FKRP / FST gene therapy. The average muscle fiber diameter (in microns) of mini ferrets was measured in cross-sections of TA muscle (A) or diaphragm (B). The error is the standard deviation (SD) of n=9 to 24 images per group in A, and n=6 to 22 images per group in B. [Figure 10] This study demonstrates induction of muscle mass exceeding wild-type levels for FST and FKRP / FST gene therapy. Muscle mass was compared to that of wild-type FKRPP448L mice treated with high doses (5 × 10¹³ vg / kg) of CMV.FST or MHCK7.FKRP.pA.CMV.FST (FKRP / FST) gene therapy. The error is the standard deviation of muscle mass (SD) for n=9–14 per group. Only single muscle FST vs. FKRP / FST comparisons are shown: TA, tibialis anterior, gastrocnemius, gastrocnemius, quad, quadriceps femoris, triceps, and triceps brachii. [Figure 11] This shows the measured gene expression levels after IV injection of AAV vectors in FKRPP448L mice. Human FKRP gene expression from AAV vectors and human FST gene expression, normalized to wild-type mouse Fkrp gene expression compared to endogenous wild-type mouse FST gene expression, were measured by qRT-PCR with reference to 18S rRNA expression. The left-hand column for each muscle group is either MHCK7.FKRP or CMV.FST. The right-hand column for each muscle group is either MHCK7.FKRP.CMV.FST or MHCK7.FKRP.CMV.FST. The error is SD for n=6-7 per group. Muscles shown: anterior tibialis (TA), gastrocnemius, quadriceps femoris, triceps, and diaphragm. [Figure 12]This study provides the in vivo distribution of tissue AAV after low-dose and high-dose IV therapy using monocistronic or bicistronic vectors. AAV vector genome (vg) per cell nucleus was calculated for low-dose (LD, 1 × 10¹³ vg / kg) and high-dose (HD, 5 × 10¹³ vg / kg) IV treatments with AAV.CMV.FST, AAV.MHCK7.FKRP, or AAV.MHCK7.FKRP.pA.CMV.FST gene therapy. Error is the standard deviation (SD) of n=5-7 mice per group. Muscles included: TA (tibialis anterior); gastrocnemius; quad (quadriceps femoris); triceps (triceps brachii). The order of the columns for each muscle group (from left to right) is as follows: LD AAV.CMV.FST, HD AAV.CMV.FST, LD AAV.MHCK7.FKRP, HD AAV.MHCK7.FKR, LD AAV.MHCK7.FKRP.pA.CMV.FST, HD AAV.MHCK7.FKRP.pA.CMV.FST. [Figure 13] This shows changes in gene expression after high-dose monocistronic or bicistronic AAV gene therapy in FKRPP448L mice. IV injection of high-dose (5 × 10¹³ vg / kg) AAV is compared for monocistronic (FKRP or FST) and bicistronic (FKRP / FST) vectors. The left column for each muscle group is MHCK7.FKRP or CMV.FST. The right column for each muscle group is MHCK7.FKRP.CMV.FST or MHCK7.FKRP.CMV.FST. All expression levels were normalized to endogenous wild-type mouse gene expression in the organ and internally referenced to 18S rRNA for each measure. (A) Shows human FKRP gene expression (hFKRP) normalized to wild-type mouse Fkrp (mFkrp) gene expression. (B) Shows human FST gene expression (hFST) normalized to wild-type mouse Fst gene expression (mFst). The error is the standard deviation (SD) of n=5-7 mice per group in A and B. [Figure 14]This study provides gene expression after low-dose IV injection of monocistronic and bicistronic AAV vectors in FKRPP448L mice. IV injection of low-dose (1 × 10¹³ vg / kg) AAV is compared for monocistronic and bicistronic vectors, all normalized to endogenous wild-type mouse gene expression in their respective organs and internally referenced to 18S rRNA for each measure. The left-hand column for each muscle group is either MHCK7.FKRP or CMV.FST. The right-hand column for each muscle group is either MHCK7.FKRP.CMV.FST or MHCK7.FKRP.CMV.FST. (A) Shows human FKRP gene expression (hFKRP) normalized to wild-type mouse Fkrp (mFkrp) gene expression. (B) Shows human FST gene expression (hFST) normalized to wild-type mouse Fst gene expression (mFst). The error is the standard deviation (SD) of n=5-6 muscles per group (A, B). [Figure 15] This study provides expression of follistatin and FKRP proteins in the liver. Western blotting of follistatin (FST) and FKRP proteins was performed using protein lysates isolated from the livers of wild-type (WT) or FKRPP448L mice treated with mock-treated (PBS) or CMV.FST(FST), MHCK7.FKRP(FKRP), or MHCK7.FKRP.pA.CMV.FST(FKRP / FST). GAPDH blotting was performed as a control. Each lane represents a sample taken from a different mouse. [Figure 16] This study provides follistatin and FKRP expression in skeletal muscle. Western blotting of follistatin (FST) and FKRP proteins was performed using protein lysates isolated from TA muscle of wild-type (WT) or FKRPP448L mice treated with mock-treated (PBS) or CMV.FST(FST), MHCK7.FKRP(FKRP), or MHCK7.FKRP.pA.CMV.FST(FKRP / FST). GAPDH blotting was performed as a control. Each lane represents a sample taken from a different mouse. [Figure 17]This report provides ELISA measurements of serum follistatin levels. Mouse-specific follistatin protein (A) and human-specific follistatin protein (B) were measured by ELISA in serum from wild-type mice (WT) or FKRPP448L mice. FKRPP448L mice were either mock-treated (PBS) or treated with low-dose (LD, 1 × 10¹³ vg / kg) or high-dose (HD, 5 × 10¹³ vg / kg) AAV, as shown. The error is SD of n=4–7 per group for A and B. [Figure 18] This paper provides Western blotting of dystroglycan glycosylation and proteins. Skeletal protein extracts from TA muscle of wild-type (WT) or FKRPP448L mice were blotted using IIH6, an aDG antibody that recognizes functional glycans required for laminin binding, and anti-peptide polyclonal antibodies against aDG and bDG proteins. FKRPP448L mice were either mock-treated (PBS) or treated with CMV.FST (FST), MHCK7.FKRP (FKRP), or MHCK7.FKRP.pA.CMV.FST (FKRP / FST). Each FKRP / FST lane represents muscle from a different mouse administered with a bicistronic vector. Only high-dose (5 × 10¹³ vg / kg) treatments are shown in all examples. Immunoblotting of GAPDH antibody is shown as a control for protein loading and transfer. [Figure 19]This study provides a quantitative method for IIH6 immunostaining. Tissue sections from the tibialis anterior muscle (A, C) or diaphragmatic muscle (B, D) were immunostained with IIH6, an antibody that recognizes the functional glycosylation of dystroglycans. The percentage of IIH6-positive muscle fibers (A, B) and violin plots (C, D) of all muscle fiber pixel intensity of IIH6-positive fibers, normalized to a median WT value set to 1, were compared in wild-type (WT) muscle and FKRPP448L muscle, either mock-treated (PBS) or treated with high-dose CMV.FST, MHCK7.FKRP, or MHCK7.FKRP.pA.CMV.FST. The error is the standard deviation (SD) of n=10-24 images per group in A and 6-22 images per group in B. The violin plot shows signals from 6,000 to 12,000 muscle fibers per group in C, and from 6,000 to 19,000 muscle fibers per group in D. [Figure 20] This provides the agreement rate of IIH6 with myofascial membrane staining. Pixel agreement between IIH6 staining and dystrophin staining of myofascial membrane was compared in wild-type and FKRPP448L muscle treated with mock-treated (PBS) or CMV.FST, MHCK7.FKRP, or MHCK7.FKRP.pA.CMV.FST. The images show the tibialis anterior (TA), diaphragm, and heart. Agreement rates are color-coded from low (blue) to high (red). Non-muscle portions are excluded from analysis by the red line. Bars are 200 μm for the heart and 100 μm for the diaphragm and TA panels. [Figure 21] This document provides IIH6 staining of wild-type and FKRPP448L muscle treated with monocistronic or bicistronic FKRP / FST AAV vectors. IIH6 staining (green, right), along with integrated dystrophin (red), IIH6 (green), and DAPI (blue) staining (left), was compared in wild-type and FKRPP448L muscle treated with mock-treated (PBS) or CMV.FST (FST), MHCK7.FKRP (FKRP), or MHCK7.FKRP.pA.CMV.FST (FKRP / FST). Staining with secondary antibody only (secondary only) is shown as a control. [Figure 22]This study provides serum creatine kinase activity before and after the walking protocol. Serum creatine kinase activity was measured before or immediately after a multi-day walking protocol in wild-type mice and FKRPP448L mice treated with mock-treated mice (PBS) or CMV.FST (FST), MHCK7.FKRP (FKRP), MHCK7.FKRP.pA.CMV.FST (FKRP / FST). The error is the standard deviation (SD) of n=2–8 mice per group (A) or n=4–7 mice (B). The order of the columns for each test group in Panel A (left to right) is as follows: WT, PBS, LD AAV.CMV.FST, LD AAV.MHCK7.FKRP, LD AAV.MHCK7.FKRP.pA.CMV.FST. The order of the columns for each test group in Panel B (from left to right) is as follows: WT, PBS, HD AAV.CMV.FST, HD AAV.MHCK7.FKRP, HD AAV.MHCK7.FKRP.pA.CMV.FST. [Figure 23] This study demonstrates the prevention of muscle injury using monocistronic FKRP and bicistronic FKRP / FST AAV vectors. The percentage of muscle fibers containing a central nucleus (A, B) and the percentage of non-muscle area (C, D) were measured in the TA (A, C) and diaphragm (B, D) muscles. The error margins were SD 9–24 images per group for A and C, and 6–22 images per group for B. [Modes for carrying out the invention]

[0034] A bicistronic gene therapy approach that adds muscle-building components to gene replacement is disclosed herein. Such therapies have the potential to reconstruct muscle loss while simultaneously halting subsequent disease progression.

