TW202424194A - Compositions and methods for the treatment of neuromuscular disorders - Google Patents

Abstract

The present disclosure relates to adeno-associated virus (AAV)-mediated delivery of nucleic acids for treating neuromuscular disorders, e.g., X-linked myotubular myopathy (XLMTM), in patients in need thereof. The AAV vectors of the disclosure may include, e.g., a transgene encoding a myotubularin protein operably linked to one or more transcription regulatory elements. In some embodiments, the one or more transcription regulatory elements are specifically active in muscle and/or liver tissue

Description

Compositions and methods for treating neuromuscular disorders

X-linked myotubular myopathy (XLMTM) is a fatal monogenic disorder of skeletal muscle caused by mutations in the myotubularin 1 (MTM1) gene. XLMTM affects approximately one in 50,000 newborn boys and typically presents with marked hypotonia and respiratory failure. In extremely rare cases, females develop severe forms of XLMTM. Survival beyond the postnatal period requires intensive support, including respiratory support (i.e., mechanical ventilation) at birth in 85%-90% of patients, continuous 24-hour ventilator dependence in nearly 50% of patients, and tracheostomy in approximately 60% of patients. Until recently, only supportive treatment options were available, such as the use of a ventilator or feeding tube. Recently, gene therapy approaches involving the delivery of MTM1 have been developed for the treatment of XLMTM. However, this technology requires improved methods of administering gene therapy to patients with XLMTM.

The present disclosure provides compositions and methods useful for treating neuromuscular disorders, particularly X-linked microtubular myopathy (XLMTM). Using the compositions and methods described herein, a viral vector, such as an adeno-associated virus (AAV) vector, having a transgene encoding myotubularin 1 (MTM1) can be administered to a patient (e.g., a human patient) suffering from XLMTM. Exemplary compositions and methods of the present disclosure are described below.

In a first aspect, the present disclosure provides a recombinant AAV vector comprising a transgene encoding MTM1, wherein the transgene is operably linked to a promoter active in liver tissue.

In some embodiments, the promoter is selectively active in liver tissue. In some embodiments, after the vector is contacted with one or more hepatocytes and one or more non-hepatocytes, respectively (e.g., in vitro or in vivo) under the same conditions, the vector causes the expression level of the transgene in the one or more hepatocytes to be higher than the expression level of the transgene in the one or more non-hepatocytes, for example, 2 to 1,000 times higher (e.g., 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 times higher than the expression level of the transgene in the one or more non-hepatocytes). In some embodiments, when the vector is in contact with one or more hepatocytes and one or more non-hepatocytes, the vector causes the expression level of the transgene in the one or more hepatocytes to be 10 to 1,000 times higher than the expression level of the transgene in the one or more non-hepatocytes. In some embodiments, when the vector is in contact with one or more hepatocytes and one or more non-hepatocytes, the vector causes the expression level of the transgene in the one or more hepatocytes to be 50 to 1,000 times higher than the expression level of the transgene in the one or more non-hepatocytes. In some embodiments, when the vector is contacted with one or more hepatocytes and one or more non-hepatocytes, respectively, the vector causes the expression level of the transgene in the one or more hepatocytes to be 100 to 1,000 times higher than the expression level of the transgene in the one or more non-hepatocytes. In some embodiments, when the vector is contacted with one or more hepatocytes and one or more non-hepatocytes, respectively, the vector causes the expression level of the transgene in the one or more hepatocytes to be at least 2 times, at least 5 times, at least 10 times, at least 50 times, or at least 100 times higher than the expression level of the transgene in the one or more non-hepatocytes.

In some embodiments, the non-liver cell is a muscle cell or a nerve cell. In some embodiments, the non-liver cell is a cardiac muscle cell.

In some embodiments, when the vector is contacted with one or more liver cells (e.g., from a patient with a neuromuscular disorder), the vector causes the expression level of the transgene in the one or more liver cells to be closer to the expression of MTM1 in wild-type/healthy liver cells, for example, the expression of the transgene can be 0.2 to 10 times (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times) the expression level of MTM1 in wild-type/healthy liver cells. In some embodiments, when the vector is contacted with one or more liver cells (e.g., from a patient with a neuromuscular disorder), the vector causes the expression level of the transgene in the one or more liver cells to be at least 0.2 times, at least 0.5 times, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the expression level of wild-type MTM1 in healthy liver cells.

In some embodiments, the promoter comprises an LP1 promoter. In some embodiments, the LP1 promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 3: GTCCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTA GGCGGGCGACTCAGATCCCAGCCAGTGcACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAA (SEQ ID NO: 3).

In some embodiments, the LP1 promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 3 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 3). In some embodiments, the LP1 promoter has the nucleic acid sequence of SEQ ID NO: 3.

In some embodiments, the promoter comprises an apolipoprotein E (ApoE) promoter. In some embodiments, the ApoE promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 8: CCCTAAAATGGGCAAACATTGCAAGCAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGGGG (SEQ ID NO: 8).

In some embodiments, the ApoE promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 8 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 8). In some embodiments, the ApoE promoter has the nucleic acid sequence of SEQ ID NO: 8.

In some embodiments, the promoter comprises an alpha-1-antitrypsin (A1AT) promoter. In some embodiments, the A1AT promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 9: AATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT (SEQ ID NO: 9).

In some embodiments, the A1AT promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 9 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 9). In some embodiments, the A1AT promoter has the nucleic acid sequence of SEQ ID NO: 9.

In some embodiments, the promoter is a chimeric promoter comprising an ApoE promoter and an A1AT promoter. In some embodiments, the chimeric promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 2: CGTGATCTAGTAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACT CGACCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAG CAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCC GTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGATCCCCCTGATCTGCGGCCTCGACGGTATCGATAAG (SEQ ID NO: 2).

In some embodiments, the chimeric promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 2 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 2). In some embodiments, the chimeric promoter has the nucleic acid sequence of SEQ ID NO: 2.

In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the constitutive promoter is a phosphoglycerate kinase (PGK) promoter, an elongation factor-1α (EF1α) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a cytomegalovirus (CMV) promoter, or a chicken-β-actin (CBA) promoter.

In some embodiments, the AAV vector further comprises a second transgene encoding MTM1. In some embodiments, the second transgene encoding MTM1 is operably linked to a promoter active in muscle tissue. In some embodiments, the promoter active in muscle tissue is selectively active in muscle tissue.

In some embodiments, a promoter that is selectively active in muscle tissue achieves (e.g., in vitro or in vivo) a level of expression of the MTM1 transgene in muscle cells that is higher than the level of expression of the MTM1 transgene achieved by the same promoter in non-muscle cells, e.g., 2-fold to 1,000-fold higher (e.g., 2-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1,000-fold higher than the level of expression of the MTM1 transgene achieved by the same promoter in non-muscle cells). In some embodiments, a promoter selectively active in muscle tissue achieves an expression level of MTM1 transgene in muscle cells that is 10-fold to 1,000-fold higher than the expression level of MTM1 transgene achieved by the same promoter in non-muscle cells. In some embodiments, a promoter selectively active in muscle tissue achieves an expression level of MTM1 transgene in muscle cells that is 50-fold to 1,000-fold higher than the expression level of MTM1 transgene achieved by the same promoter in non-muscle cells. In some embodiments, a promoter that is selectively active in muscle tissue achieves an expression level of the MTM1 transgene in muscle cells that is 100-fold to 1,000-fold higher than the expression level of the MTM1 transgene achieved by the same promoter in non-muscle cells. In some embodiments, a promoter that is selectively active in muscle tissue achieves an expression level of the MTM1 transgene in muscle cells that is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold higher than the expression level of the MTM1 transgene achieved by the same promoter in non-muscle cells.

In some embodiments, when the vector is contacted with one or more liver cells (e.g., from a patient with a neuromuscular disorder), the vector causes the expression level of the transgene in the one or more liver cells to be closer to the expression of MTM1 in wild-type/healthy liver cells, for example, the expression of the transgene can be 0.2 to 10 times (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times) the expression level of MTM1 in wild-type/healthy liver cells. In some embodiments, when the vector is contacted with one or more liver cells (e.g., from a patient with a neuromuscular disorder), the vector causes the expression level of the transgene in the one or more liver cells to be at least 0.2 times, at least 0.5 times, at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the expression level of wild-type MTM1 in healthy liver cells.

In some embodiments, the transgene encoding MTM1 is operably linked to the muscle creatine kinase (MCK) promoter or the desmin (DES) promoter.

In some embodiments, each transgene encoding MTM1 independently has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1. In some embodiments, each transgene encoding MTM1 independently has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1). In some embodiments, each transgene encoding MTM1 has a nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1.

In some embodiments, each transgene encoding MTM1 independently has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, each transgene encoding MTM1 independently has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 6 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 6). In some embodiments, each transgene encoding MTM1 has a nucleic acid sequence of SEQ ID NO: 6.

In some embodiments, each transgene encoding MTM1 independently has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, each transgene encoding MTM1 independently has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 7 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 7). In some embodiments, each transgene encoding MTM1 has a nucleic acid sequence of SEQ ID NO: 7.

In some embodiments, the MTM1 sequence is codon-optimized.

In some embodiments, the AAV is serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74. In some embodiments, the AAV vector is a pseudotyped AAV. In some embodiments, the pseudotyped AAV is AAV2/8 or AAV2/9.

In another aspect, the present disclosure provides a method for treating XLMTM in a human patient in need thereof, by administering to the patient a therapeutically effective amount of an AAV vector of any of the above aspects or embodiments of the present disclosure.

In some embodiments, the patient is five years old or younger when the AAV vector is administered. In some embodiments, the patient is four years old or younger when the AAV vector is administered, optionally wherein the patient is three years old or younger, two years old or younger, one year old or younger, or six months or younger.

In some embodiments, the AAV vector is administered to a patient in an amount of less than 3 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of less than 2.5 x 10 ¹⁴ vg/kg, optionally wherein the AAV vector is administered to a patient in an amount of less than 2 x 10 ¹⁴ vg/kg, less than 1.5 x 10 ¹⁴ vg/kg, or less than 1.4 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of 3 x 10 ¹³ vg/kg to 2.3 x 10 ¹⁴ vg/kg, optionally wherein the AAV vector is administered to a patient in an amount of 8 x 10 ¹³ vg/kg to 1.8 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg to 1.6 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg to 1.5 x 10 ¹⁴ vg/kg, or 1.2 x 10 ¹⁴ vg/kg to 1.4 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.3 x 10 ¹⁴ vg/kg.

In some embodiments, the AAV vector is administered to a patient by intravenous, intramuscular, intrahepatic, intradermal, or subcutaneous administration.

In some embodiments, the patient is further administered an anticholestatic agent. In some embodiments, the anti-cholestatic agent is selected from the group consisting of bile acid, farnesoid X receptor (FXR) ligand, fibroblast growth factor 19 (FGF-19) mimetic, Takeda-G protein receptor 5 (TGR5) agonist, peroxisome proliferator-activated receptor (PPAR) agonist, PPAR-α agonist, PPAR-δ agonist, dual PPAR-α and PPAR-δ agonist, apical sodium-dependent bile acid transporter (ASBT) inhibitor, immunomodulatory drug, antifibrotic chemotherapy and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor. In some embodiments, (i) the FXR ligand is obeticholic acid, cilofexor, tropifexor, tretinoin, or EDP-305; (ii) the FGF-19 mimetic is aldafermin; (iii) the TGR5 agonist is INT-777 or INT-767; (iv) the PPAR agonist is bezafibrate, fenofibrate, seladelpar, or elafibrinor; (v) the PPAR-α agonist is fenofibrate; (vi) the PPAR-δ agonist is seladelpar; (vii) The dual PPAR-α and PPAR-δ agonist is elafibranor; (viii) the ASBT inhibitor is odevixibat, maralixibat or linerixibat; (ix) the immunomodulatory drug is rituximab, abatacept, ustekinumab, infliximab, baricitinib or FFP-104; (x) the antifibrotic chemotherapy is a vitamin D receptor agonist or simtuzumab; and/or (xi) the NOX inhibitor is setanaxib.

In some embodiments, the bile acid is ursodeoxycholic acid, norursodeoxycholic acid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the patient has no history of cholestasis or hyperbilirubinemia. In some embodiments, the patient has no history of any underlying liver disease.

In some embodiments, the method comprises administering to the patient a therapeutically effective amount of: (i) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in liver tissue, and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in muscle tissue.

In some embodiments, the promoter active in liver tissue comprises an LP1 promoter. In some embodiments, the LP1 promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the LP1 promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 3 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 3). In some embodiments, the LP1 promoter has a nucleic acid sequence of SEQ ID NO: 3.