[0035] To inhibit disease through FKRP gene substitution while simultaneously constructing new muscle mass and strength, the disclosed gene therapy approach utilizes follistatin (FST) as a second gene component in a bicistronic AAV vector. FST encodes a secreted myostatin inhibitor protein that binds to and inhibits myostatin protein, which binds to muscle cells (Amthor et al., Dev Biol, 270(1):p.19-30, 2004). Myostatin is a secreted muscle nutrient that negatively modulates muscle growth and strength. Myostatin removal in mice, cattle, or humans can double skeletal muscle size with minimal or no effect on cardiac or non-muscle tissue. Myostatin removal in mdx mice significantly increased muscle size and strength but did not improve weight-normalized grip strength or specific (weight-normalized) tetanic muscle strength. Therefore, myostatin inhibition does not stabilize the muscle membrane or prevent muscle damage, but instead increases muscle strength by increasing muscle mass. In this sense, myostatin inhibitor therapy alone may be ineffective in the long term, as the muscle expressing the inhibitor is ultimately destroyed if effective gene replacement is not provided. In fact, recent clinical trials of myostatin inhibitor therapy using a blocking antibody approach have failed to achieve any of the clinically important stages in the LGMDR9 clinical trial (Leung et al., Muscle Nerve, 2021.64(2):p.172-179, 2021).

[0036] The FST genotype, FS344, used in the exemplary approaches described herein has been tested in two clinical trials: IM delivery of rAAV1.CMV.FST bilaterally to the quadriceps femoris in patients with Becker muscular dystrophy (BMD) resulted in improvement in the 6-minute walk test (6MWT, up to 125 meters) in 4 out of 6 patients one year after treatment. Muscle biopsies from treated BMD patients suggested muscle hypertrophy, reduced endomysial fibrosis, and more uniform muscle fiber size (Mendell et al., Mol Ther, 23(1):p.192-201, 2015). Similar IM delivery of rAAV1.CMV.FST in patients with inclusion body myositis (IBM) resulted in a mean improvement of +56 meters (and up to +153 m) in the 6MWT at 1 year after treatment in 6 subjects. Muscle biopsies from treated IBM patients showed improved muscle hypertrophy, reduced fibrosis, and regeneration (Mendell et al., Mol.Ther.23(1):192-201, 2015). Therefore, the main appeal of using FST gene therapy in a bicistronic vector is that this gene has already been tested in humans and shown to be safe and effective. Another main appeal is that FST gene therapy is more potent than myostatin inhibition alone (Lee et al., PLoS One 2(8):e789, 2007) and can activate additional muscle growth signaling pathways (Winbanks et al., J.Cell Biol.197(7):997-1008, 2012).

[0037] In particular, the data provided herein show that the binding of gene substitution (FKRP) to a second gene (FST) known to build muscle mass and muscle strength is equivalent to the FKRP of LGMDR9. P448L In mouse models, this provides evidence that lost muscle function can be restored and new muscle strength can be built. In fact, this gene therapy treats FKRP beyond what is normally seen in wild-type mice. P448LMuscle strength was constructed in mice. FST constructed new muscle mass and strength by inducing both muscle cell growth (hypertrophy) and the formation of new muscle cells (hyperplasia). The use of a constitutive promoter (CMV) to drive FST expression in bicistronic FKRP / FST AAV increased serum protein levels to approximately 11 times higher than normal endogenous levels. This may have enabled the induction of trans-muscle growth in cells where gene therapy was not present.

[0038] The use of the more muscle-specific MHCK7 promoter to drive FKRP expression simultaneously enabled the induction of functional glycosylation on dystroglycan (aDG). Deficiency in functional glycosylation of aDG is a molecular deficiency that causes disease in LGMDR9. FKRP / FST therapy not only increased the number of muscle cells with aDG glycosylation but also increased the intensity and consistency of such glycosylation along the muscle fiber membrane, which also exceeded the levels seen in wild-type muscle. MHCK7 expression also enabled some degree of non-muscle expression of FKRP. The FKRP protein was identified in Western blotting of liver proteins, which may partially reflect large-scale induction of FKRP gene expression by a bicistronic vector.

[0039] The data provided herein suggest that manipulating both genes within a single AAV vector has clear advantages. One advantage is that both genes are guaranteed to be delivered to the same cells in all cases. A second advantage is that half the AAV dose is required to perform the treatment. The data provided herein also demonstrate additional unexpected advantages of bicistronic AAV. Firstly, the use of bicistronic AAV amplified gene expression for both transgenes compared to single AAV alone using the same promoters. While part of this increase is attributable to the loss of the single FST AAV vector due to muscle injury, part of the effect is attributable to higher transcription induction due to the presence of two promoters, and it is likely that different enhancers from each of these promoters acted to amplify the expression of both genes. Secondly, bicistronic FKRP / FST had a greater impact on muscle growth than single FST vector alone. FKRP / FST and FST induced similar amounts of muscle growth in several limb muscles (triceps, quad, TA), but FKRP / FST doubled gastrocnemius muscle growth compared to FST alone, while maintaining this muscle growth induction at a level comparable to the induced growth seen in other limb muscles. More uniform muscle growth may provide more effective limb muscle balance, preventing contraction and improving gait. FKRP / FST also significantly increased the size of diaphragmatic muscle fibers, which FST alone did not. Given that recent studies suggest reduced respiratory capacity as a phenotype in FKRPP448L mice, improved diaphragmatic strength may also contribute to improved walking endurance.

[0040] Another important, perhaps underestimated, aspect of bisistron therapy is its potential to expand the patient population that can participate in clinical trials. If only disease prevention is expected, only patients with the matching disease may be used to effectively evaluate therapeutic efficacy. Ideally, such a patient cohort would include only young patients not severely affected by the disease. LGMDR9 is a slowly progressing, genetically heterogeneous disease. An MRI study of LGMDR9 patients by Straub et al. showed fat replacement of muscle mass at a rate of 0–2% per year across 14 different leg muscles (Xiao et al. J Virol 72:2224-2232). While muscle mass loss was evident in that MRI study over one year, measurements of hip, knee, or ankle flexion / extension or adduction / abduction, or any time to walk or stand up, differed significantly when comparing 32 subjects over the same period (Willis et al., PLoS One 8:e70993, 2013). Therefore, due to the slow and variable clinical progression of LGMDR6, it is extremely difficult, perhaps impossible, to demonstrate clinically significant improvement in motor function over a typical one-year period commonly used in clinical trials. The ability of bisistron vectors to improve strength beyond baseline should allow for comparison of patients with different baseline strengths with the patient themselves after treatment. This should open up clinical possibilities, including more variable and significantly affected patients, in which such bisistron therapy may still yield clinical improvement.

[0041] Limb-girdle muscular dystrophy Limb-girdle muscular dystrophy (LGMD) is a rare condition, and symptoms vary from person to person in terms of age of onset, area of ​​muscle weakness, involvement of the heart and respiratory system, rate of progression, and severity. LGMD can begin in childhood, adolescence, young adulthood, or later. Both sexes are equally affected. LGMD causes weakness in the shoulder and pelvic girdle, and the muscles near the upper limbs and arms may also weaken over time. Leg weakness often appears before arm weakness. Facial muscles are usually unaffected. As the condition progresses, people may have difficulty walking and may need to use a wheelchair over time. When the shoulder and arm muscles are involved, it may become difficult to raise the arms overhead and lift objects. Depending on the type of LGMD, the heart and respiratory muscles may also be involved.

[0042] LGMD has at least 19 forms, which are classified according to the associated genetic defect.