In some embodiments, the promoter active in liver tissue comprises an ApoE promoter. In some embodiments, the ApoE promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the ApoE promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 8 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 8). In some embodiments, the ApoE promoter has the nucleic acid sequence of SEQ ID NO: 8.

In some embodiments, the promoter active in liver tissue comprises an A1AT promoter. In some embodiments, the A1AT promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the A1AT promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 9 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 9). In some embodiments, the A1AT promoter has the nucleic acid sequence of SEQ ID NO: 9.

In some embodiments, the promoter active in liver tissue is a chimeric promoter comprising an ApoE promoter and an A1AT promoter. In some embodiments, the chimeric promoter has a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the chimeric promoter has a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 2 (e.g., a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 2). In some embodiments, the chimeric promoter has the nucleic acid sequence of SEQ ID NO: 2.

In another aspect, the present disclosure provides a method for treating XLMTM in a human patient in need thereof, by administering to the patient a therapeutically effective amount of: (i) a non-viral composition comprising a nucleic acid encoding MTM1 operably linked to a promoter that is active (e.g., selectively active) in liver tissue, and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in muscle tissue.

In some embodiments, the non-viral composition is a liposome, a vesicle, a synthetic vesicle, an exosome, a synthetic exosome, a dendrimer, or a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle that can deliver DNA constructs similar in composition and size to those delivered via AAV capsids.

In some embodiments, the promoter is a DES promoter. In some embodiments, the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently have a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently have a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently have a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each have a nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1.

In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each have a nucleic acid sequence of SEQ ID NO: 6.

In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each have a nucleic acid sequence of SEQ ID NO: 7.

In some embodiments, the AAV vector is resamirigene bilparvovec.

In another aspect, the present disclosure provides a kit comprising an AAV vector of any one of the above aspects or embodiments of the present disclosure. The kit may further include a package insert instructing a user to administer the AAV vector to a patient diagnosed with XLMTM.

In another aspect, the present disclosure provides a kit comprising: (i) a non-viral composition comprising a nucleic acid encoding MTM1 (e.g., the non-viral composition described above), and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in muscle tissue (e.g., the AAV vector described above). The kit may further include a packaging insert that instructs a user to administer the non-viral composition and the AAV vector to a patient diagnosed with XLMTM.

Definition

As used herein, the term "about" refers to a value that is within 10% above or below the described value. For example, "100 pounds" as used in the context of weights described herein includes amounts within 10% above or below 100 pounds. In addition, when used in the context of a list of numerical quantities, it is understood that the term "about" when preceding a list of numerical quantities applies to each individual amount listed in the list.

As used herein, the terms "administering" and "administration" refer to directly giving a therapeutic agent (e.g., a pharmaceutical composition comprising a viral vector comprising a nucleic acid sequence encoding the myotubularin 1 (MTM1) gene operably linked to a promoter) to a patient by any effective route. Exemplary routes of administration are described herein and include systemic routes of administration, such as intravenous injection, and routes of administration directly to the patient's central nervous system, such as by intrathecal injection or intraventricular injection, and routes of administration directly to the patient's liver, etc.

As used herein, the term "age-adjusted norming" refers to the process of normalizing data by age, which is a technique used to allow comparisons of groups of subjects when the age profiles of the groups are different. As used herein, the term "normative" refers to data that have not been normalized by age because the age profiles of the groups of subjects are similar.

As used herein, the terms "alanine aminotransferase" and "ALT" refer to proteins whose amino acid sequences comprise or consist of the amino acid sequences of naturally occurring wild-type ALT proteins (e.g., ALT1 and ALT2), and proteins whose amino acid sequences comprise or consist of the amino acid sequences of naturally occurring ALT allelic variants (GPT or GPT2, such as splice variants or allelic variants). The human GPT nucleic acid sequence is provided as NCBI RefSeq Accession No. NM_005309.2, and an exemplary wild-type ALT1 amino acid sequence is provided as NCBI RefSeq Accession No. NP_005300.1. The human GPT2 nucleic acid sequence is provided as NCBI RefSeq Accession No. NM_001142466.2, and an exemplary wild-type ALT2 amino acid sequence is provided as NCBI RefSeq Accession No. NP_001135938.1.

As used herein, the terms "alkaline phosphatase" and "ASP" refer to a protein whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring wild-type ASP protein, and a protein whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring ASP allelic variant (e.g., a splice variant or an allelic variant). The human ASP nucleic acid sequence is provided as NCBI RefSeq Accession No. NM_000478.5, and an exemplary wild-type ASP amino acid sequence is provided as NCBI RefSeq Accession No. NP_000469.3.

As used herein, the term "anticholestatic agent" refers to a substance, such as a small molecule, used to increase bile formation and/or antagonize the effects of hydrophobic bile acids on biological membranes. As used herein, the term "antagonist" with respect to a protein refers to a molecule that reduces signal transduction resulting from the interaction of the protein with one or more of its binding partners. An antagonist can result in a reduction in the binding of a protein to one or more of its binding partners relative to the binding of the two proteins in the absence of the antagonist.

As used herein, the terms "aspartate aminotransferase" and "AST" refer to a protein whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring wild-type AST protein, and a protein whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring AST allelic variant (e.g., a splice variant or an allelic variant). The human AST nucleic acid sequence is provided as NCBI RefSeq Accession No. NM_002079.2, and an exemplary wild-type ASP amino acid sequence is provided as NCBI RefSeq Accession No. NP_002070.1.

As used herein, the terms "bile acid test" and "serum bile acid test" refer to a procedure in which a preprandial (i.e., before eating) blood sample is collected as a baseline, followed by a meal, and then about two hours later a postprandial (i.e., after eating) blood sample is collected. Both blood samples are tested for bile acid levels, and the preprandial sample is used as a reference. As used herein, "bile acid" refers to a steroid acid found primarily in the bile of mammals and other vertebrates.

As used herein, the terms "Philadelphia Children's Hospital Neuromuscular Disorders Infant Test" and "CHOP INTEND" refer to a validated motor outcome measure developed for the assessment of frail infants, such as infants with skeletal muscle diseases (e.g., X-linked microtubular myopathy (XLMTM)). CHOP INTEND uses a 0-64 subscale, with higher scores indicating better motor function. As used herein, the term "motor function score" refers to the score on the 0-64 subscale of CHOP INTEND (e.g., a CHOP INTEND scale >45).

As used herein, the term "cholestasis" refers to a condition in which bile cannot flow from the liver to the duodenum. Two clinical distinctions are "obstructive" types of cholestasis, in which there is a mechanical blockage of the duct system that may result from gallstones or malignant tumors, and "metabolic" types of cholestasis, which are disturbances in bile formation that may result from genetic defects or be obtained as a side effect of many medications. As used herein, the term "bile" refers to the digestive fluid secreted by the liver to help digest fats.

As used herein, "combination therapy" means administering two (or more) different agents or treatments to a subject as part of a treatment regimen defined for a particular disease or disorder (e.g., a neuromuscular disorder). In some embodiments, "combination therapy" may include surgery. The treatment regimen defines the dosage and administration cycle of each agent so that the effects of the individual agents on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or synchronous and the agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescription regimen. In some embodiments, the combined administration of two or more agents or treatments results in a greater reduction in symptoms or other parameters associated with the disorder than observed with one agent or treatment delivered alone or in the absence of the other agent or treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent may be achieved by any appropriate route, including, but not limited to, oral, intravenous, intramuscular, intrahepatic, and direct absorption through mucosal tissues. The therapeutic agents may be administered by the same route or by different routes. For example, the first therapeutic agent of the combination can be administered by intravenous injection, and the second therapeutic agent of the combination can be administered enterally. In another example, the agents of the therapeutic combination can be administered by intravenous injection, and the surgical procedure for the therapeutic combination (e.g., nasobiliary drainage (NBD)) can be performed.

As used herein, the term "dose" refers to the amount of a therapeutic agent, such as a viral vector described herein, which is administered to a subject in a specific case to treat a disorder, such as to treat or ameliorate one or more symptoms of a neuromuscular disorder (e.g., XLMTM) described herein. A therapeutic agent as described herein may be administered in a single dose or multiple doses during a treatment period, as defined herein. In each case, a therapeutic agent may be administered using one or more unit dosage forms of the therapeutic agent, a term referring to one or more discrete compositions containing the therapeutic agent, which together constitute a single dose of the agent.

As used herein, the terms "effective amount," "therapeutically effective amount," and the like, when used with respect to therapeutic compositions such as the carrier constructs described herein, refer to an amount sufficient to produce a beneficial or desired outcome (e.g., a clinical outcome) when administered to a subject (including a mammal, such as a human). For example, in the context of treating neuromuscular disorders such as XLMTM, these terms refer to an amount of the composition sufficient to achieve a therapeutic response compared to the response obtained when the composition of interest is not administered. An "effective amount," "therapeutically effective amount," and the like of a composition such as the carrier constructs disclosed herein also includes an amount that produces a beneficial or desired outcome in a subject compared to a control.

As used herein, the terms "γ-glutamyl transferase" and "GGT" refer to proteins whose amino acid sequences include or consist of the amino acid sequences of naturally occurring wild-type GGT proteins, and proteins whose amino acid sequences include or consist of the amino acid sequences of naturally occurring GGT allelic variants (GGT1, GGT2, and GGT3, e.g., splice variants or allelic variants). The human GGT1 nucleic acid sequence is provided as NCBI RefSeq Accession No. NM_001288833.1, and an exemplary wild-type GGT1 amino acid sequence is provided as NCBI RefSeq Accession No. NP_001275762.1.

As used herein, the term "hyperbilirubinemia" refers to a condition in which the level of bilirubin in the blood is higher than normal. As used herein, the term "bilirubin" refers to a compound that occurs in the normal metabolic pathway that breaks down heme in vertebrates. This metabolism is a necessary process for the body to eliminate waste products produced by the destruction of aging or abnormal red blood cells. As used herein, "bilirubin test" refers to measuring the amount of bilirubin in a patient's blood.

As used herein, the term "level" refers to the level of a protein compared to a reference. A reference can be any useful reference as defined herein. "Decreased levels" and "increased levels" of a protein mean a decrease or increase in the level of a protein compared to a reference (e.g., a decrease or increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more; a decrease or increase of greater than about 100% compared to a reference). The level of protein can be expressed as mass/volume (e.g., g/dL, mg/mL, μg/mL, ng/mL) or as a percentage of the total protein in the sample.

As used herein, the terms "liver function tests" and "LFTs" refer to a liver panel (e.g., a set of blood tests that provide information about the status of a patient's liver). A liver panel may include measuring gamma-glutamicin transferase levels, alkaline phosphatase levels, aspartate aminotransferase levels, alanine aminotransferase levels, albumin levels, bilirubin levels, prothrombin time, activated partial thromboplastin time, or a combination thereof.

As used herein, the terms "maximum inspiratory pressure" and "MIP" refer to variables in mechanical ventilation, including the total airway pressure delivered, which is usually used to overcome respiratory system compliance and airway resistance. In pressure control mode, MIP includes the sum of positive end-expiratory pressure and "differential pressure". As used herein, the term "differential pressure" refers to variables in mechanical ventilation, including the difference between MIP and positive end-expiratory pressure.

As used herein, the term "mechanical ventilatory support" refers to the medical term for artificial ventilation in which a mechanical device is used to assist or replace spontaneous breathing. As used herein, the term "invasive mechanical ventilatory support" refers to the medical term for artificial ventilation in which air is delivered through a tube inserted into the patient's trachea through the mouth or nose, and a mechanical device is used to assist or replace spontaneous breathing. As used herein, the term "non-invasive mechanical ventilatory support" refers to mechanical ventilatory support in which air is delivered to the patient through a sealed mask that can be placed over the mouth, nose, or entire face.

As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, wherein the molecules are arranged so that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single adjacent molecule and may or may not be adjacent. For example, if a promoter regulates the transcription of a transcribable polynucleotide molecule of interest in a cell, the promoter is operably linked to the transcribable polynucleotide molecule. In addition, if the two parts of a transcriptional regulatory element are linked so that the transcriptional activation function of one part is not adversely affected by the presence of the other part, the two parts are operably linked to each other. Two transcriptional regulatory elements can be operably linked to each other by means of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or can be operably linked to each other in the absence of an intervening nucleotide in the absence of an intervening nucleotide.

As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic compound that is intended for administration to a subject (e.g., a mammal, such as a human) to prevent, treat, or control a specific disease or disorder that affects or may affect the subject.

As used herein, the term "pharmaceutically acceptable" means that the compounds, materials, compositions and/or dosage forms are suitable for contact with tissues of subjects such as mammals (e.g., humans) without excessive toxicity, irritation, allergic response, and other problematic complications commensurate with a reasonable benefit/risk ratio.

As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of the transgene. Exemplary promoters suitable for use with the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which is incorporated herein by reference because it relates to nucleic acid regulatory elements. In addition, the term "promoter" may refer to synthetic promoters, which are regulatory DNA sequences that do not naturally occur in biological systems. Synthetic promoters contain a combination of portions of naturally occurring promoters and polynucleotide sequences that do not occur in nature, and can be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types.

As used herein in the context of promoters, the term "selectively active" refers to a promoter that preferentially drives gene expression in one cell or tissue type as compared to another cell or tissue type. For example, a promoter that is "selectively active" in hepatocytes can achieve a gene expression level in hepatocytes that is higher, e.g., 2-fold to 1,000-fold higher (e.g., 2-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1,000-fold higher expression level) than the gene expression level achieved by the same promoter in one or more non-hepatocytes under substantially the same conditions. Exemplary methods for measuring gene expression are known in the art and described herein.

As used herein, a therapeutic agent is considered to be "provided" to a patient if the therapeutic agent is administered directly to the patient, or if a substance that is processed or metabolized in the body to endogenously produce the therapeutic agent is administered to the patient. For example, a nucleic acid molecule encoding a therapeutic protein (e.g., MTM1) can be provided to a patient, such as a patient suffering from a neuromuscular disorder described herein, by direct administration of the nucleic acid molecule or by administration of a substance (e.g., a viral vector or a cell) that is processed in vivo to produce the desired nucleic acid molecule.

As used herein, the terms "patient" and "subject" refer to an organism that is being treated for a particular disease or condition as described herein (e.g., a neuromuscular disorder, e.g., XLMTM). Examples of subjects and patients include mammals, such as humans, that are being treated for a disease or condition as described herein.

"Reference" means any useful reference for comparison of protein levels associated with cholestasis, hyperbilirubinemia, or one or more symptoms thereof. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or a reference standard or level. A "reference sample" can be, for example, a control, such as a predetermined negative control value, such as a "normal control" or a previous sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject who does not have cholestasis, hyperbilirubinemia, or one or more symptoms thereof; a sample from a subject diagnosed with cholestasis, hyperbilirubinemia, or one or more symptoms thereof; a sample from a subject who has been treated for cholestasis, hyperbilirubinemia, or one or more symptoms thereof; or a sample of a purified protein (e.g., any of the proteins described herein) of known normal concentration. "Reference standard or level" means a value or numerical value derived from a reference sample. "Normal control value" is a predetermined value indicative of a non-disease state, such as a value expected in a healthy control subject. Typically, a normal control value is expressed as a range ("between X and Y"), an upper threshold ("not higher than X"), or a lower threshold ("not lower than X"). Subjects whose measured values are within the normal control value for a particular biomarker are often referred to as being "within the normal limit for that biomarker." A normal reference standard or level can be a value or numerical value derived from a normal subject who does not have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In preferred embodiments, the reference sample, standard or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage and general health status. A standard curve of purified protein (e.g., any of the proteins described herein) levels within the normal reference range can also be used as a reference.

As used herein, the term "full-term age" refers to the age of a patient (e.g., a newborn) born between 37 weeks of gestational age and 42 weeks of gestational age. For example, if a patient is born at 35 weeks of gestational age, the patient's full-term age is 14 days.

As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA or cDNA) encoding a gene product (e.g., a gene product described herein). The gene product may be RNA, a peptide, or a protein. In addition to the coding region of the gene product, the transgene may include or be operably linked to one or more elements to promote or enhance expression, such as a promoter, enhancer, destabilizing domain, response element, reporter element, insulator element, polyadenylation signal, and/or other functional elements. The embodiments of the present disclosure may utilize any known suitable promoter, enhancer, destabilizing domain, response element, reporter element, insulator element, polyadenylation signal, and/or other functional elements.

As used herein, the terms "treat" and "treatment" refer to therapeutic treatment, in which the purpose is to prevent or slow down (lessen) undesirable physiological changes or disorders, such as the progression of neuromuscular disorders, such as XLMTM, etc. Beneficial or desired clinical results include, but are not limited to, relief of symptoms (e.g., stiffness and/or joint spasms); reduction in disease severity; stabilization (i.e., not worsening) of the disease state; delay or slow progression of the disease; improvement or alleviation of the disease state; and remission (whether partial or complete), whether detectable or undetectable. In the setting of a neuromuscular disorder such as XLMTM, treatment of a patient can manifest one or more detectable changes, such as an increase in the concentration of MTM1 protein or a nucleic acid encoding MTM1 (e.g., DNA or RNA, such as mRNA), or an increase in MTM1 activity (e.g., an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or more. The concentration of MTM1 protein can be determined using protein detection assays known in the art, including ELISA assays described herein. The concentration of nucleic acids encoding MTM1 can be determined using nucleic acid detection assays described herein (e.g., RNA Seq assays). In addition, treatment of patients suffering from neuromuscular disorders (such as XLMTM) can be manifested as improvements in the patient's muscle function (e.g., skeletal muscle function) and improvements in muscle coordination. For example, the improved manifestations may include increased diaphragm and/or respiratory muscle development.

As used herein, the terms "X-linked myomicrotubular myopathy" and "XLMTM" refer to a hereditary neuromuscular disorder caused by mutations in the MTM1 gene and characterized by symptoms including mild to severe muscle weakness, hypotonia (decreased muscle tone), feeding difficulties, and/or severe respiratory complications. Human MTM1 has NCBI Gene ID No. 4534. An exemplary wild-type human MTM1 nucleic acid sequence is provided as NCBI RefSeq Accession No. NM_000252.3, and an exemplary wild-type myotubularin 1 amino acid sequence is provided as NCBI RefSeq Accession No. NP_000243.1.

As used herein, the term "vector" refers to a nucleic acid, such as DNA or RNA, that can be used as a medium for delivering a gene of interest into a cell (e.g., a mammalian cell, such as a human cell), for example, for the purpose of replication and/or expression. Exemplary vectors that can be used in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994/11026, the disclosure of which is incorporated herein by reference. The expression vectors described herein contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and/or integrating the polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the transgenes described herein include vectors containing regulatory sequences (e.g., promoter and enhancer regions) that direct gene transcription. Other vectors that can be used to express transgenes contain polynucleotide sequences that enhance the translation rate of the genes or improve the stability or nuclear export of the mRNA produced by gene transcription. Such sequence elements include, for example, 5' and 3' non-translated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites that direct efficient transcription of the genes carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, concomitant or nourseothricin .

This application contains a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on September 14, 2023, is named "51037-075WO2_Sequence_Listing_9_14_23", and is 27,146 bytes in size.

The present disclosure provides compositions and methods useful for treating neuromuscular disorders, particularly X-linked microtubular myopathy (XLMTM). According to the compositions and methods described herein, a viral vector, such as an adeno-associated virus (AAV) vector, containing a transgene encoding myotubularin 1 (MTM1) can be administered to a patient (e.g., a human patient) suffering from XLMTM. The AAV vector can be, for example, a pseudotyped AAV vector, such as an AAV vector containing an AAV2 inverted terminal repeat sequence packaged in a capsid protein from AAV8 (AAV2/8).

In some embodiments, the MTM1 transgene is operably linked to a transcriptional regulatory element, such as a promoter that drives gene expression in hepatocytes. This aspect of the present disclosure is based at least in part on the discovery that expression of the MTM1 transgene in liver tissue of XLMTM patients can improve the safety of MTM1 gene therapy. In some embodiments, the promoter contains an LP1 promoter, an apolipoprotein E (ApoE) promoter, and/or an alpha-1-antitrypsin (A1AT) promoter (e.g., the LP1 promoter, ApoE promoter, and/or A1AT promoter described herein).

In some embodiments, the MTM1 transgene is operably linked to a constitutive transcriptional regulatory element, such as a phosphoglycerate kinase (PGK) promoter, an elongation factor-1α (EF1α) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a cytomegalovirus (CMV) promoter, or a chicken-β-actin (CBA) promoter. In some embodiments, the viral vector includes two MTM1 transgenes: one under the control of a promoter active in liver tissue and the other under the control of a promoter active in muscle tissue. These features of the present disclosure are based at least in part on the discovery that simultaneous expression of the MTM1 transgene in multiple tissues (e.g., liver tissue and muscle tissue) can improve the safety of MTM1 gene therapy.

Also described herein are methods of treating XLMTM in a patient in need thereof by administering to the patient two compositions: (i) a non-viral composition containing a nucleic acid encoding MTM1 operably linked to a promoter that is active (e.g., selectively active) in liver tissue, and (ii) a viral vector containing an MTM1 transgene operably linked to a muscle-specific promoter. Non-viral compositions that can be used in conjunction with this aspect of the disclosure include, but are not limited to, liposomes, vesicles, synthetic vesicles, exosomes, synthetic exosomes, dendrimers, and nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles. Viral vectors that can be used in conjunction with this aspect of the disclosure include, but are not limited to, birelin.

In some embodiments, the present disclosure describes methods of reducing stiffness and/or joint spasm in a human patient diagnosed with XLMTM by administering one or more compositions described herein.

In some embodiments, the present disclosure describes methods of increasing diaphragm and/or respiratory muscle development in a human patient diagnosed with XLMTM by administering one or more of the compositions described herein.

In some embodiments, the present disclosure describes methods of preventing cholestasis or hyperbilirubinemia in a human patient diagnosed with XLMTM by administering one or more of the compositions described herein.

The following section provides a description of therapeutic agents and parameters for evaluating cholestasis, hyperbilirubinemia, or one or more symptoms thereof. The following section also describes various transduction agents that can be used in conjunction with the compositions and methods of the present disclosure. X- linked microtubular myopathy

XLMTM is a rare, life-threatening congenital myopathy caused by mutations in the MTM1 gene and characterized in most patients by severe muscle weakness and hypotonia at birth, leading to severe respiratory insufficiency, inability to sit, stand or walk, and early death.

The myopathy associated with XLMTM impairs the development of motor skills, such as sitting, standing, and walking. Affected infants may also have difficulty feeding due to muscle weakness. Individuals with this disorder typically do not have the muscle strength to breathe on their own and must rely on mechanical ventilation for support. Some affected individuals require mechanical ventilation only periodically, such as during sleep, while others require continuous ventilation. People with XLMTM may also experience weakness of the muscles that control eye movement (ophthalmoplegia), weakness of other facial muscles, and loss of reflexes (areflexia).

In XLMTM, muscle weakness often disrupts normal bone development and may lead to weak bones, abnormal curvature of the spine (scoliosis), and deformities of the hip and knee joints (knees). People with XLMTM may have a large head, a narrow, elongated face, and a high, arched mouth (palpectomy). People may also have liver disease, recurrent ear and respiratory infections, or seizures.

Due to their severe dyspnea, patients with XLMTM typically survive only into early childhood; however, some patients with the disorder have survived into adulthood. The compositions and methods disclosed herein provide an important medical benefit in that they are able to prolong the life of such patients by restoring functional MTM1 expression. In addition, the compositions and methods described herein may be used to improve a patient's quality of life following treatment (e.g., reducing stiffness and/or joint spasms, or increasing diaphragmatic and/or respiratory muscle development) because the disclosure provides a series of guidelines that may be used to determine a patient's eligibility for weaning from mechanical ventilation. Treatment Methods

In some embodiments, the patient is a newborn (e.g., 0-4 months), an infant (e.g., 0-5 months), a toddler (e.g., 6-12 months), a child 1-⁠3 years old, or a child 3-⁠5 years old at the time of administration of the viral vector.

In some embodiments, the patient is a newborn (e.g., 0-4 months) at the time of administration of the viral vector. For example, in some embodiments, the patient is a newborn of about 0 to about 4 months (e.g., 0 months to about 4 months, 1 month to about 4 months, 2 months to about 4 months, or 3 months to 4 months). In some embodiments, the patient is 0 months. In some embodiments, the patient is 1 month. In some embodiments, the patient is 2 months. In some embodiments, the patient is 3 months. In some embodiments, the patient is 4 months.

In some embodiments, the patient is a newborn (e.g., less than about 4 months old) at the time of administration of the viral vector. For example, in some embodiments, the patient is a newborn less than about 4 months old. In some embodiments, the patient is less than about 4 months old. In some embodiments, the patient is less than about 3 months old. In some embodiments, the patient is less than about 2 months old. In some embodiments, the patient is less than about 1 month old.