[0043] [Table 1]

[0044] Specialized testing for LGMD is now available through the National Commissioning Group (NCG), a nationwide program for diagnosis. The gene therapies offered are for limb-girdle GNE myopathy, Duchenne and Becker muscular dystrophy (DMD and BMD), as well as LGMD2A (CAPN3), LGMDB (DYSF), LGMD2C (SGCG), LGMD2D (SGCA), LGMD2E (SGCB), LGMD2F (SGCD), LGMD2G (TCAP), LGMD2H (TRIM32), LGMDR9 (FKRP), LGMD2J (TTN), LGMD2K (POMT1), LGMD2L (ANO5), LGMD2M (FKTN), LGMD2N (POMT2), LGMD2O (POMT2), LGMD2P (DAG1), and LGMD2Q. It is useful in the treatment of limb-girdle muscular dystrophy (LGMD) such as PLEC1), LGMD2R (DES), LGMD2S (TRAPPPCII), LGMD2T (GMPPB), LGMD2U (ISPD), LGMD2V (GAA), LGMD2X (BVES), LGMD2Y (TOR1AIP1), LGMD2Z (POGLUT1), LGMD1A (TTID, MYOT), LGMD1B (LMNA), LGMD1C (CAV3), LGMD1D (DES), LGMD1F (TNPO3), and LGMD1G (HNRPDL). In either case, the first transgene may be used for gene replacement or alternative gene replacement of a gene missing in the disease, while the second transgene encodes muscle-building proteins or muscle growth factors such as FS344, FS315, FS317, FS288, HB-EGF1, IGF1, or SMAD7, which reverse the symptoms of the disease by building new muscle growth and strength.

[0045] Muscle building protein Muscle-building proteins include growth factors that induce muscle growth or increase muscle strength, such as IGF, HB-EGF, Pax7, HGF (hepatocyte growth factor), HGH (human growth hormone), FGF19 (fibroblast growth factor 19), FGF21 (fibroblast growth factor 21), VEGF (vascular endothelial growth factor), IL6 (interleukin 6), IL15 (interleukin 15), and SMAD7 (mother to decapentaplesic homolog 7 (MADH7)).

[0046] Growth factors that induce muscle growth or increase muscle strength also include follistatin (FST). Follistatin is a secreted protein that inhibits the activity of TGF-β family members such as GDF-11 / BMP-11. Follistatin-344 is a follistatin precursor that undergoes peptide cleavage to form the circulating follistatin-315 isoform, which contains a C-terminal acidic region. It circulates with myostatin propeptide in a complex containing two other proteins, follistatin-associated gene (FLRG) and GDF-associated serum protein (GASP-1). Follistatin-317 is another follistatin precursor that undergoes peptide cleavage to form the membrane-bound follistatin-288 isoform.

[0047] The DNA and amino acid sequences of the follistatin-344 precursor are shown in SEQ ID NOs. 4 and 5 (and nucleotides 5393-6427 of SEQ ID NO. 1), respectively. FS344 contains a C-terminal protein domain lacking FS288. The presence of this C-terminal domain reduces binding to activin and heparan sulfate glycosaminoglycans, thereby reducing non-muscle effects. The follistatin-288 isoform, lacking the C-terminal acidic region, exhibits strong affinity for heparin sulfate proteoglycans, is a potent inhibitor of pituitary follicle-stimulating hormone, is found in ovarian follicular fluid, and shows high affinity for ovarian granule cells. The testes also produce follistatin-288. Follistatin deficiency results in reduced muscle mass at birth.

[0048] Examples of follistatin are presented in Shimasaki et al., U.S. Patent No. 5,041,538; other follistatin-like proteins are presented in U.S. Patents No. 5,942,420, 6,410,232, 6,537,966, and 6,953,662; FLRG is presented in Hill et al., J. Biol. Chem., 277(43):40735-40741 (2002); and GASP-1 is presented in Hill et al., Mol Endocrinol, 17:1144-1154 (2003).

[0049] SMAD7 is known to inhibit the TGF-β activated signaling response by associating with the active TGF-β complex, thereby reducing TGF-β signaling. Myostatin and TGF-β signaling induce SMAD7 expression, establishing a negative feedback loop that inhibits TGF-β signaling. In particular, SMAD7 is known to regulate myogenesis using this negative feedback loop (Kollias et al. Mol. Cell Biol. 26(16):6248-6260, 2006). The nucleotide sequence encoding SMAD7 is presented under Genbank acceptance number NM_005904.4, and the amino acid sequence is presented under Genbank acceptance number NP_005895.

[0050] Conversion factors are factors that convert non-muscle cells into muscle cells or induce their differentiation. For example, MyoD is known to convert several cell types, including dermal fibroblasts, chondrocytes, smooth muscle cells, retinal pigment epithelial cells, adipocytes, and cells of melanoma, neuroblastoma, osteosarcoma, and hepatocytoma, into muscle cells (Abraham & Tapscott, Curr. Opin. Genet. Dev. 23(5):568-573, 2013). Other examples of conversion factors include Myocd (myocardindin), Mef2C (muscle cell enhancer factor 2C), Mef2B (muscle cell enhancer factor 2B), Mkl1 (MKL [megakaryoblastic leukemia] / Myocd-like 1), Gata4 (GATA-binding protein 4), Gata5 (GATA-binding protein 5), Gata6 (GATA-binding protein 6), and Ets1 (E26 avian leukemia oncogene 1, 5' domain).

[0051] AAV gene therapy As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only within cells, provided with certain functions by co-infecting helper viruses. Currently, there are 13 characterized serotypes of AAV. General information and an overview of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is quite expected that these same principles may apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, all of which possess three related capsid proteins, such as the one expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along genome length and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0052] As used herein, “AAV vector” means one or more target polynucleotides (or transgenes) adjacent to an AAV terminal repeat sequence (ITR). Such an AAV vector can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing rep and cap gene products.

[0053] An "AAV virion," "AAV virus particle," or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector enclosed within the capsid. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than those in the wild-type AAV genome, such as transgenes delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Therefore, since such vectors are contained within AAV vector particles, the production of AAV vector particles inevitably involves the production of AAV vectors.

[0054] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains inverted end repeats (ITRs). Exemplary ITR sequences can be 130 or 141 base pairs long. AAV has multiple serotypes. The nucleotide sequences of the AAV serotype genomes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome was presented in Srivastava et al., J Virol, 45:555-564 (1983), as modified by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is presented under GenBank access number NC_002077, the complete genome of AAV-3 is presented under GenBank access number NC_1829, the complete genome of AAV-4 is presented under GenBank access number NC_001829, the genome of AAV-5 is presented under GenBank access number AF085716, the complete genome of AAV-6 is presented under GenBank access number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are presented under GenBank access numbers AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 relating to AAV-8), and the genome of AAV-9 is presented by Gao et al. The genome for AAV-10 was presented in al., J. Virol., 78:6381-6388 (2004), the genome for AAV-10 was presented in Mol. Ther., 13(1):67-76 (2006), and the genome for AAV-11 was presented in Virology, 330(2):375-383 (2004). Cloning of AAVrh.74 serotypes is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007). The Cis action sequence, which directs viral DNA replication (rep), capsid formation / packaging, and integration into host cell chromosomes, is contained within the ITR.Three AAV promoters (named p5, p19, and p40 after their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Coupled with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 of AAV2), the two rep promoters (p5 and p19) result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3). Alternative splicing and a non-consensus translation initiation site contribute to the production of the three associated capsid proteins. A single-consensus polyadenylation site is located at map location 95 of the AAV genome. The life cycle and genetics of AAV are outlined in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0055] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytotoxic, and natural infections in humans and other animals are asymptomatic. Furthermore, AAV infects many mammalian cells, offering the potential to target many different tissues in vivo. Additionally, AAV can transduce slow-dividing and non-dividing cells and essentially persist throughout the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in a plasmid, enabling the construction of recombinant genomes. Furthermore, because signals directing AAV replication, genomic capsid formation, and integration are contained within the ITR of the AAV genome, some or all of the approximately 4.3 kb inside the genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing the promoter, the DNA of interest, and polyadenylation signals. The rep and cap proteins can be supplied trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. This means it easily withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), reducing the importance of chilling AAV. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to co-infection.

[0056] The recombinant AAV genome of this disclosure comprises the nucleic acid molecule of this disclosure and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA in the rAAV genome may be derived from any AAV serotype capable of inducing recombinant viruses, including but not limited to AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, AAVMyo, MYOAAV, and their derivatives). The generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background technology section above, the nucleotide sequences of various AAV serotype genomes are known in the field of this technology.

[0057] The provided recombinant AAV (i.e., infectious capsidized rAAV particles) contains an rAAV genome. The term “rAAV genome” refers to a polynucleotide sequence derived from a modified native AAV genome. In some embodiments, the rAAV genome is modified to remove native cap and rep genes. In some embodiments, the rAAV genome contains endogenous 5' and 3' inverted end repeats (ITRs). In some embodiments, the rAAV genome contains ITRs derived from an AAV serotype different from the AAV serotype from which the AAV genome is derived. In some embodiments, the rAAV genome contains a target transgene flanked at the 5' and 3' ends by inverted end repeats (ITRs). In some embodiments, the rAAV genome contains a “gene cassette”. In exemplary embodiments, both rAAV genomes lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome.

[0058] The DNA plasmid of this disclosure comprises the rAAV genome of this disclosure. The DNA plasmid is transferred to a cell tolerant of infection with an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles that provide the AAV genome, rep gene and cap gene, and helper virus function to be packaged are standard in the art. The production of rAAV requires that the following components, the rAAV genome, the AAV rep gene and cap gene isolated from (i.e., not present in) the rAAV genome, and helper virus function, be present in a single cell (represented herein as a packaging cell). The AAV rep and cap genes may originate from any AAV serotype capable of inducing recombinant viruses, including but not limited to AAV-9, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-10, AAV-11, AAV-12, AAVrh.10, AAV-13, Anc80, AAV7m8, AAVMyo, or MYOAAV, and may originate from an AAV serotype different from the rAAV genome ITR. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692 (which is incorporated herein by reference in its entirety).