In some embodiments, the patient is an infant (e.g., 0-5 months) when the viral vector is administered. For example, in some embodiments, the patient is an infant of about 0 months to about 5 months (e.g., 0 months to about 5 months, 1 month to about 5 months, 2 months to about 5 months, 3 months to about 5 months, or 4 months to about 5 months). In some embodiments, the patient is 0 months. In some embodiments, the patient is 1 month. In some embodiments, the patient is 2 months. In some embodiments, the patient is 3 months. In some embodiments, the patient is 4 months. In some embodiments, the patient is 3 months. In some embodiments, the patient is 5 months.

In some embodiments, the patient is an infant (e.g., less than about 5 months) at the time of administration of the viral vector. For example, in some embodiments, the patient is an infant less than about 5 months. In some embodiments, the patient is less than about 5 months. In some embodiments, the patient is less than about 4 months. In some embodiments, the patient is less than about 3 months. In some embodiments, the patient is less than about 2 months. In some embodiments, the patient is less than about 1 month.

In some embodiments, the patient is a toddler (e.g., 6-12 months) when the viral vector is administered. For example, in some embodiments, the patient is an infant of about 6 months to about 12 months (e.g., 6 months to about 12 months, 7 months to about 12 months, 8 months to about 12 months, 9 months to about 12 months, 10 months to about 12 months, or 11 months to about 12 months). In some embodiments, the patient is 6 months. In some embodiments, the patient is 7 months. In some embodiments, the patient is 8 months. In some embodiments, the patient is 9 months. In some embodiments, the patient is 10 months. In some embodiments, the patient is 11 months. In some embodiments, the patient is 12 months.

In some embodiments, the patient is a toddler (e.g., less than about 12 years old) when the viral vector is administered. For example, in some embodiments, the patient is a toddler less than about 12 months. In some embodiments, the patient is less than about 12 months. In some embodiments, the patient is less than about 11 months. In some embodiments, the patient is less than about 10 months. In some embodiments, the patient is less than about 9 months. In some embodiments, the patient is less than about 8 months. In some embodiments, the patient is less than about 7 months. In some embodiments, the patient is less than about 6 months. In some embodiments, the patient is less than about 5 months. In some embodiments, the patient is less than about 4 months. In some embodiments, the patient is less than about 3 months. In some embodiments, the patient is less than about 2 months. In some embodiments, the patient is younger than about 1 month.

In some embodiments, the patient is a child of 1-3 years of age at the time of administration of the viral vector. For example, in some embodiments, the patient is a child of about 1 to about 3 years of age (e.g., 1 to about 3 years of age, or 2 to about 3 years of age). In some embodiments, the patient is 1 year old. In some embodiments, the patient is 2 years old. In some embodiments, the patient is 3 years old.

In some embodiments, the patient is a child (e.g., less than about 3 years old) when the viral vector is administered. For example, in some embodiments, the patient is a child less than about 3 years old. In some embodiments, the patient is less than about 3 years old. In some embodiments, the patient is less than about 2 years old. In some embodiments, the patient is less than about 1 year old. In some embodiments, the patient is less than about 12 months. In some embodiments, the patient is less than about 11 months. In some embodiments, the patient is less than about 10 months. In some embodiments, the patient is less than about 9 months. In some embodiments, the patient is less than about 8 months. In some embodiments, the patient is less than about 7 months. In some embodiments, the patient is less than about 6 months. In some embodiments, the patient is less than about 5 months. In some embodiments, the patient is less than about 4 months. In some embodiments, the patient is less than about 3 months. In some embodiments, the patient is less than about 2 months. In some embodiments, the patient is less than about 1 month.

In some embodiments, the patient is a child of 3-5 years of age when the viral vector is administered. For example, in some embodiments, the patient is a child of about 3 to about 5 years of age (e.g., 3 to about 5 years of age, or 4 to about 5 years of age). In some embodiments, the patient is 3 years of age. In some embodiments, the patient is 4 years of age. In some embodiments, the patient is 5 years of age.

In some embodiments, the patient is a child (e.g., less than about 5 years old) when the viral vector is administered. For example, in some embodiments, the patient is a child less than about 5 years old. In some embodiments, the patient is less than about 5 years old. In some embodiments, the patient is less than about 4 years old. In some embodiments, the patient is less than about 3 years old. In some embodiments, the patient is less than about 2 years old. In some embodiments, the patient is less than about 1 year old. In some embodiments, the patient is less than about 12 months. In some embodiments, the patient is less than about 11 months. In some embodiments, the patient is less than about 10 months. In some embodiments, the patient is less than about 9 months. In some embodiments, the patient is less than about 8 months. In some embodiments, the patient is less than about 7 months. In some embodiments, the patient is less than about 6 months. In some embodiments, the patient is less than about 5 months. In some embodiments, the patient is less than about 4 months. In some embodiments, the patient is less than about 3 months. In some embodiments, the patient is less than about 2 months. In some embodiments, the patient is less than about 1 month.

In some embodiments, the patient is between about 1 month and about 5 years of age (e.g., between about 1 month and about 5 years of age, between about 2 months and about 5 years of age, between about 3 months and about 5 years of age, between about 4 months and about 5 years of age, between about 5 months and about 5 years of age, between about 6 months and about 5 years of age, between about 1 year of age and about 5 years of age, between about 2 years of age and about 5 years of age, between about 3 years of age and about 5 years of age, or between about 4 years of age and about 5 years of age) at the time of administration of the viral vector.

In some embodiments, the patient is greater than or equal to 35 weeks gestational age (e.g., 35 weeks gestational age, 36 weeks gestational age, 37 weeks gestational age, 38 weeks gestational age, 39 weeks gestational age, 40 weeks gestational age, 41 weeks gestational age, and 42 weeks gestational age) at birth, and is between an adjusted full-term age (e.g., 37 weeks gestational age or older) and about 5 years old when the viral vector is administered. For example, if the patient is born at 35 weeks gestational age, the patient's full-term age is 14 days.

In some embodiments, the patient is at 35 weeks gestational age at birth and is between an adjusted full-term age to about 5 years old (e.g., 14 days to about 5 years old, 15 days to about 5 years old, 16 days to about 5 years old, 17 days to about 5 years old, 18 days to about 5 years old, 19 days to about 5 years old, 20 days to about 5 years old, 25 days to about 5 years old, one month to about 5 years old, two months to about 5 years old, three months to about 5 years old, four months to about 5 years old, five months to about 5 years old, six months to about 5 years old, one year to about 5 years old, two years to about 5 years old, three years to about 5 years old, and four years to about 5 years old) at the time of administration of the viral vector.

In some embodiments, the patient is born at 36 weeks gestational age and is between an adjusted term age and about 5 years old (e.g., 7 days to about 5 years old, 8 days to about 5 years old, 9 days to about 5 years old, 10 days to about 5 years old, 11 days to about 5 years old, 12 days to about 5 years old, 13 days to about 5 years old, 14 days to about 5 years old, 15 days to about 5 years old, 16 days to about 5 years old) at the time of administration of the viral vector. 5 years, 1 year to about 5 years, 2 years to about 5 years, 3 years to about 5 years, 4 years to about 5 years, 5 months to about 5 years, 6 months to about 5 years, 1 year to about 5 years, 2 years to about 5 years, 3 years to about 5 years, and 4 years to about 5 years).

In some embodiments, the patient is born at 37 weeks gestational age and is between an adjusted term age and about 5 years old (e.g., 1 day to about 5 years old, 2 days to about 5 years old, 3 days to about 5 years old, 4 days to about 5 years old, 5 days to about 5 years old, 6 days to about 5 years old, 7 days to about 5 years old, 8 days to about 5 years old, 9 days to about 5 years old, 10 days to about 5 years old, 11 days to about 5 years old, 12 days to about 5 years old, 13 days to about 5 years old) at the time of administration of the viral vector. , 14 days to about 5 years, 15 days to about 5 years, 16 days to about 5 years, 17 days to about 5 years, 18 days to about 5 years, 19 days to about 5 years, 20 days to about 5 years, 25 days to about 5 years, one month to about 5 years, two months to about 5 years, three months to about 5 years, four months to about 5 years, five months to about 5 years, six months to about 5 years, one year to about 5 years, two years to about 5 years, three years to about 5 years, and four years to about 5 years).

In some embodiments, the patient is male.

In some embodiments, the patient is female. Cholestasis and hyperbilirubinemia

Cholestasis is any condition in which the outflow of bile acid from the liver is slowed or blocked, while hyperbilirubinemia refers to a condition in which bilirubin accumulates in the blood and serum bile acid appears to remain normal. In contrast, cholestatic syndromes are characterized by marked cholestatic hyperactivity with normal to slightly elevated bilirubin levels.

In some embodiments, the patient is monitored for the development of cholestasis. In some embodiments, the patient is monitored for the development of hyperbilirubinemia. In some embodiments, the patient is monitored for the development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In some embodiments, the patient is monitored for the development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof by evaluating a parameter in a blood sample obtained from the patient, wherein the parameter is found to be above a reference level to identify the patient as suffering from cholestasis, hyperbilirubinemia, or one or more symptoms thereof.

In some embodiments, a patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient exhibits one or more parameters (e.g., total bile acid levels, gamma-glutamyl transferase (GGT) levels, alkaline phosphatase (ASP) levels, aspartate aminotransferase (AST) levels, and/or alanine aminotransferase (ALT) levels) greater than or less than age-adjusted norms as measured in a serum bile acid test and/or a blood test (e.g., liver function tests (LFTs)).

In some embodiments, a patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof when the patient exhibits bilirubin levels greater than norm, as measured in a blood test (e.g., a bilirubin test).

In some embodiments, the patient has no history of cholestasis or hyperbilirubinemia. In some embodiments, the patient has no history of any underlying liver disease. Vectors for delivering exogenous nucleic acids to target cells Viral vectors for nucleic acid delivery

Recombinant viral genomes provide a rich source of vectors that can be used to efficiently deliver genes of interest (e.g., transgenes encoding MTM1) to the genome of target cells (e.g., mammalian cells, such as human cells). Recombinant viral genomes are particularly useful vectors for gene delivery because they deliver genes of interest to the nucleus of target cells. To select viruses, polynucleotides contained within such genomes can be incorporated into the genome of target cells by general or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents. Examples of recombinant viral vectors for delivering genes of interest include AAV, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-stranded RNA viruses (e.g., orthomyxoviruses, such as influenza virus), rhabdoviruses (e.g., rabies and stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-stranded RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses (including adenoviruses, herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), chickenpox, and canarypox). Other viruses that can be used to deliver polynucleotides encoding the antibody light and heavy chains or antibody fragments of the present invention include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus. Examples of retroviruses include: avian leukosis sarcoma, mammalian C virus, B virus, D virus, HTLV-BLV group, lentivirus, foamy virus

(Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, 3rd edition, BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described in, for example, U.S. Patent No. 5,801,030, which is incorporated herein by reference for its disclosure of viral vectors for use in gene therapy. AAV vectors for nucleic acid delivery

In some embodiments, nucleic acids of the compositions and methods described herein are incorporated into recombinant AAV (rAAV) vectors and/or virions to facilitate their introduction into cells. rAAV vectors that can be used in the present invention are recombinant nucleic acid constructs that include (1) a transgene to be expressed (e.g., a polynucleotide encoding an MTM1 protein) and (2) a viral nucleic acid that promotes stability and expression of the heterologous gene. The viral sequences can include those AAV sequences required for sequential replication and packaging of the DNA (e.g., functional ITRs) into the virion. In a typical application, the transgene encodes MTM1, which can be used to correct MTM1 mutations in patients with neuromuscular disorders (e.g., XLMTM). The rAAV vectors can also contain markers or reporter genes. Useful rAAV vectors have one or more AAV wild-type genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITRs can be of any serotype suitable for a particular application (e.g., derived from serotype 2). Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000) and Monahan and Samulski, Gene Delivery 7:24-30 (2000) , each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate introduction of the nucleic acid or vector into cells. The capsid proteins of AAV constitute the outer, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for example, in US 5,173,414; US 5,139,941; US 5,863,541; US 5,869,305; US 6,057,152; and US 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

rAAV virions useful in conjunction with the compositions and methods described herein include virions derived from a variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, and 9. For targeting muscle cells, rAAV virions that include at least one serotype 1 capsid protein may be particularly useful. rAAV virions that include at least one serotype 6 capsid protein may also be particularly useful, as serotype 6 capsid proteins are structurally similar to serotype 1 capsid proteins and are therefore expected to also result in high expression of MTM1 in muscle cells. rAAV serotype 9 has also been found to be an effective sensor for muscle cells. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther . 2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol . 72:2224-2232 (1998); Halbert et al., J. Virol . 74:1524-1532 (2000); Halbert et al., J. Virol . 75:6615-6624 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081. (2001) , each of which is incorporated herein by reference for its disclosure of AAV vectors for gene delivery.

Pseudotyped rAAV vectors can also be used in conjunction with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with capsid genes derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). For example, a representative pseudotyped vector is an AAV8 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 2. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert et al., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001) .

AAV virions with mutations in the virion capsid can infect specific cell types more efficiently than non-mutated capsid virions. For example, a suitable AAV mutant can have a ligand insertion mutation that helps AAV target a specific cell type. The construction and characterization of AAV capsid mutants (including insertion mutants, alanine selection mutants, and antigenic determinant tag mutants) are described in Wu et al., J. Virol. 74:8635-45 (2000). Other rAAV virions that can be used in the methods of the present invention include those capsid hybrids generated by molecular breeding of the virus and by exon shuffling. See, for example, Soong et al., Nat. Genet ., 25:436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol . 19:423-428 (2001).

As described herein, the pseudotyped AAV vector includes a nucleic acid sequence encoding the MTM1 gene, which is operably linked to the desmin promoter flanked by AAV2 ITRs and packaged within capsid proteins from AAV8 (AAV2/8), and the other genetic components listed in Table 1 , which refer to compounds known by the International Proprietary Name (INN) of pyrrolidine.

Birelase is a non-replicating recombinant AAV8 vector that expresses a non-codon-optimized human MTM1 cDNA under the control of the muscle-specific human desmin promoter. The MTM1 expression cassette was constructed by cloning a synthetic DNA sequence complementary to the coding portion (nucleotides 43-1864) of the wild-type human MTM1 transcript (NCBI Ref. Seq NM_000252.3) downstream of the 1.05 kb human desmin enhancer/promoter region. The second intron and polyadenylation sequence of the human β-globin gene ( HBB ) were inserted upstream and downstream of the MTM1 synthetic cDNA, respectively, to mediate RNA processing. The expression cassette is flanked by AAV serotype 2 (AAV2) inverted terminal repeats (ITRs). The vector was produced in AAV8 capsids by binoplasmic transfection in HEK293 cells grown in bioreactor suspension culture in a complete GMP process.

In some embodiments, a method of treating a disorder (e.g., XLMTM) in a human patient in need thereof or alleviating one or more symptoms (e.g., stiffness and/or joint contractions or the need for diaphragmatic and/or respiratory muscle development) of a disorder (e.g., XLMTM) in a human patient in need thereof comprises administering to the patient a therapeutically effective amount of bireludinyl during a treatment period.

In some embodiments, a method of weaning a human patient from mechanical ventilation comprises administering to the patient a therapeutically effective amount of Birrellen. The composition of Birrellen is shown in Table 1 below: Table 1. Birrellen nucleic acid sequence (SEQ ID NO: 1) Range (nucleotides, relative to SEQ ID NO: 1) Length (nucleotides) Genetic components 3080-3198 119 AAV2 ITR 3199-3256 58 Connector sequence 3257-4316 1,060 Human desmin promoter 4317-4354 38 Connector sequence 4355-4460 106 Human β-globin intron 4373-4848 476 Human β-globin intron 4458-4902 445 Human β-globin intron 4927-6748 1,822 Human MTM1 coding sequence 6749-6759 11 Connector sequence 6760-7519 760 Human β-globin polyadenylation sequence 7520-7551 32 Connector sequence 7552 7,696 AAV2 ITR

As described herein, birelin refers to an AAV vector having a nucleic acid sequence of SEQ ID NO: 1, as shown below: Transcription control element

Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein can contain various parts that are operably linked to each other. For example, the transcriptional regulatory elements described herein can contain ApoE and/or A1AT promoters, such as chimeric promoters shown in SEQ ID NO: 2, or functional portions thereof. Additional nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9% or more sequence identity) relative to the above nucleic acid sequences.

Additionally or alternatively, the transcriptional regulatory elements described herein may contain the LP1 promoter or a functional portion thereof. For example, the regulatory element may contain the LP1 promoter or a functional portion thereof as set forth in SEQ ID NO: 3. Additional nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9% or more sequence identity) relative to the above nucleic acid sequences.

Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include promoters that stimulate the expression of a transgenic gene operably linked to a liver-specific promoter. Examples of such promoters are ApoE/A1At and LP1 promoters or variants thereof (e.g., variants having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity) to the nucleic acid sequence of the wild-type promoter locus and capable of stimulating transcription of a transgenic gene operably linked thereto after introduction into a cell) or a functional portion thereof.

Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include promoters that stimulate the expression of a transgenic gene operably linked to a muscle-specific promoter. Examples of such promoters are the desmin or MCK promoters or variants thereof (e.g., variants that have at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity) to the nucleic acid sequence of the wild-type promoter locus and are capable of stimulating transcription of a transgenic gene operably linked thereto upon introduction into a cell) or a functional portion thereof.

Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include promoters that stimulate the expression of a transgenic gene operably linked to a ubiquitous promoter. Examples of such promoters are PGK, Ef1a, and GAPDH, or variants thereof (e.g., variants having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity) to the nucleic acid sequence of the wild-type promoter locus and capable of stimulating transcription of a transgenic gene operably linked thereto upon introduction into a cell) or a functional portion thereof.

The transcriptional regulatory elements described herein may contain the SV40 enhancer or a functional portion thereof. For example, the regulatory element may contain the SV40 promoter or a functional portion thereof as shown in SEQ ID NO: 4. Additional nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9% or more sequence identity) to the above nucleic acid sequences.

The transcriptional regulatory elements described herein may contain a β-globin enhancer or a functional portion thereof. For example, the regulatory element may contain a β-globin enhancer or a functional portion thereof as shown in SEQ ID NO: 5. Additional nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9% or more sequence identity) relative to the above nucleic acid sequences.

Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include enhancers that enhance the expression of the transgenic gene. Examples of such promoters are PGK, Ef1a, and GAPDH, or variants thereof (e.g., variants having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity) with the nucleic acid sequence of the wild-type promoter locus, and capable of stimulating transcription of the transgenic gene to which it is operably linked after introduction into a cell) or a functional portion thereof.

The aforementioned nucleic acid regulatory elements are summarized in Table 2 below. Table 2. Exemplary nucleic acid regulatory elements SEQ ID NO. Description of transcription control elements Nucleic acid sequence 2 ApoE/A1AT promoter (775 nt) CGTGATCTAGTAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACT CGACCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAG CAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCC GTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTCAGGCACCACCACTGACCTGGGACAGTGAATGATCCCCCTGATCTGCGGCCTCGACGGTATCGATAAG 3 LP1 promoter (449 nt) GTCCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTA GGCGGGCGACTCAGATCCCAGCCAGTGcACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAA 4 SV40 enhancer (66 nt) AGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTT 5 β-globin enhancer (476 nt) AGGGTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTTTGCACCATTCTAAAGAATAACAGTGATA ATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCA

Additional nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity ( e.g. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity) to the nucleic acid sequences shown in Table 2 .

The pseudotyped AAV vectors described herein may include a nucleic acid sequence encoding an MTM1 gene, which is operably linked to a promoter flanked by AAV2 ITRs and packaged in a capsid protein from AAV8 (AAV2/8). In some embodiments, the nucleic acid sequence encoding the MTM1 gene encodes a human MTM1 gene. In some embodiments, the nucleic acid sequence encoding the MTM1 gene encodes a mouse MTM1 gene. In some embodiments, the MTM1 gene is codon optimized. In some embodiments, the promoter is a liver-specific promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the promoter is a ubiquitously expressed promoter. In some embodiments, two MTM1 genes can be expressed simultaneously using different promoters on the same AAV vector.

The transgene that can be used in conjunction with the compositions and methods described herein can contain the human MTM1 transgene, as shown in SEQ ID NO: 6, or a functional portion thereof. Additional transgenes that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9% or more sequence identity) relative to the above nucleic acid sequence.

The transgene that can be used in conjunction with the compositions and methods described herein can contain the mouse MTM1 transgene, as shown in SEQ ID NO: 7, or a functional portion thereof. Additional transgenes that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9% or more sequence identity) relative to the above nucleic acid sequence.

In some embodiments, a method of treating a disorder (e.g., XLMTM) in a human patient in need thereof or alleviating one or more symptoms (e.g., stiffness and/or joint contraction or the need for diaphragmatic and/or respiratory muscle development) of a disorder (e.g., XLMTM) in a human patient in need thereof comprises administering to the patient during a treatment period a therapeutically effective amount of a pseudotyped AAV vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific or muscle-specific promoter flanked by AAV2 ITRs and packaged within a capsid protein from AAV8 (AAV2/8), and other genetic components listed in Table 2 .

In some embodiments, a method of weaning a human patient from mechanical ventilation comprises previously administering to the patient a therapeutically effective amount of a pseudotyped AAV vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific or muscle-specific promoter flanked by AAV2 ITRs and packaged within capsid proteins from AAV8 (AAV2/8), and other genetic components listed in Table 2 .