[0059] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from the rAAV genome, and a selection marker (e.g., a neomycin resistance gene) is incorporated into the cell's genome. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Next, the packaging cell line is infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a preferred method is to use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0060] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described below: Ratschin et al., Mol.Cell.Biol.4:2072(1984), Hermonat et al., Proc.Natl.Acad.Sci.USA,81:6466(1984), Tratschin et al., Mol.Cell.Biol.5:3251(1985), McLaughlin et al., J.Virol.,62:1963(1988), and Lebkowski et al., Mol.Cell.Biol.,7:349(1988). Samulski et al., J. Virol., 63:3822-3828 (1989), U.S. Patent No. 5,173,414, WO95 / 13365, and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13:1244-1250 (1995), Paul This is described in Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996), U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on the section concerning documents relating to rAAV production.

[0061] Accordingly, this disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 strain). In another embodiment, the packaging cells may be non-transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus macaque fetal lung cells).

[0062] rAAV can be purified by methods standard in the art (e.g., by column chromatography or cesium chloride gradient). Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0063] In another embodiment, the Disclosure envisions a composition comprising the rAAV of the Disclosure. The composition of the Disclosure comprises the rAAV and a pharmaceutically acceptable carrier. The composition may also include other components such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to the recipient, preferably inactive at the dosage and concentration used, and include, but are not limited to, buffers such as phosphoric acid [e.g., phosphate-buffered saline (PBS)], citric acid, or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparaginine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®; copolymers such as poloxamer 188, Pluronic® (e.g., Pluronic F68), or polyethylene glycol (PEG).

[0064] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired components from its previously sterile filtered solution.

[0065] The titer and dosage of rAAV administered by the method of this disclosure will vary depending, for example, on the specific rAAV, mode of administration, therapeutic target, individual, timing of administration, and target cell type, and may be determined by standard methods in the art. The titer of rAAV is approximately 1 × 10⁶ per ml.6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 and above, or can be in the range of more than DNase-resistant particles (DRP). The dosage can be expressed in units of viral genome (vg). These dosages of rAAV are about 1×10 9 vg or more, about 1×10 10 vg or more, about 1×10 11 vg or more, about 1×{10} 12 vg or more, about 6×10 12 and above, about 1×10 13 vg or more, about 1.3×10 13 vg or more, about 1.4×10 13 vg or more, about 2×10 13 vg or more, about 3×10 13 vg or more, about 6×10 13 vg or more, about 1×10 14 vg or more, about 3×10 14 and above, about 6×10 14 and above, about 1×10 15 vg or more, about 3×10 15 and above, about 6×10 15 and above, about 1×10 16 and above, about 3×10 16 and above, or about 6×10 16 and above. In the case of newborns, the dosage of rAAV is about 1×10 9 vg or more, about 1×10 10 vg or more, about 1×10 11 vg or more, about 1×10 12 vg or more, about 6×10 12 and above, about 1×10 13 vg or more, about 1.3×10 13 vg or more, about 1.4×10 13 vg or more, about 2×10 13 vg or more, about 3×10 13 vg or more, about 6×10 13 vg or more, about 1×10 14 vg or more, about 3×1014 The above is approximately 6 x 10 14 In summary, approximately 1 x 10 15 vg or more, approximately 3×10 15 The above is approximately 6 x 10 15 In summary, approximately 1 x 10 16 The above is approximately 3 x 10 16 The above, or approximately 6 x 10 16 It could be within the above range.

[0066] A method for transducing target cells with rAAV in vivo or in vitro is contemplated by this disclosure. The in vivo method comprises the step of administering a composition comprising an effective dose or effective multiple doses of the rAAV of this disclosure to an animal (including humans) in need thereof. If the dose is administered before the onset of the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. In embodiments of this disclosure, the effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition being treated, delays or prevents progression to the disorder / disease condition, delays or prevents progression to the disorder / disease condition, reduces the severity of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival. Examples of diseases for which prevention or treatment by the method of this disclosure is contemplated are limb-girdle muscular dystrophy, e.g., LGMDR9 or congenital muscular dystrophy 1C.

[0067] The term "transduction" is used to refer to the administration / delivery of the coding regions of transgenes, e.g., FKRP and FST genes, to recipient cells, either in vivo or in vitro, via the replication-deficient rAAV of this disclosure, resulting in the expression of FKRP and FST proteins in recipient cells.

[0068] Transduction of cells with rAAV as disclosed herein results in sustained expression of proteins encoded by first and second transdextrose genes. Thus, the disclosure provides methods for administering / delivering rAAV to animals, preferably humans. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs as disclosed herein. Transduction may be carried out with a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present disclosure includes the actin and myosin gene families such as the myoD gene family (Weintraub et al., Science, 251:761-766 (1991)), muscle cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11:4854-4862 (1991)), regulatory elements derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)), cardiac actin gene, muscle creatine kinase sequence elements (see Johnson et al., Mol Cell Biol, 9:3393-3399 (1989)), and mouse creatine kinase enhancer (MCK) elements, MHCK7, regulatory elements derived from the skeletal fast-twitch muscle troponin C gene, slow-twitch muscle cardiac troponin C gene, and slow-twitch muscle troponin I gene: hypoxia-inducible nuclear factor (Semenza et al.) This invention provides a method for transducing muscle cells and muscle tissue directed by muscle-specific promoter elements, including, but not limited to, those derived from steroid-inducible elements and promoters, including glucocorticoid response elements (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), as well as other regulatory elements.

[0069] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. "Muscle cells" or "muscle tissue" means cells or groups of cells derived from any type of muscle (e.g., skeletal and smooth muscle derived from the digestive tract, bladder, blood vessels, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, including myoblasts, myocytes, myotubes, cardiomyocytes, and cardiac muscle cells.

[0070] Combination therapies are also contemplated in this disclosure. The combinations used herein include both concurrent and sequential therapies. In particular, combinations of the methods disclosed herein with standard medical treatments, such as in combination with novel therapies, are contemplated. In some embodiments, combination therapy includes administering an immunosuppressant in combination with a gene therapy disclosed herein.

[0071] The effective dose of the composition may be administered by a standard route in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, intrarectal, or vaginal. The route of administration and serotype of the AAV components of the rAAV of this disclosure (in particular, AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the disease condition being treated and the target cells / tissues expressing the proteins encoded by the first and / or second transgenes.

[0072] This disclosure provides topical and systemic administration of effective doses of the rAAV and compositions of this disclosure. For example, systemic administration means administration to the circulatory system so that the whole body is affected. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration through injection, infusion, or transplantation.

[0073] Immunosuppressants Immunosuppressants may be administered before or after the onset of the immune response to rAAV in subjects following gene therapy. In addition, immunosuppressants may be administered concurrently with gene therapy or protein replacement therapy. The immune response in subjects may include adverse immune or inflammatory responses that follow or are triggered by the administration of rAAV to the subjects. The immune response may be the production of antibodies in the subjects in response to the administered rAAV.

[0074] Examples of immunosuppressants include glucocorticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, cell proliferation inhibitors such as purine analogs, methotrexate, and cyclophosphamide, inosine monophosphate dehydrogenase (IMDH) inhibitors, and biologics such as monoclonal antibodies or fusion proteins.

[0075] Immunosuppressants may be anti-inflammatory steroids, which are steroids that reduce inflammation and suppress or modulate the target immune system. Exemplary anti-inflammatory steroids are glucocorticoids such as prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, budesonide, or prednisone.

[0076] Janus kinase inhibitors are inhibitors of the JAK / STAT signaling pathway that target one or more enzymes in the Janus kinase family. Exemplary Janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib.

[0077] Calcineurin inhibitors bind to cyclophylline and inhibit the activity of calcineurin. Examples of calcineurin inhibitors include cyclosporine, tacrolimus, and piceclorimus.

[0078] mTOR inhibitors reduce or inhibit the serine / threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include sirolimus, everolimus, and temsirolimus.

[0079] Immunosuppressants include immunosuppressive macrolides. The term "immunosuppressive macrolide" refers to macrolide agents that suppress or modulate the target immune system. Macrolides are a class of drugs that contain a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desamine, are attached. The lactone ring is usually 14, 15, or 16 members. Macrolides belong to the polyketide class of drugs and can be natural products. Examples of immunosuppressive macrolides include tacrolimus, pimecrolimus, and sirolimus.

[0080] Purine analogs inhibit nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolic acid, and lefnomide. Examples of immunosuppressive biologics include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, isekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinenumab, vedolizumab, basiliximab, belatacept, and daclizumab.

[0081] Specifically, immunosuppressants are anti-CD20 antibodies. The term anti-CD20-specific antibody refers to an antibody that specifically binds to CD20 or inhibits or reduces the expression or activity of CD20. Examples of anti-CD20 antibodies include rituximab, ocrelizumab, or ofatumumab.

[0082] Examples of additional immunosuppressive antibodies include anti-CD25 antibodies (or anti-IL2 antibodies or anti-TAC antibodies) such as basiliximab and daclizumab, as well as anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teprizumab and bicilizumab, and anti-CD52 antibodies such as alemtuzumab.

[0083] The following examples are provided as illustrations, not as limitations. The numerical ranges described include each integer value within each range, including the minimum and maximum integers described. [Examples]

[0084] Example 1 Construction of a bicistronic AAV vector The disclosed plasmid pAAV.MHCK7.FKRP.spA.CMV.FST344.spA is shown as Sequence ID No. 1. This plasmid contains expression cassettes adjacent to the AAV2 inverted terminal repeat (ITR), and these expression cassettes may also contain a first transcriptional regulatory sequence operably ligated to a transgene encoding the FKRP protein, a second transcriptional regulatory sequence operably ligated to a transgene encoding MHCK7 and FST344, and a CMV promoter / enhancer.

[0085] Human FKRP (NM_001039885.3) cDNA was synthesized by Twist Biosciences (San Francisco, CA) using the 5'NheI / 3'SphI-SpeI-NotI restriction enzyme cut site and the 5'Kozak consensus sequence (GCCGCCACCATG). Human FST (NM_013409.3) cDNA was synthesized by GeneArt (Thermo Fisher; Waltham, MA) using the 5' and 3'NotI restriction enzyme cut sites and the 5'Kozak consensus sequence (ACCATGG). FKRP, FST, SMAD7, or GFP were cloned into pAAV vectors under various promoters including MCK (CK7-like), MHCK7, CMV, or CBH, with or without the presence of an internal ribosome entry site (IRES) from FGF1 (Dulluc-Clavieres et al. Gene Ther 15:1090-1098, 2008; Gray et al. Hum Gene Ther 22:1143-1153, 2011; Xu et al., Mol Ther Methods Clin Dev 10:89-104, 2011; Salva et al., Mol Ther 15:320-329, 2007).

[0086] The bisistronic expression cassette contained a kanamycin resistance gene and an optimized Kozak sequence enabling more accurate and robust protein translation. The rAAV vector was produced by a modified cross-packaging approach that allows the AAV2-type vector genome to be packaged into multiple AAV capsid serotypes (Rabinowitz et al., J Virol. 76(2):791-801 (2002)). Production was achieved using a standard 3 plasmid DNA / CaPO4 precipitation method with HEK293 cells. HEK293 cells were maintained in 10% fetal bovine serum (FBS) and DMEM supplemented with penicillin and streptomycin. The produced plasmids were (i) a plasmid encoding a therapeutic protein, (ii) a rep2-capX modified AAV helper plasmid encoding a cap serotype AAVrh74 isolate, and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I / II RNA genes. AAV was purified using iodixanol density centrifugation and anion exchange chromatography (Clark et al., Human Gene Therapy 10:1031-1039, 1999). Capsid-formed vector genome (vg) titers were determined using a quantitative PCR-based titration method with a Prism 7500 Taqman detector system (PE Applied Biosystems). [Clark et al., Hum Gene Ther. 10(6):1031-1039 (1999)]. Final titer (vg ml) -1 The virus was identified by quantitative reverse transcriptase PCR using specific primers and probes with a Prism 7500 real-time detector system (PE Applied Biosystems, Grand Island, NY, USA). The divided viruses were stored at -80°C until production.

[0087] All plasmids used to construct the packaged AAV genome also contain a kanamycin resistance gene (KanR) outside the ITR sequence used for packaging the genome. This allows the DNA encoding the AAV genome to be transformed into bacteria, which will produce large amounts of DNA in the presence of kanamycin, killing all untransformed bacteria. KanR is not packaged in the AAV capsid in the AAV genome used to treat patients, but its presence enables DNA production in bacteria.

[0088] The description for pAAV.MHCK7.FKRP.spA.CMV.FST344.spA is shown as sequence number 1 and is presented in the table below.

[0089] [Table 2]

[0090] Example 2 Screening of bisistron-induced AAV for muscle mass increase in wild-type mice We screened various bicistronic (two-gene) formulations to identify the one that best utilized follistatin to induce muscle growth when positioned at the second location in the AAV gene therapy vector. At 2 months of age, 1 × 10¹⁶ follistatin was administered to the pretibial (TA) muscle of wild-type C57Bl / 6J mice. 11 AAV of vg (vg is vector genome), and 5 × 10 in the gastrocnemius muscle. 11The VG vector was administered intramuscularly. Two months after injection, the mice were euthanized, and muscle mass was measured to determine the increase in muscle mass. Muscle was dissected tendon to tendon, and its weight was measured to determine the increase in muscle mass (Figure 2). Two types of vectors were compared. One set utilized a single promoter and positioned an internal ribosome entry site (IRES) from FGF1A, an element known to function well in skeletal muscle (Delluc-Clavieres et al. Gene Therp. 15(15):1090-8, 2008), driving the expression of a second follistatin protein. We previously characterized this IRES element and demonstrated its ability to drive GFP expression in non-muscle cells (ns).

[0091] The second configuration compared the placement of two promoters to drive the mRNA transcripts of each gene separately, with poly(A) placed at the end of each cDNA. Here, the use of a strong constitutive promoter (either cytomegalovirus (CMV) or chicken beta-actin (Cbh) with a hybrid intron) or a strong muscle-specific promoter (muscle creatine kinase (type 7-like) (MCK) or MCK with a hybrid enhancer (MHCK7)) was compared. In all cases, the human FKRP gene was placed at the first position of the bicistronic vector, and the human follistatin (FST) gene forming FS344 (Rodino-Klapac et al. Muscle Nerve 39(3):283-96, 2009) was placed at the second position. These were compared to monocistronic constructs in which only a single gene was expressed. With two exceptions, all AAVs were produced using the rhesus macaque 74 (rh74) serotype, which performed well in terms of muscle transduction in DMD clinical trials (Mendell et al. JAMA Neurol. 77(9):1122-1131, 2020). rAAV.CMV.FST utilized AAV9, which also transduced muscle well based on our previous research (Rodino-Klapac et al. Muscle Nerve 39:283-296, 2009), while AAV.MHCK7.FKRP.pA.CBH.FST utilized either AAVrh74 or AAVMYO3a (Myo3a). Because AAVMYO3a is approximately 10 times more muscle-building than rAAV9 (Weinmann et al., Nat Commun 11:5432, 2020), rAAVMyo3a was injected at one-tenth the dose of other vectors.

[0092] AAV.CMV.FST doubled the size of TA or gastroc muscle within two months of treatment in these experiments. Regarding bisistronic formulations, AAV.MHCK7.FKRP.pA.CMV.FST and AAV.MHCK7.FKRP.pA.Cbh.FST induced similar growth to AAV.CMV.FST, with CMV showing slightly better results than Cbh. The use of rAAVMYO3a with rAAV.MHCK7.FKRP.pA.CBH.FST yielded similar results in TA but not in gastroc. AAV.MHCK7.FST and AAV.CMV.FKRP.pA.MHCK7.FST showed less muscle growth than the CMV vector, while AAV.CMV.FKRP.IRES.FST showed even less growth, and AAV.MHCK7.FKRP.IRES.FST showed no growth at all during this period. Vectors expressing only FKRP showed no growth. When MCK was used as the promoter, FST was superior to SMAD7 in inducing muscle growth, and as previously shown, FST was superior to IGF1 in similar assays (ns). These data suggest that rAAV.MHCK7.FKRP.pA.CMV.FST can have a significant impact on muscle growth within two months of treatment, and that the use of two promoter systems can produce functional changes as favorable as AAV expressing only a single FST gene.

[0093] The in vivo distribution of AAV vector genomes (vg) (Figure 3) and FKRP and FST gene expression (Figure 4) was assayed in muscle after IM injection. The bicistronic vectors that produced the greatest muscle growth actually had the lowest relative levels of muscle transduction. Therefore, these AAVs were not more potent simply because more gene therapies were available. For FKRP and FST gene expression, CMV was a more potent promoter than MHCK7 or MCK. However, bicistronic vectors utilizing MHCK7 for FKRP gene expression showed 3–8 times stronger FKRP gene expression in TA or Gastroc muscle than monocistronic vectors using the same MHCK7 promoter, despite the latter AAVs having more vg. Similarly, bicistronic vectors using CMV for FST gene expression showed 3–6 times stronger FST gene expression than monocistronic vectors using the same CMV promoter. Again, there was no significant difference in the vg of the two vectors in muscle in TA or Gastroc. Therefore, the presence of a dual promoter in the bicistronic vector amplified the expression of both FKRP and FST compared to monocistronic AAVs using the same promoter.

[0094] Example 3 Changes in muscle function after bicistronic or monocistronic AAV therapy in FKRPP44L mice Gene expression of the most potent bicistronic vectors and their monocistronic equivalents after IM injection in wild-type muscle was analyzed, and the data are presented in Figure 6. All AAVs were serotype rhesus 74 (rh74). IM injection was performed as described in Example 2.