The aforementioned transgenic genes are summarized in Table 3 below. Table 3. Exemplary transgenic genes SEQ ID NO. Description of transgenic genes Nucleic acid sequence 6 Human MTM1 coding sequence (1812 nt) ATGGCTTCTGCATCAACTTCTAAATATAATTCACACTCCTTGGAGAATGAGTCTATTAAGAGGACGTCTCGAGATGGAGTCAATCGAGATCTCACTGAGGCTGTTCCTCGACTTCCAGGAGAAACACTAATCACTGACAAAGAAGTTATTTACATATGTCCTTTCAATGGCCCCATTAAGGGAAGAGTTTACATCACAAATTATCGTCTTTTTAAGAAGTTTGGAAACGGATTCTTCTCTAATACTTGATGTTCCTCTGG GTGTGATCTCGAGAATTGAAAAAATGGGAGGCGCGACAAGTAGAGGAGAAAATTCCTATGGTCTAGATATTACTTGTAAAGACATGAGAAACCTGAGGTTCGCTTTGAAACAGGAAGGCCACAGCAGAAGAGATATGTTTGAGATCCTCACGAGATACGCGTTTCCCCTGGCTCACAGTCTGCCATTATTT GCATTTTTAAATGAAGAAAAGTTTAACGTGGATGGATGGACAGTTTACAATCCAGTGGAAGAATACAGGAGGCAGGGCTTGCCCAATCACCATTGGAGAATAACTTTTATTAATAAGTGCTATGAGCTCTGTGACACTTACCCTGCTCTTTTGGTGGTTCCGTATCGTGCCTCAGATGATGACCTCCGGAGAGTTGCAACTTTTAGGTCCCGAAATCGAATTCCAGTGCTGTCATGGATTCATCCAGAAAATAAGACGGTC ATTGTGCGTTGCAGTCAGCCTCTTGTCGGTATGAGTGGGAAACGAAATAAAGATGATGAGAAATATCTCGATGTTATCAGGGAGACTAATAAACAAATTTCTAAACTCACCATTTATGATGCAAGACCCAGCGTAAATGCAGTGGCCAACAAGGCAACAGGAGGAGGATATGAAAGTGATGATGCATATCAT AACGCCGAACTTTTCTTCTTAGACATTCATAATATTCATGTTATGCGGGAATCTTTAAAAAAAGTGAAGGACATTGTTTATCCTAATGTAGAAGAATCTCATTGGTTGTCCAGTTTGGAGTCTACTCATTGGTTAGAACATATCAAGCTCGTTTTGACAGGAGCCATTCAAGTAGCAGACAAAGTTTCTTCAGGGAAGAGTTCAGTGCTTGTGCATTGCAGTGACGGATGGGACAGGACTGCTCAGCTGACATCCTTGGCCAT GCTGATGTTGGATAGCTTCTATAGGAGCATTGAAGGGTTCGAAATACTGGTACAAAAAGAATGGATAAGTTTTGGACATAAATTTGCATCTCGAATAGGTCATGGTGATAAAAACCACACCGATGCTGACCGTTCTCCTATTTTTCTCCAGTTTATTGATTGTGTGTGGCAAATGTCAAAACAGTTCCCT ACAGCTTTTGAATTCAATGAACAATTTTTGATTATAATTTTGGATCATCTGTATAGTTGCCGATTTGGTACTTTCTTATTCAACTGTGAATCTGCTCGAGAAAGACAGAAGGTTACAGAAAGGACTGTTTCTTTATGGTCACTGATAAACAGTAATAAAGAAAAATTCAAAAACCCCTTCTATACTAAAGAAATCAATCGAGTTTTATATCCAGTTGCCAGTATGCGTCACTTGGAACTCTGGGTGAATTACTACATTAGATGGAA CCCCAGGATCAAGCAACAACAGCCGAATCCAGTGGAGCAGCGTTACATGGAGCTCTTAGCCTTACGCGACGAATACATAAAGCGGCTTGAGGAACTGCAGCTCGCCAACTCTGCCAAGCTTTCTGATCCCCCAACTTCACCTTCCAGTCCTTCGCAAATGATGCCCCATGTGCAAACTCACTTCTGA 7 Mouse MTM1 coding sequence (1812 nt) ATGGCTTCTGCATCAGCATCTAAGTATAATTCACACTCCTTGGAGAATGAATCCATTAAGAAAGTGTCTCAAGATGGAGTCAGTCAGGATGTGAGTGAGACTGTCCCTCGGCTCCCAGGGGAGTTACTAATTACTGAAAAAGAAGTTATTTACATATGTCCTTTCAATGGCCCCATTAAGGGAAGAGTTTACATCACAAATTATCGTCTTTTTAAGAAGTTTGGAAACGGATTCTGCTCTAATACTTGATGTTCTCTGGG TGTGATATCAAGAATTGAAAAAATGGGAGGCGCGACAAGTAGAGGAGAAAATTCCTATGGTCTAGATATTACTTGTAAAGATTTGAGAAACCTGAGGTTTGCATTGAAGCAAGAAGGCCACAGCAGAAGAGATATGTTTGAGATCCTTGTAAAACATGCCTTTCCTCTGGCACACAATCTGCCATTATTT GCATTTGTAAATGAAGAGAAGTTTAACGTGGATGGGTGGACTGTTTATAATCCAGTTGAAGAATATAGAAGGCAGGGCCTGCCCAATCACCATTGGAGGATAAGTTTTATTAACAAGTGCTATGAGCTCTGTGAGACATACCCTGCTCTTTTGGTGGTTCCCTATCGGACCTCAGATGATGATCTTAGGAGGATCGCAACGTTTAGATCCCGAAATCGGCTTCCTGTACTGTCGTGGATTCACCCAGAAAACAAAATGGT CATTATGCGCTGCAGTCAGCCTCTTGTCGGTATGAGTGGTAAAAGAAATAAAGATGACGAGAAATACCTGGATGTGATCAGGGAAACTAACAAACAAACTTCTAAGCTCATGATTTATGATGCACGACCCAGTGTAAATGCAGTCGCCAACAAGGCAACAGGAGGAGGATATGAAAGTGATGACGCATATCAA AACTCAGAACTTTCCTTCTTAGACATTCATAATATTCATGTTATGCGAGAATCTTTAAAAAAAGTGAAAGATATTGTTTATCCCAACATAGAAGAATCTCATTGGTTGTCCAGTTTGGAGTCTACTCATTGGTTAGAACATATCAAGCTTGTTCTGACCGGTGCCATTCAAGTGGCAGACCAAGTGTCTTCAGGAAAGAGCTCGGTACTTGTGCACTGCAGTGACGGATGGGACAGGACCGCTCAGCTGACATCCTTGGC CATGCTGATGTTGGACAGCTTCTACAGAACTATTGAAGGCTTTGAGATATTGGTACAGAAAGAGTGGATAAGTTTTGGCCATAAATTTGCATCTAGAATAGGTCATGGTGATAAAAACCATGCTGATGCTGATCGATCTCCTATTTTTCTTCAGTTTATTGACTGTGTGTGGCAGATGTCGAAACAGTTCCCC ACAGCTTTGAGTTCAATGAAGGCTTTTTGATTACCGTTTTGGATCATCTGTATAGCTGTCGATTTGGTACTTTCTTATTCAACTGTGACTCGGCTCGAGAAAGACAGAAACTTACAGAAAGAACAGTTTCTCTATGGTCGCTAATTAACAGCAATAAAGACAAATTCAAAAACCCCTTCTATACAAAAGAAATCAATCGGGTTTTGTATCCAGTTGCCAGCATGCGTCACTTGGAACTGTGGGTGAATTACATCCGATG GAATCCCAGGGGTCAAGCAGCAACAGCCCAACCCAGTGGAGCAGCGTTACATGGAGCTTTTGGCCTTGCGTGACGATTATATAAAGAGGCTCGAGGAATTGCAGCTGGCCAACTCCGCCAAGCTTGCTGATGCCCCCGCTTCGACTTCCAGTTCGTCACAGATGGTGCCCCATGTGCAGACGCACTTCTGA

Additional transgenes that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules having at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity) to the nucleic acid sequences listed in Table 3. Methods for delivering exogenous nucleic acids to target cells Transfection techniques

Techniques that can be used to introduce a transgene (e.g., the MTM1 transgene described herein) into a target cell (e.g., a mammalian cell) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying a static electric potential to the cells of interest. Mammalian cells (e.g., human cells) subjected to an external electric field in this manner are then susceptible to taking up exogenous nucleic acids (e.g., nucleic acids that can be expressed in, for example, neurons, neuroglia, or non-neural cells such as colon and kidney cells). Electroporation of mammalian cells is described in detail in, for example, Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, NUCLEOFECTION™, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. NUCLEOFECTION™ and protocols that can be used to implement this technique are described in detail in, for example, Distler et al., Experimental Dermatology 14:315 (2005) and US 2010/0317114, the disclosures of each of which are incorporated herein by reference.

An additional technique that can be used to transfect target cells is the extrusion-poration method. This technique induces rapid mechanical deformation of cells to stimulate the uptake of foreign DNA through membrane pores formed in response to applied stress. The advantage of this technique is that a vector is not required to deliver nucleic acids into cells, such as human target cells. Extrusion-poration is described in detail in, for example, Sharei et al. , J. Vis. Exp. 81:e50980 (2013) , the disclosure of which is incorporated herein by reference.

Lipofection represents another technique that can be used to transfect target cells. This method involves loading nucleic acids into liposomes, which typically present cationic functional groups, such as quaternary or protonated amines, facing the exterior of the liposomes. This promotes electrostatic interactions between the liposomes and the cells due to the anionic nature of the cell membrane, ultimately taking up exogenous nucleic acids, for example by inducing fusion of the liposomes with the cell membrane or by endocytosis of the complex. Lipofection is described in detail in, for example, US 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to cause uptake of foreign nucleic acids is to contact the cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that conjugate to polynucleotides to impart a positive charge that facilitates interaction with cell membranes are activated dendrimers (described, e.g., in Dennig, Top Curr Chem. 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and DEAE-polydextrose, the use of which as transfection agents is described in detail, e.g., in Gulick et al., Curr Protoc Mol Biol . 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference.

Another tool that can be used to induce the uptake of exogenous nucleic acids by target cells is laser transfection, also known as phototransfection, which involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to pass through the cell membrane. The biological activity of this technique is similar to that of electroporation, and in some cases has been found to be superior to electroporation.

Piercing transfection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene to be delivered into the cell is attached to the surface of the nanostructure. A chip with an array of these needles is then pressed against a cell or tissue. Cells pierced by the nanostructure can express the delivered gene. An example of this technique is described in Shalek et al., PNAS 107:25 1870 (2010), the disclosure of which is incorporated herein by reference.

Nucleic acids can also be delivered to target cells using MAGNETOFECTION™. The principle of MAGNETOFECTION™ is to combine nucleic acids with cationic magnetic nanoparticles. Magnetic nanoparticles are made of completely biodegradable iron oxide and coated with specific cationic molecules that vary according to the application. Their combination with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells by affecting the external magnetic field generated by the magnet. This technology is described in detail in Scherer et al., Gene Ther. 9:102 (2002), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, since this method utilizes an applied magnetic field to guide the uptake of nucleic acids. This technology is described in detail in, for example, US2010/0227406, the disclosure of which is incorporated herein by reference.

Another tool that can be used to induce uptake of exogenous nucleic acids by target cells is sonoporation, which is a technique that involves the use of sound (usually ultrasound frequencies) to modify the permeability of the plasma membrane of the cell to permeate the cell and allow polynucleotides to pass through the cell membrane. This technique is described in detail in, for example, Rhodes et al., Methods Cell Biol. 82:309 (2007), the disclosure of which is incorporated herein by reference.

Microvesicles represent another potential medium that can be used to modify the genome of a target cell according to the methods described herein. For example, microvesicles that have been induced by co-expression of the glycoprotein VSV-G and, for example, genome modifying proteins (such as nucleases) can be used to efficiently deliver proteins into cells, which then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of the cell for covalent incorporation of a target polynucleotide (such as a gene or regulatory sequence). The use of such vesicles (also called gesicles) for genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract], in Methylation changes in early embryonic genes in cancer [Abstract], and in Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, Abstract No. 122.

In addition to the above, a variety of tools have been developed that can be used to incorporate genes of interest into target cells, such as human cells. One method that can be used to incorporate a polynucleotide encoding a target gene into a target cell involves the use of a transposon. A transposon is a polynucleotide that encodes a transposase and contains a polynucleotide sequence or a gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered to the cell, expression of the transposase gene begins and produces active enzymes that cut the gene of interest from the transposon. The activity is mediated by site-specific recognition of the transposon excision site by the transposase. In some cases, the excision sites may be terminal repeat sequences or inverted terminal repeat sequences. Once excised from the transposon, the gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the genome of the cell nucleus. This allows the gene of interest to be inserted into the cleaved nuclear DNA at the complementary excision site, followed by covalent attachment of the phosphodiester bonds that connect the gene of interest to the DNA of the genome of the mammalian cell to complete the incorporation process. In some cases, the transposon can be a retrotransposon, so that the gene encoding the target gene is first transcribed into an RNA product and then reversely transcribed into DNA before being incorporated into the genome of the mammalian cell. Exemplary transposon systems are the piggybac transposon (described in detail, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail, e.g., US 2005/0112764), each of which is incorporated herein by reference for its disclosure of transposons for delivering genes into cells of interest.

Another tool for integrating target genes into the genome of target cells is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, which was originally developed as an adaptive defense mechanism for bacteria and archaea against viral infection. The CRISPR/Cas system consists of palindromic repeat sequences within plastid DNA and the associated Cas9 nuclease. This collection of DNA and proteins directs site-specific DNA cleavage of a target sequence by first incorporating foreign DNA into the CRISPR locus. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are then transcribed in the host cell to produce guide RNAs, which can then bind to the target sequence and localize the Cas9 nuclease to that site. In this way, highly site-specific cas9-mediated DNA cleavage can be generated in exogenous polynucleotides because the interaction that brings cas9 close to the target DNA molecule is controlled by RNA:DNA hybridization. Therefore, one can design a CRISPR/Cas system to cleave any target DNA molecule of interest. The technology has been used to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an efficient means of site-specifically editing target cell genomes to cleave DNA prior to incorporation of the gene encoding the target gene. The use of CRISPR/Cas to regulate gene expression has been described, for example, in U.S. Patent No. 8,697,359, which is incorporated herein by reference for its disclosure of the use of CRISPR/Cas systems for gene editing. Alternative methods for site-specific cleavage of genomic DNA prior to incorporating the gene of interest into target cells include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes do not contain a guide polynucleotide for localization to a specific target sequence. Instead, target specificity is controlled by a DNA binding domain within the enzymes. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al., Nat. Rev. Genet. 11:636 (2010); and Joung et al., Nat. Rev. Mol. Cell Biol . 14:49 (2013), each of which is incorporated herein by reference for its disclosure of compositions and methods for genome editing.

Other genome editing techniques that can be used to incorporate polynucleotides encoding target genes into the genome of target cells include the use of ARCUS™ meganucleases, which can be rationally designed to site-specifically cleave genomic DNA. In view of the well-established structure-activity relationships that have been established for these enzymes, it is advantageous to use these enzymes to incorporate genes encoding target genes into the genome of mammalian cells. Single-chain meganucleases can be modified at certain amino acid positions to produce nucleases that selectively cleave DNA at desired positions, thereby enabling site-specific incorporation of target genes into the nuclear DNA of target cells. Such single-chain nucleases have been extensively described in, for example, U.S. Patent Nos. 8,021,867 and 8,445,251, each of which is incorporated herein by reference for its disclosure of compositions and methods for genome editing .

Gene therapy agents described herein may contain a transgene, such as a transgene encoding MTM1, and may be incorporated into a vehicle for administration to a patient, such as a human patient suffering from a neuromuscular disorder (e.g., XLMTM). Pharmaceutical compositions containing a vector (e.g., a viral vector) containing a transcriptional regulatory element described herein (e.g., ApoE/A1AT promoter) operably linked to a therapeutic transgene may be prepared using methods known in the art. For example, such compositions may be prepared using, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980); incorporated herein by reference) and in a desired form (e.g., in the form of a lyophilized formulation or aqueous solution).