[0095] Human FKRP and FST gene expression from AAV vectors normalized to endogenous mouse Fkrp gene expression, compared to endogenous mouse Fst gene expression, were measured by qRT-PCR with reference to 18S rRNA expression in the tibialis anterior (TA) and gastrocnemius (Gastroc) muscles. CMV is a stronger promoter than MHCK7. However, the use of a two-promoter bicistronic vector (either AAV.MHCK7.FKRP.pA.CMV.FST or AAV.MHCK7.FKRP.pA.Cbh.FST) resulted in greater FKRP gene expression compared to AAV.MHCK7.FKRP alone, and greater FST gene expression compared to CMV.FST alone. The IRES construct was inferior to the two-promoter construct.

[0096] Each promoter contained its own enhancer element, and both enhancers in such a bicistronic construct amplified gene expression independently of their positions relative to the promoter (which is part of the definition of how enhancers function). AAV.MHCK7.FKRP.pA.CMV.FST showed stronger gene expression with respect to FKRP than AAV.MHCK7.FKRP alone, and stronger gene expression with respect to FST gene expression than CMV.FST. This was also true for Cbh used in a second position utilizing the same CMV enhancer element. FST gene expression was also strong in the two-promoter bicistronic construct. Therefore, the presence of dual promoters in a bicistronic vector amplified the transgene expression of both transgenes.

[0097] Example 4 FKRP P448L Changes in muscle function after bicistronic or monocistronic AAV therapy in mice LGMD2I's FKRPP 448LAAV.MHCK7.FKRP.pA.CMV.FST vector and its monocistronic equivalent (AAV.MHCK7.FKRP and AAV.CMV.FST) in a mouse model. High dose (HD, 5 × 10⁻¹) in mice. 13 (vg / kg) or low dose (LD, 1 x 10) 13 These vectors (vg / kg) were administered intravenously into the tail vein at 1 month of age (Figure 6). One week before euthanasia at 7 months of age, the mice underwent a 60-minute walking test at 15 m / min for 5 days. In this test, the mice were trained for 3 days, and then the test was performed once daily for 5 consecutive days, with a single walking measurement obtained by averaging over all 5 days. The data for all 5 days were averaged for each indicated data point.

[0098] During the evaluation week, mice were run on a treadmill for a 75-minute test (5 meters / min for 5 minutes, increasing by 1 meter / min every 10 minutes, to 15 meters / min for 60 minutes). Mice were run for 5 days before euthanasia. Blood was collected via the submandibular vein before and after walking. The collected blood was allowed to coagulate at room temperature for 1 hour, then centrifuged at 1000 × g for 10 minutes to separate the serum. Serum CK was measured using the creatine kinase (CK)-SL kit (Sekisui Diagnostics; Burlington, MA) according to the manufacturer's protocol.

[0099] In our experience, all wild-type mice can walk uniformly for one hour in this assay, and this was also the case in this experiment. In contrast, untreated mutant (PBS-treated) FKRPP 448L Mice showed an average 60% decrease in walking endurance. None of the untreated mutant mice showed normal levels of walking endurance. FKRPP treated with high doses of AAV.MHCK7.FKRP. 448L Mice showed a 50% improvement in walking endurance compared to PBS treatment, but only one out of six mice exhibited normal walking, and this result was not statistically significant. Low doses of AAV.MHCK7.FKRP and both doses of AAV.CMV.FST showed no improvement in walking.

[0100] As shown in Figure 4, the LD bicistronic (Bi) AAV.MHCK7.FKRP.pA.CMV.FST showed an average 60% improvement in gait compared to the Mut, with 3 out of 6 mice exhibiting normal gait. The HD Bi AAV.MCHK7.FKRP.pA.CMV.FST mice showed completely normal gait (7 out of 7) in all subjects of this assay. Therefore, gait, one of the most clinically important measures of motor function in muscular dystrophy, was completely restored in all mice treated with the high-dose AAV.MHCK7.FKRP.pA.CMV.FST bicistronic vector, a finding that did not occur with monogenetic therapy alone at any dose.

[0101] After completing the gait study, maximal tetanic force and force falloff during repetitive eccentric contractions were measured in situ in the tibialis anterior (TA) muscle (Figures 7A, B, 8A-D). Mice were anesthetized with ketamine / xylazine, and the hindlimb skin was removed to expose the tibialis anterior (TA). The distal tendon was dissected and sutured to a force transducer (Aurora Scientific, Aurora, ON). Muscle contraction was generated by sciatic nerve stimulation with bipolar platinum electrodes, and the optimal length was determined by stretching the muscle until the maximum typonic force was induced. Maximum force was determined by continuous stimulation at 50, 100, 150, and 200 Hz with a 1-minute rest period between each stimulation, and the specific force was determined by dividing the maximum force by the muscle cross-sectional area. Finally, the TA underwent 10 cycles of eccentric contraction, with each TA stimulated for a total of 350 ms, and this last 200 ms was stretched by 10% before returning to the optimal length. In these measurements, the maximum force generated in the first cycle before muscle lengthening is designated as 100%.

[0102] Absolute strength increased with high doses of rAAV.CMV.FST or HD rAAV.MHCK7.FKRP.pA.CMV.FST compared to PBS-treated FKRPP448L mice, and rAAV.MHCK7.FKRP.pA.CMV.FST-treated FKRPP448L mice exhibited significantly higher absolute strength than wild-type mice. For high-dose bisistronic FKRP / FST treatment, muscle mass increased 1.9±0.4 times compared to PBS-treated FKRPP448L muscle, cross-sectional muscle area increased 1.8±0.4 times, and cross-sectional muscle area increased 2.2±0.5 times and 2.1±0.9 times, respectively, compared to wild-type muscle (Figures 8A, 8C). Neither low-dose rAAV.CMV.FST nor LD rAAV.MHCK7.FKRP.pA.CMV.FST significantly altered absolute strength, muscle mass, or cross-sectional area, and neither did LD rAAV.MHCK7.FKRP significantly alter these parameters. Interestingly, HD rAAV.MHCK7.FKRP muscle showed similar results to mock-treated FKRP in terms of both mass and cross-sectional area. P448L It was significantly smaller than muscle (Figures 8A, 8C). In addition, high doses of AAV, CMV, and FST in muscle showed a difference compared to mock-treated FKRP. P448L Furthermore, it showed a significant increase in muscle length compared to wild-type muscles (Figure 8B).

[0103] Both absolute force and specific force (absolute force normalized to muscle cross-sectional area (CSA)) were compared to untreated FKRP mice in age- and sex-matched wild-type mice. P448L FKRP was reduced in mutant mice at 7 months of age. P448L Mice exhibited a 25% reduction in specific force and a 20% reduction in absolute force at either 150 Hz (Figures 7A and 7D) or 200 Hz (ns). HD rAAV.CMV.FST and rAAV.MHCK7.FKRP.pA.CMV.FST therapy significantly increased absolute force (Figure 8A), but this was attributed to increased muscle mass (Figure 8A). Therefore, this did not result in a significant increase in specific force that takes relative muscle mass into account (Figure 8D).

[0104] FKRP P448LMice exhibited a 27±4% force reduction after 10 contractions in the eccentric contractile muscle injury paradigm. While not significant, this was greater than the 11±1% force reduction observed in wild-type mice (Figure 7B). The rAAV.MHCK7.FKRP.pA.CMV.FST treatment maintained a force reduction close to that of wild-type mice (17±1% vs. 11±1%) after 10 contractions. In contrast, HD rAAV.CMV.FST and HD rAAV.MHCK7.FKRP showed force reductions of 34±3% and 24±6%, respectively, compared to the mock-treated FKRP. P448L The results were comparable to those in mice. This trend suggested some degree of prevention of muscle damage by rAAV, MHCK7, FKRP, pA, CMV, and FST during repeated stimulation, but this improvement in muscle weakness was not statistically significant.

[0105] Example 5 Heterogeneous induction of limb muscle growth by single FST gene therapy Average mini-ferret muscle fiber diameter was quantified for cross-sections of the TA and diaphragmatic muscles (Figures 9A, B), as well as for the total number of muscle fibers in the cross-section of the TA muscle (ns). rAAV.MHCK7.FKRP.pA.CMV.FST therapy increased both the mean size (+43±4%, Figure 9A) and the number of muscle fibers (49±19%) of the TA in the WT. rAAV.CMV.FST gene therapy showed a comparable increase to bicistronic therapy in the TA, but similarly did not increase muscle fiber size in the diaphragm (Figure 9C). The muscle fiber diameter of the diaphragmatic muscle in FKRP / FST treatment increased significantly by 31±2% compared to FST alone, but FST alone was not comparable to wild-type or mock-treated FKRP. P448L It did not show an increase compared to muscle mass.

[0106] In the muscles of TA (+82±17% vs. +87±30%), Quad (+62±22% vs. +59±14%), and Triceps (+37±16% vs. +45±18%), equivalent percentage increases in muscle mass were observed with rAAV.CMV.FST and rAAV.MHCK7.FKRP.pA.CMV.FST treatments, exceeding those of wild-type muscles. However, there was a significant difference in muscle growth in Gastroc (+16±11% for FST vs. +40±12% for FKRP / FST) (Figure 10). Therefore, the induction of growth among individual muscles was more uniform with FKRP / FST bicistron therapy than with FST therapy alone, with significant differences observed in the gastrocnemius and diaphragm.

[0107] Example 6 Comparison of bicistronic vector and monocistronic vector in vivo distribution and AAV-induced gene expression. FKRP treated IV with various AAV vectors P448L Gene expression in mice was also high-dose (5 × 10 13 Measurements were taken using skeletal muscle (tibialis anterior, gastrocnemius, quadriceps femoris, triceps brachii, and diaphragm), heart, and liver isolated from vg / kg mice. Human FKRP gene expression and human FST gene expression from the AAV vector, normalized to wild-type mouse FKRP gene expression compared to endogenous wild-type mouse FST gene expression, were measured by qRT-PCR with reference to 18S rRNA expression. As shown in Figure 11, amplification of human FKRP and human FST gene expression using the AAV.MHCK7.FKRP.pA.CMV.FST vector is greater than that of either AAV.MHCK7.FKRP or AAV.CMV.FST alone. Higher doses allow for levels exceeding normal levels of therapeutic gene expression in the skeletal muscle of the entire regimen. Therefore, the effect of therapeutic gene expression amplification is greater in FKRP than in wild-type mice. P448LThe effect was significant in mice. These data clearly demonstrate the great advantage of having two enhancers that can act synergistically to amplify dual transgene expression in skeletal muscle with muscular dystrophy. Such therapy can restore lost walking ability, essentially reversing the disease in ways that single-gene replacement therapy has not been shown to do. In addition, this bicistronic formulation amplifies the expression levels of both therapeutic genes compared to the use of a single promoter alone.

[0108] In addition, FKRP treated with various AAV vectors P448L In mice, the in vivo distribution of AAV (Figure 12) and the expression of FKRP and FST genes (Figures 13 and 14) were measured. Analysis was performed on specific limb muscles (TA, gastric, quad, triceps), diaphragm, heart, and liver. Several interesting findings were observed. Firstly, rAAV.CMV.FST therapy showed approximately 10 times lower overall transduction in skeletal muscle compared to rAAV.MHCK7.FKRP or MHCK7.FKRP.pA.CMV.FST, but there were no significant differences in vg levels of the three AAVs in the heart or liver (Figure 12). FKRP showed... P448L FKRP is known to prevent skeletal muscle damage in mice, which leads to prolonged muscle transduction, but FST fails to do so, thereby causing the removal of transduced muscle fibers. Vg levels for rAAV.MHCK7.FKRP and rAAV.MHCK7.FKRP.pA.CMV.FST were similar in all tissues studied, with the single FKRP vector showing slightly higher levels in all cases. At high doses, amplification of FKRP and FST gene expression was observed with rAAV.MHCK7.FKRP.pA.CMV.FST compared to either rAAV.MHCK7.FKRP or rAAV.CMV.FST alone (Figure 7). Low-dose results were more varied (Figure 13). These data suggest that the presence of two enhancers within two individual promoter elements can act synergistically to amplify the expression of both transgenes in both FKRPP448L muscle (Figure 7) and wild-type muscle (Figure 4).

[0109] Example 7 Induction of FKRP and FST protein expression in muscle, liver, and serum. Using antiserum against FKRP protein, we identified increased FKRP protein expression in both liver and muscle tissue treated with rAAV.MHCK7.FKRP and rAAV.MHCK7.FKRP.pA.CMV.FST (Figures 15, 16). Although differences in FST protein expression were more difficult to observe by Western blotting, FST protein appeared to be elevated in both liver and muscle by HD bisistronic treatment (Figures 15, 16). FST is a secreted protein that can have trans-effects after secretion into serum. Therefore, ELISA assays were performed to measure serum levels of human FST protein derived from delivered gene therapy (Figure 17A) and endogenous mouse FST protein (Figure 17B). Endogenous mouse FST protein was 2.2 ± 0.5 ng / mL mean in wild-type mouse serum, and PBS-treated FKRP P448L The mean level in mouse serum was 3.2 ± 1 ng / mL. Serum levels of mouse FST were maintained within this range for all other treatment groups. Serum human FST protein levels increased to 6.4 ± 2.2 ng / mL in HD rAAV.CMV.FST, 5.1 ± 2.5 ng / mL in LD rAAV.MHCK7.FKRP.pA.CMV.FST, and 39.7 ± 8 ng / mL in HD rAAV.MHCK7.FKRP.pA.CMV.FST (12 ± 3 times higher than endogenous mouse FST).

[0110] Example 8 AAV-induced aDG glycosylation induction Immunoblotting was performed using IIH6, an antibody that recognizes the functional glycosylation of aDG (Ervasti et al., J Cell Biol 122:809-823, 1993), and an anti-peptide polyclonal antibody that recognizes aDG and J3DG proteins (Figure 18). IIH6 identified the expression of matriglycan necessary for laminin binding to aDG (Goddeeris et al., Nature 503:136-140, 2013), while the anti-peptide polyclonal antiserum detected total protein expression for both aDG and J3DG. WT muscle showed a strong IIH6 immunoblot signal at 156 kDa, the native molecular weight of functionally glycosylated aDG in skeletal muscle (Ervasti et al., Cell 66:1121-1131, 1991), while PBS-treated and FST-treated FKRP showed a strong signal. P448L Muscle tissue did not show IIH6 signaling, and for aDG polypeptide, it showed reduced molecular weight, consistent with reduced glycosylation (Michele et al., Nature 418:417-422, 2002). Muscle samples from both rAAV.MHCK7.FKRP and multiple rAAV.MHCK7.FKRP.pA.CMV.FST showed increased expression of IIH6 with a protein molecular weight of 156 kDa. The 43 kDa band of the bDG protein was present in all samples, as expected based on previous studies (Brockington et al., Am J Hum Genet 69:1198-1209, 2001, Brown et al., Am J Pathol 164:727-737, 2004).

[0111] Quantification of IIH6 immunostaining was also performed to assess the expression of functional aDG glycosylation in the TA, diaphragm, and heart (Figures 19-22). Dystrophin and DAPI co-staining were performed to identify the muscle fiber membrane and nucleus, respectively. The percentage of muscle fibers with at least 50% matching IIH6 membrane staining was compared to WT for both TA and diaphragm muscles with PBS-treated FKRP. P448LIt was significantly reduced in muscle, and significantly increased in rAAV.MHCK7.FKRP.pA.CMV.FST-treated muscle in both cases. This is because the P448L mutation is a partial loss-of-function mutation (Chan et al. Hum Mol Genet 19:3995-4006, 2010), and because regenerated muscle resulting after muscle fiber injury typically has greater IIH6 expression (Cohn et al., Cell 110:639-648, 2002, Krag et al. Skelet Muscle 1:31, 2011), FKRP P448L Positively stained muscle fibers were still present in the muscle. Both the percentage of positively stained muscle fibers and the intensity of IIH6 staining significantly increased to near or above WT levels in rAAV.MHCK7-FKRP.pA.CMV.FST-treated FKRPP448L TA and diaphragm (Figure 19). The percentage of membranes showing continuous IIH6 membrane staining dramatically increased in rAAV.MHCK7-FKRP.pA.CMV.FST-treated FKRPP448L muscle compared to all other conditions (Figure 20). These data suggest that functional aDG glycosylation is not only expressed along more muscle fibers after bicistronic FKRP / FST gene therapy, but this glycosylation is also stronger per unit membrane area.

[0112] Example 9 Impact on muscle pathology In mice with muscular dystrophy, damaged or destroyed muscle fibers can regenerate to form new muscle fibers. When such regeneration occurs, the nucleus typically remains within the center of the newly formed skeletal muscle fiber, providing an indelible marker of such degeneration and regeneration cycles. Mock (PBS) treated FKRP P448LBoth the TA and diaphragm muscles of mice showed a dramatic increase in the percentage of muscle fibers with centrally located nuclei compared to age- and sex-matched wild-type mice (Figure 23A, B). While rAAV.CMV.FST-treated FKRPP448L muscles also showed a significant increase in muscle fibers with central nuclei, rAAV.MHCK.FKRP and rAAV.MHCK7.FKRP.pA.CMV.FST treatments significantly reduced this level in the TA and diaphragm.

[0113] The release of creatine kinase into the serum is another measure of muscle damage in dystrophy mice. Serum CK activity levels were higher in FKRP mice compared to wild-type mice both before and after a 5-day walking protocol. P448L In mice, levels were elevated, and the elevations in both WT and FKRPP448L were greater after walking (Figure 22). Neither low-dose (LD) nor high-dose (HD) CMV.FST showed improvement in serum CK levels. HD rAAV.MHCK7.FKRP and HD rAAV.MHCK7.FKRP.pA.CMV.FST showed a tendency toward decreased serum CK levels in the pre-walking group of HD, but not in LD. This tendency toward decreased serum CK levels with HD FKRP / FST therapy disappeared in the post-walking group.