Viral vectors, such as AAV vectors and other vectors described herein, containing transcriptional regulatory elements operably linked to a therapeutic transgene can be administered to a patient (e.g., a human patient) by a variety of routes of administration. The route of administration can vary, for example, depending on the onset and severity of the disease, and can include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intrahepatic, intranasal, subcutaneous, transcutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, infusion, lavage, and oral administration. Intravascular administration includes delivery into the vascular system of the patient. In some embodiments, administration is into a blood vessel that is considered a vein (intravenous), and in some administrations, administration is into a blood vessel that is considered an artery (intraarterial). Veins include, but are not limited to, the internal cervical vein, peripheral vein, coronary vein, hepatic vein, portal vein, great sphenoid vein, pulmonary vein, superior vena cava, inferior vena cava, gastric vein, splenic vein, inferior mesenteric vein, superior mesenteric vein, capillary vein, and/or femoral vein. Arteries include, but are not limited to, the coronary arteries, pulmonary arteries, brachial arteries, internal cervical arteries, aortic arch, femoral arteries, peripheral arteries, and/or ciliary arteries. Delivery is expected to occur via or to arterioles or capillaries.

The mixture of nucleic acid and viral vector described herein can be prepared in a suitable mixture of water and one or more excipients, carriers or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oils. Under ordinary storage and use conditions, these preparations may contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference). In either case, the formulation can be sterile and can be a fluid that is easily injected. The formulation can be stable under manufacturing and storage conditions and can prevent the contamination of microorganisms (such as bacteria and fungi). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of a surfactant. The action of microorganisms may be prevented by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In many cases, it will be preferred to include an isotonic agent, for example, a sugar or sodium chloride. Prolonged absorption of the injectable composition may be achieved by the use in the composition of an agent that delays absorption, for example, aluminum monostearate and gelatin.

For example, if necessary, a solution containing the pharmaceutical composition described herein may be appropriately buffered and the liquid diluent may first be isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, intrahepatic, subcutaneous, and intraperitoneal administration. In view of this, according to this disclosure, those skilled in the art will know that sterile aqueous media may be used. For example, a dose may be dissolved in 1 mL of NaCl isotonic solution and added to 1000 mL of subcutaneous perfusion fluid or injected at the proposed infusion site. Depending on the disease of the subject being treated, some variation in dose will inevitably occur. The person responsible for administration will determine the appropriate dose for the individual subject in any case. In addition, for human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics Standards.

The compositions described herein may be provided in a kit for treating a neuromuscular disorder (e.g., XLMTM). In some embodiments, the kit may include one or more viral vectors as described herein. The kit may include a package insert that directs the user of the kit (e.g., a physician familiar with the art) to perform any of the methods described herein. The kit may optionally include a syringe or other device for administering the composition. In some embodiments, the kit may include one or more additional therapeutic agents.

The kit may include a package insert that instructs the kit user (e.g., a physician skilled in the art) to perform any of the methods described herein. The kit may optionally include a syringe or other device for administering the composition. In some embodiments, the kit may include one or more additional therapeutic agents. Dosage regimens involving dosing regimens of AAV-MTM1 vectors

Using the compositions and methods disclosed herein, an AVV vector containing a transgene encoding MTM1 can be administered to a patient suffering from a neuromuscular disorder (e.g., XLMTM) in an amount of about 1.3 x 10 ¹⁴ vg/kg. Administration of the vector to the patient in this amount can achieve the beneficial effect of increasing the expression of MTM1 in the patient, for example, to within 50% or 200% of the wild-type level, without causing toxic side effects.

In some embodiments, the AVV carrier is present in an amount of less than about 3 x 10 ¹⁴ vg/kg (e.g., less than about 3 x 10 ¹⁴ vg/kg, 2.9 x 10 ¹⁴ vg/kg, 2.8 x 10 ¹⁴ vg/kg, 2.7 x 10 ¹⁴ vg/kg, 2.6 x 10 ¹⁴ vg/kg, 2.5 x 10 ¹⁴ vg/kg, 2.4 x 10 ¹⁴ vg/kg, 2.3 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 1.8 x 10 14 vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg, or less) are administered to a patient. For example, AAV vectors can be about 3 x 10 ¹⁴ vg/kg, 2.9 x 10 ¹⁴ vg/kg, 2.8 x 10 ¹⁴ vg/kg, 2.7 x 10 ¹⁴ vg/kg, 2.6 x 10 ¹⁴ vg/kg, 2.4 x 10 ¹⁴ vg/kg, 2.3 x 10 ¹⁴ vg/ kg, 2.2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/ ^{kg, 1.9 x 10 14 vg/kg, 1.8 x 10 14 vg/kg, 1.7 x 10 14 vg / kg} , 1.6 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ^{The amounts of 11 vg/kg, 1 x 10 10 vg/kg, 1 x 10 9 vg / kg} , and 1 x 10 ⁸ vg/kg are administered to the patient.

In some embodiments, the AVV carrier is present in an amount of less than about 2.5 x 10 ¹⁴ vg/kg (e.g., less than about 2.5 x 10 ¹⁴ vg/kg, 2.4 x 10 ¹⁴ vg/kg, 2.3 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 14 vg/kg, 1.2 x 10 ¹⁴ vg/ ^kg , vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg, or less) are administered to a patient. For example, AAV vectors can be about 2.5 x 10 ¹⁴ vg/kg, 2.4 x 10 ¹⁴ vg/kg, 2.3 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/ kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg are administered to the patient.

In some embodiments, the AVV carrier is present in an amount of less than about 2 x 10 ¹⁴ vg/kg (e.g., less than about 2 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 11 vg/ ^kg , vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg, or less) are administered to a patient. For example, AAV vectors can be about 2 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/ kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x ^{10 12 vg} /kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg are administered to patients.

In some embodiments, the AVV vector is administered to a patient in an amount of less than about 1.5 x 10 ¹⁴ vg/kg (e.g., less than about 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg, or less). For example, the AAV vector can be administered to a patient in an amount of about 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg.

In some embodiments, the AVV vector is administered to a patient in an amount less than about 1 x 10 ¹⁴ vg/kg (e.g., less than about 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg, or less). For example, the AAV vector can be administered to a patient in an amount of about 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg.

In some embodiments, the AAV vector is administered to a patient in an amount of about 3 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg (e.g., in an amount of about 3 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg). For example, the AVV vector can be about 3 x 10 ¹³ vg/kg, 3.1 x 10 ¹³ vg/kg, 3.2 x 10 ¹³ vg/ ^kg , 3.3 x 10 ¹³ vg/kg, 3.4 x 10 ¹³ vg/ ^kg , 3.6 x 10 ¹³ vg/kg, 3.7 x 10 ¹³ vg/ kg, 3.8x1013vg/ ^{kg ,} 3.9x1013vg/kg, 4x1013vg/kg, ^4.1x1013vg /kg, 4.2x1013vg/kg, ^4.3x1013vg / ^kg , ^4.4x1013vg /kg, ^4.5x1013 vg/kg, 4.7 x 10 ¹³ vg/kg, 4.8 x 10 ¹³ vg/kg, 4.9 x 10 ¹³ vg/kg, 5 x 10 ¹³ vg/kg, 5.1 x 10 ¹³ vg/kg, 5.2 x 10 ¹³ vg/kg, 5.3 x 10 ¹³ vg/ ^{kg, 5.4 x 10 13 vg/kg, 5.5 x 10 13 vg/kg, 5.6 x 10 13 vg / kg} , 5.7 x 10 ¹³ vg/kg, 5.8 x 10 ¹³ vg/kg, 5.9 x 10 ¹³ vg/kg, 6 x 10 ¹³ vg/kg, 6.1 x 10 ¹³ vg/kg, 6.2 ^{10 13 vg} / ^kg , ^6.3 x ^{10 13 vg} / ^{kg , 6.5 3 vg / kg , 7.1} vg/kg、7.9 x 10 ¹³ vg/kg、8.1 x 10 ¹³ vg/kg、8.2 x 10 ¹³ vg/ ^{kg、8.3 x 10 13 vg/kg、8.5 x 10 13 vg /} kg 8.7 x 10 ¹³ vg/kg, 8.8 x 10 ¹³ vg/kg, 8.9 x 10 ¹³ vg/kg, 9 x 10 ¹³ vg/kg, ^9.1 x ^{10 13 vg} /kg, 9.3 x 10 ¹³ vg/kg, 9.4 x 10 ¹³ vg/ ^kg , 9.5 10 ¹³ vg/kg, 9.6 ⁴ vg/kg、1.4 x 10 ¹⁴ vg/kg、 ^1.5 x 10 ¹⁴ vg/kg、1.6 x 10 ¹⁴ vg/ ^{kg 、} 1.7 x 10 ¹⁴ vg ^{/kg、1.8 x 10 14 vg/kg、1.9 x 10 14 vg / kg 、 2 x 10 14 vg} /kg、2.1 x 10 ¹⁴ 10 vg/kg, 2.2 x 10 ¹⁴ vg/kg, or 2.3 x 10 ¹⁴ vg/kg is administered to the patient.

In some embodiments, the AVV carrier is present in an amount of about 4 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg, such as about 4 x 10 ¹³ vg/kg, 4.1 x 10 ¹³ vg/kg, 4.2 x 10 ¹³ vg/kg, 4.3 x 10 ¹³ vg/kg, 4.4 x 10 ¹³ vg/kg, 4.5 x 10 ¹³ vg/kg, 4.6 x 10 ¹³ vg/kg, 4.7 x 10 ¹³ vg/kg, 4.8 x 10 ¹³ vg/kg, 4.9 x 10 ¹³ vg/kg, 5 x 10 ¹³ vg/kg, 5.1 x 10 13 vg/kg, 5.2 x 10 ¹³ vg/kg, 5.3 x 10 ¹³ vg/kg, 5 10 ¹³ vg/kg、5.4 x 10 ¹³ vg/kg、5.5 x 10 ¹³ vg/kg、5.6 x ^{10 13} vg/kg、5.7 x 10 ¹³ vg/ ^{kg 、} 5.8 x 10 ¹³ vg/ ^{kg、5.9 x 10 13 vg/kg、6 x 10 13 vg / kg} 、6.1 ^{3 vg} /kg ^{, 6.2} vg/kg, 7 x 10 ¹³ vg/kg, 7.1 x 10 ¹³ vg/kg, 7.2 x 10 ¹³ vg/kg, 7.3 x 10 ¹³ vg/kg, 7.4 x 10 ¹³ vg/kg, 7.6 x ^{10 13 vg} /kg, 7.8 x 10 ¹³ vg/kg, 7.9 x 10 ¹³ vg/kg, ^{8 x 10 13 vg/kg, 8.1 x 10 13 vg/kg, 8.2 x 10 13 vg/kg, 8.3 x 10 13 vg / kg ,} 8.5 x ^{10 13 vg} /kg, 8.6 x ^{10 13 vg / kg , 8.7 3 vg / kg 、 9.5 ​ ​ ​ ​} 14 vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, or 2.3 x 10 ¹⁴ vg/kg is administered to a patient.

In some embodiments, the AVV carrier is present in an amount of about 5 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg, such as about 5 x 10 ¹³ vg/kg, 5.1 x 10 ¹³ vg/kg, 5.2 x 10 13 vg/kg, 5.3 x 10 ¹³ vg/kg, 5.4 x 10 ¹³ vg/kg, 5.5 x 10 13 vg/kg, 5.6 x 10 ¹³ vg/kg, 5.7 x 10 ¹³ vg/kg, 5.8 x 10 ¹³ vg/kg, 5.9 x 10 ^{13 vg/kg, 6 x 10 13 vg/kg, 6.1 x 10 13} vg/kg, 6.2 x 10 ¹³ vg/kg, 6.3 x 10 ¹³ vg/kg, 6.4 x 10 13 vg/kg, 6.5 x 10 13 vg/kg, 6.6 x 10 ¹³ vg/kg, 6.7 x 10 13 vg/kg, 6.8 x 10 ¹³ vg/kg, 6.9 x 10 ¹³ vg/kg, 7 ^{10 13 vg / kg 、 6.4 3 vg / kg , 7.2 ​ ​ ​ ​} vg/kg, 8 x 10 ¹³ vg/kg, 8.1 x 10 ¹³ vg/kg, 8.2 x 10 ¹³ vg/kg, 8.3 x 10 ¹³ vg/kg, 8.5 x 10 ¹³ vg/kg, 8.6 x 10 ¹³ vg/kg, 8.7 x 10 ¹³ vg/kg, 8.8 x 10 ¹³ vg/kg, 8.9 x 10 ¹³ vg/ ^{kg, 9 x 10 13 vg/kg, 9.1 x 10 13 vg/kg, 9.2 x 10 13 vg / kg , 9.3 x 10 13 vg/kg, 9.5 x 10 13 vg /} kg, 9.6 ^{10 13 vg / kg , 9.7 4 vg / kg , 1.5 ​ ​ ​ ​} vg/kg, or 2.3 x 10 ¹⁴ vg/kg is administered to the patient.