[0114] Next, the percentage of non-muscle area within the muscle was measured (Figure 23). This is a measure of muscle wasting, as non-muscle tissue replaces muscle cells as the severity of muscular dystrophy progresses. In both the TA (Figure 23C) and diaphragm (Figure 22D), PBS-treated FKRP compared to wild-type (WT) P448LThe percentage of non-muscle area increased in mice. For both the TA and diaphragm, the percentage of non-muscle area showed an inverted pattern compared to the induction of muscle growth (Figure 9). The percentage of non-muscle area decreased in the TA with both rAAV.CMV.FST and rAAV.MHCK7.FKRP.pA.CMV.FST (both induced muscle growth in that muscle), but for the diaphragm, the percentage of non-muscle area was reduced to normal levels only with FKRP / FST bisistron therapy. This is also consistent with the fact that only bisistron therapy induced diaphragmatic muscle growth.

[0115] array pAAV.MHCK7.FKRP.spA.CMV.spA KanR(Sequence ID 1) FKRP DNA sequence, Sequence ID No. 2 FKRP amino acid sequence (495aa) NP_077277.1 Sequence ID 3 MRLTRCQAALAAAITLNLLVLFYVSWLQHQPRNSRARGPRRASAAGPRVTVLVREFEAFDNAVPELVDSF LQQDPAQPVVVAADTLPYPPLALPRIPNVRLALLQPALDRPAAASRPETYVATEFVALVPDGARAEAPGL LERMVEALRAGSARLVAAPVATANPARCLALNVSLREWTARYGAAPAAPRCDALDGDAVVLLRARDLFNL SAPLARPVGTSLFLQTALRGWAVQLLDLTFAAARQPPLATAHARWKAEREGRARRAALLRALGIRLVSWE GGRLEWFGCNKETTRCFGTVVGDTPAYLYEERWTPPCCLRALRETARYVVGVLEAAGVRYWLEGGSLLGA ARHGDIIPWDYDVDLGIYLEDVGNCEQLRGAEAGSVVDERGFVWEKAVEGDFFRVQYSESNHLHVDLWPF YPRNGVMTKDTWLDHRQDVEFPEHFLQPLVPLPFAGFVAQAPNNYRRFLELKFGPGVIENPQYPNPALLS LTGSG FST 344 DNA Sequence ID 4 atggtccgcgcgaggcaccagccgggtgggctttgcctcctgctgctgctgctctgccag ttcatggaggaccgcagtgcccaggctgggaactgctggctccgtcaagcgaagaacggc cgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccgg ctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgatt ttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgt ggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccg gattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgc aatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtac caaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgtgtg gtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgct tcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctg agaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatc aaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttc aaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggat gagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagct gcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcg gaagacaccgaggaagaggaggaagatgaagaccaggactacagctttcctatatcttct attctagagtgg FST344 protein Sequence ID 5 MVRARHQPGGLCLLLL LLCQFMEDRSAQAGNC WLRQAKNGRCQVLYKT ELSKEECCSTGRLSTS WTEEDVNDNTLFKWMI FNGGAPNCIPCKETCE NVDCGPGKKCRMNKKN KPRCVCAPDCSNITWK GPVCGLDGKTYRNECA LLKARCKEQPELEVQY QGRCKKTCRDVFCPGS STCVVDQTNNAYCVTC NRICPEPASSEQYLCG NDGVTYSSACHLRKAT CLLGRSIGLAYEGKCI KAKSCEDIQCTGGKKC LWDFKVGRGRCSLCDE LCPDSKSDEPVCASDN ATYASECAMKEAACSS GVLLEVKHSGSCNSIS EDTEEEEEDEDQDYSF PISSILEW MHCK7 Promoter Sequence ID 6 AGCTTGCATGTCTAAGCTAGACCCTTCAGATTAAAAATAACTGAGGTAAGGGCCTGGGTAGGGGAGGTGGTGTGAGACGCTCCTGTCTCTCCTCTATCTGCCCATCGGCCCTTTGGGGAGGAGGAATGTGCCCAAGGACTAAAAAAAGGCCATGGAGCCAGAGGGGCGAGGGCAACAGACCTTTCATGGGCAAACCTTGGGGCCCTGCTGTCTAGCATGCCCCACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAGATGCCTGGTTATAATTAACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGTCCCTGGTGGATCCCCTGCATGCGAAGATCTTCGAACAAGGCTGTGGGGGACTGAGGGCAGGCTGTAACAGGCTTGGGGGCCAGGGCTTATACGTGCCTGGGACTCCCAAAGTATTACTGTTCCATGTTCCCGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGGCTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACACTCAGGAGCAGCCAGC [[ID=PROBLEM]]CMV promoter SEQ ID NO: 7 It should be noted that in the original text, the content in is "CMVプロモーター" which is Japanese. I translated it into English as "CMV promoter" in the translation. If there is a more accurate English expression according to specific context requirements, it can be adjusted accordingly.CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGA References 1. High, KA, and Anguela, XM (2016). Adeno-associated viral vectors for the treatment of hemophilia. Hum Mol Genet 25: R36 - 41. 2. Samelson-Jones, BJ, and Arruda, VR (2020). 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Claims

1. A polynucleotide sequence comprising, in the order 5' to 3', AAV ITR, a first transcriptional regulatory sequence, an enhancer and / or intron, a first transgene sequence, a polyadenylation signal sequence, a second transcriptional regulatory sequence, a second transgene sequence, a polyadenylation signal sequence, and AAV ITR.

2. The polynucleotide sequence according to claim 1, wherein the first transcriptional regulatory sequence is a muscle-specific promoter.

3. The polynucleotide sequence according to claim 2, wherein the muscle-specific promoter comprises one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer binding factor MEF binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MHC enhancer-promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast muscle troponin C gene element, slow muscle cardiac troponin c gene element, slow muscle troponin I gene element, hypoxia-inducible nuclear factor (HIF)-response element (HRE), steroid-inducible element, and glucocorticoid-response element (GRE).

4. The polynucleotide sequence according to any one of claims 1 to 3, wherein the first transgene sequence encodes a protein that is reduced or eliminated in subjects suffering from degenerative muscle disease.

5. The polynucleotide sequence according to any one of claims 1 to 4, wherein the second introduced gene sequence encodes a muscle-building protein.

6. A polynucleotide sequence comprising AAV ITR, a first transcriptional regulatory sequence operably ligated to a nucleotide sequence encoding fukutin-related protein (FKRP), a second transcriptional regulatory sequence operably ligated to a nucleotide sequence encoding FST (follistatin 344), and AAV ITR, in the order of 5' to 3'.

7. The polynucleotide sequence according to claim 6, wherein the nucleotide sequence encoding the FKRP protein includes nucleotides 2056 to 2846 of SEQ ID NO:

1.

8. The polynucleotide sequence according to claim 6 or 7, wherein the nucleotide sequence encoding FST (follistatin 344) includes nucleotides 5393 to 6427 of SEQ ID NO:

1.

9. The polynucleotide sequence according to any one of claims 6 to 8, wherein the first transcriptional regulatory sequence is the MHCK7 promoter.

10. The polynucleotide sequence according to any one of claims 6 to 9, wherein the second transcriptional regulatory sequence is a CMV promoter and / or a CMV enhancer.

11. A polynucleotide sequence according to any one of claims 6 to 10, further comprising an SV40 enhancer and / or an intron.

12. A polynucleotide sequence containing a nucleotide sequence that is at least 95% identical to nucleotides 1847-6672 of SEQ ID NO:

1.

13. The polynucleotide sequence according to claim 12, wherein the nucleotide sequence includes nucleotides 1847 to 6672 of sequence number 1.

14. Recombinant adeno-associated virus (rAAV) comprising the polynucleotide sequence described in any one of claims 1 to 13.

15. The rAAV according to any one of claims 14, wherein the rAAV comprises AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAVrh. 74, AAVrh. 10, Anc80, AAV7m8, AAVMyo, MYOAAV capsid protein, or variants thereof.

16. Recombinant AAV particles comprising a polynucleotide sequence according to any one of claims 1 to 13, or rAAV according to claim 14 or 15.

17. A composition comprising the rAAV described in claim 14 or 15, or the rAAV particles described in claim 16.

18. A method for treating muscular dystrophy, comprising administering to a subject in need of treatment the rAAV described in claim 14 or 15, the rAAV particles described in claim 16, or the composition described in claim 17.

19. The method according to claim 18, wherein the muscular dystrophy is limb-girdle muscular dystrophy 2I or congenital muscular dystrophy 1C.

20. The method according to claim 17 or 18, wherein the rAAV, rAAV particles, or composition is administered by systemic administration, intramuscular injection, or intravenous injection.

21. A composition for treating muscular dystrophy in a subject requiring treatment, comprising rAAV according to claim 14 or 15, rAAV particles according to claim 16, or the composition according to claim 17.

22. The composition according to claim 21, wherein the muscular dystrophy is limb-girdle muscular dystrophy 2I or congenital muscular dystrophy 1C.

23. The composition according to claim 21 or 22, wherein the composition is formulated for systemic administration, intramuscular injection, or intravenous injection.

24. Use of the rAAV according to claim 14 or 15, the rAAV particles according to claim 16, or the composition according to claim 17 for the preparation of a drug for treating muscular dystrophy.

25. The use according to claim 24, wherein the muscular dystrophy is limb-girdle muscular dystrophy 2I or congenital muscular dystrophy 1C.

26. The use according to claim 24 or 25, wherein the drug is formulated for systemic administration, intramuscular injection, or intravenous injection.

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