In some embodiments, the AVV carrier is present in an amount of about 6 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg, such as about 6 x 10 ¹³ vg/kg, 6.1 x 10 ¹³ vg/kg, 6.2 x 10 ¹³ vg/kg, 6.3 x 10 ¹³ vg/kg, 6.4 x 10 ¹³ vg/kg, 6.5 x 10 ¹³ vg/kg, 6.6 x 10 ¹³ vg/kg, 6.7 x 10 ¹³ vg/kg, 6.8 x 10 ¹³ vg/kg, 6.9 x 10 ¹³ vg/kg, 7 x 10 ¹³ vg/kg, 7.1 x 10 13 vg/kg, 7.2 x 10 ¹³ vg/kg, 7.3 x 10 ¹³ vg/kg, 7 ^{10 13 vg / kg , 7.4 3 vg / kg , 8.2 ​ ​ ​ ​} vg/kg, 9 x 10 ¹³ vg/kg, 9.1 x 10 ¹³ vg/kg, 9.2 x 10 ¹³ vg/kg, 9.3 x 10 ¹³ vg/kg, 9.4 x 10 ¹³ vg/kg, 9.6 x 10 ¹³ vg/kg, 9.7 x 10 ¹³ vg/kg, 9.8 x 10 ¹³ vg/kg, 9.9 x 10 ¹³ vg/kg, ^{1 x 10 14 vg/kg, 1.1 x 10 14 vg/kg, 1.2 x 10 14 vg/kg, 1.3 x 10 14 vg / kg ,} 1.5 x ^{10 14 vg} /kg, 1.6 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, or 2 x 10 ¹⁴ vg/kg are administered to the patient.

In some embodiments, the AVV carrier is present in an amount of about 7 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg, such as about 7 x 10 ¹³ vg/kg, 7.1 x 10 ¹³ vg/kg, 7.2 x 10 ¹³ vg/kg, 7.3 x 10 ¹³ vg/kg, 7.4 x 10 ¹³ vg/kg, 7.5 x 10 ¹³ vg/kg, 7.6 x 10 ¹³ vg/kg, 7.7 x 10 ¹³ vg/kg, 7.8 x 10 ¹³ vg/kg, 7.9 x 10 ¹³ vg/kg, 8 x 10 ¹³ vg/kg, 8.1 x 10 13 vg/kg, 8.2 x 10 ¹³ vg/kg, 8.3 x 10 ¹³ vg/kg, 8 ^{10 13 vg / kg , 8.4 3 vg / kg , 9.2 ​ ​ ​ ​} 14 vg/kg, 1 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, or 2.3 x 10 ¹⁴ vg/kg is administered to a patient.

In some embodiments, the AVV carrier is present in an amount of about 8 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg, such as about 8 x 10 ¹³ vg/kg, 8.1 x 10 ¹³ vg/kg, 8.2 x 10 13 vg/kg, 8.3 x 10 ¹³ vg/kg, 8.4 x 10 ¹³ vg/kg, 8.5 x 10 13 vg/kg, 8.6 x 10 ¹³ vg/kg, 8.7 x 10 ¹³ vg/kg, 8.8 x 10 ¹³ vg/kg, 8.9 x 10 ¹³ vg/kg, 9 x 10 13 vg/kg, 9.1 x 10 13 vg/kg, 9.2 x 10 ¹³ vg/kg, 9.3 x 10 ¹³ vg/kg, 9.4 x 10 13 vg/kg, 9.5 x 10 13 vg/kg, 9.6 x 10 ¹³ vg/kg, 9.7 x 10 13 vg/kg, 9.8 x 10 ¹³ vg/kg, 9.9 x 10 ¹³ vg/kg, 10 10 ¹³ vg/kg, 9.4 4 vg/kg、 ^1.2 x 10 ¹⁴ vg/ ^kg 、1.3 x 10 ¹⁴ vg/kg、1.4 x 10 ¹⁴ vg/ ^{kg 、} 1.5 x 10 ¹⁴ vg/ ^{kg 、1.6 x 10 14 vg/kg、1.7 x 10 14 vg / kg 、1.8 x 10 14 vg} /kg、1.9 x 10 ¹⁴ The amount of 10 vg/kg, 2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, or 2.3 x 10 ¹⁴ vg/kg is administered to the patient.

In some embodiments, the AVV carrier is in an amount of about 9 x 10 ¹³ vg/kg to about 2.3 x 10 ¹⁴ vg/kg, such as 9 x 10 ¹³ vg/kg, 9.1 x 10 ¹³ vg/kg, 9.2 x 10 ¹³ vg/kg, 9.3 x 10 ¹³ vg/kg, 9.4 x 10 ¹³ vg/kg, 9.5 x 10 ¹³ vg/kg, 9.6 x 10 ¹³ vg/kg, 9.7 x 10 ¹³ vg/kg, 9.8 x 10 ¹³ vg/kg, 9.9 x 10 ¹³ vg/kg, 1 x 10 14 vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, or 2.3 x 10 ¹⁴ vg/kg is administered to a patient.

In some embodiments, the AVV carrier is present in an amount of about 1 x 10 ¹⁴ vg/kg to about 2.3 x 10 ¹⁴ vg/kg, such as 1 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2.2 x 10 ^{14 vg/kg, or 2.3 x 10 14} vg/kg. 10 ¹⁴ vg/kg is administered to the patient.

In some embodiments, the AAV vector is administered to a patient in an amount of about 3 x 10 ¹³ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 4 x 10 ¹³ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 5 x 10 ¹³ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 6 x 10 ¹³ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 7 x 10 ¹³ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 8 x 10 ¹³ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 9 x 10 ¹³ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.1 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.2 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.3 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.4 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.5 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.6 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.7 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.8 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 1.9 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.1 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.2 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.3 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.4 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.5 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.6 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.7 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.8 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 2.9 x 10 ¹⁴ vg/kg. In some embodiments, the AAV vector is administered to a patient in an amount of about 3 x 10 ¹⁴ vg/kg.

In some embodiments, the AAV vector is administered to the patient in a single dose comprising such amount (e.g., less than about 3 x ¹⁰¹⁴ vg/kg).

In some embodiments, the AAV vector is administered to the patient in two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) doses ^that together comprise the amount (e.g., less than about 3 x 10 14 vg/kg).

In some embodiments, the AAV vector is administered ^to the patient in two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) doses that each independently comprise the amount (e.g., less than about 3 x 10 14 vg/kg).

In some embodiments, two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) doses are separated from each other by one year or more (e.g., one year and one day, one year and one month, one year and six months, two years, three years, four years, or five years).

In some embodiments, two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) doses are administered to a patient within about 12 months (e.g., about 12 months, about 11 months, about 10 months, about 9 months, about 8 months, about 7 months, about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, or about 1 month) of each other. Combination Therapy

AAV vectors containing the transgene encoding MTM1 described herein can be combined with one or more additional AAV vectors containing the transgene encoding MTM1 described herein or administered in combination with one or more therapeutic procedures (e.g., nasobiliary drainage (NBD)) and/or agents (e.g., anticholestatic agents) for the treatment of neuromuscular disorders (e.g., XLMTM).

In some embodiments, one or more of the additional treatment procedures is NBD. NBD is a treatment procedure that helps drain bile (e.g., when the bile ducts are blocked, a biliary drain can help bile flow from the liver into the intestines). In some embodiments, NBD is performed with a biliary drain (also called a biliary stent), which is a thin, hollow, flexible tube with several small holes along the side. The biliary drain can be inserted into the patient's bile duct to drain it. Treatment Agents

In some embodiments, the one or more additional therapeutic agents are anticholestatic agents (e.g., bile acid, farnesoid X receptor (FXR) ligands, fibroblast growth factor 19 (FGF-19) mimetics, Takeda-G protein receptor 5 (TGR5) agonists, peroxisome proliferator-activated receptor (PPAR) agonists, PPAR-α agonists, PPAR-δ agonists, dual PPAR-α and PPAR-δ agonists, apical sodium-dependent bile acid transporter (ASBT) inhibitors, immunomodulatory drugs, antifibrotic chemotherapy, and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitors) or combinations thereof.

In some embodiments, the anticholestatic agent is administered to the patient in one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty, and seventy) doses beginning within about six weeks before or after administration of the viral vector to the patient (e.g., about six weeks before or after administration, about five weeks before or after administration, about four weeks before or after administration, about three weeks before or after administration, about two weeks before or after administration, or about one week before or after administration).

In some embodiments, the anticholestatic agent is administered to the patient in one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty, and seventy) doses beginning within about five weeks before or after administration of the viral vector to the patient (e.g., about five weeks before or after administration, about four weeks before or after administration, about three weeks before or after administration, about two weeks before or after administration, or about one week before or after administration).

In some embodiments, the anticholestatic agent is administered to the patient in one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty, and seventy) doses beginning within about one week before or after administration of the viral vector to the patient (e.g., about one week before or after administration, about six days before or after administration, about five days before or after administration, about four days before or after administration, about three days before or after administration, about two days before or after administration, or about one day before or after administration).

In some embodiments, the anticholestatic agent is administered to the patient on the same day as the viral vector is administered to the patient (e.g., 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 60 minutes, 59 minutes, 58 minutes, 57 minutes, 56 minutes, 55 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, or the same minute). ) is administered to the patient in one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty, and seventy) doses starting within a certain period of time.

In some embodiments, the anticholestatic agent is bile acid. In some embodiments, the bile acid is ursodeoxycholic acid or a derivative thereof or norsoursodeoxycholic acid. In some embodiments, the bile acid is ursodiol.

In some embodiments, the anticholestatic agent is a FXR ligand. In some embodiments, the FXR ligand is obeticholic acid, cilofaciolide, trofasol, tretinoin, or EDP-305.

In some embodiments, one or more anticholestatic agents are FGF-19 mimetics. In some embodiments, the FGF-19 mimetic is aldafermin.

In some embodiments, the anticholestatic agent is a TGR5 agonist. In some embodiments, the TGR5 agonist is INT-777 or INT-767.

In some embodiments, the anticholestatic agent is a PPAR agonist. In some embodiments, the PPAR agonist is bezafibrate, seradepa, or arabeno.

In some embodiments, the anticholestatic agent is a PPAR-alpha agonist. In some embodiments, the PPAR-alpha agonist is fenofibrate.

In some embodiments, the anticholestatic agent is a PPAR-delta agonist. In some embodiments, the PPAR-delta agonist is serradrepa.

In some embodiments, the anticholestatic agent is a dual PPAR-α and PPAR-δ agonist. In some embodiments, the dual PPAR-α-δ agonist is elafinol.

In some embodiments, one or more anticholestatic agents are ASBT inhibitors. In some embodiments, the ASBT inhibitor is odevixibat, malalixibat, or linexibat.

In some embodiments, the anticholestatic agent is an immunomodulatory drug. In some embodiments, the immunomodulatory drug is rituximab, abatacept, ustekinumab, infliximab, baricitinib, or FFP104.

In some embodiments, the anticholestatic agent is an antifiber chemotherapy. In some embodiments, the antifiber chemotherapy is a vitamin D receptor (VDR) agonist or sintuzumab.

In some embodiments, the anticholestatic agent is a NOX inhibitor. In some embodiments, the NOX inhibitor is setanacoxib. Recommended clinical parameters for monitoring the development of cholestasis or hyperbilirubinemia in patients

In some embodiments, a patient is monitored for the development of cholestasis by serum bile acid testing and/or blood tests (e.g., LFTs), as described herein.

In some embodiments, a patient is monitored for the development of hyperbilirubinemia by a blood test (e.g., a bilirubin test), as described herein.

In some embodiments, a patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is monitored for the development of hyperbilirubinemia, and if the patient exhibits hyperbilirubinemia or one or more symptoms thereof, an anticholestatic agent is administered to the patient.

In some embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by a blood test (e.g., a serum bile acid test or a liver function test). In some embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by a blood test (e.g., a serum bile acid test or a liver function test), and an anti-cholestatic agent is administered to the patient if the patient exhibits cholestasis or hyperbilirubinemia or one or more symptoms thereof.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof by finding that the patient exhibits an increase in a parameter (e.g., serum bile acid level) relative to a reference level in a blood test (e.g., a serum bile acid test).

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof by finding that the patient exhibits an increase in serum bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) relative to a reference level in a blood test (e.g., a serum bile acid test).

In some embodiments, the blood test is a liver function test.

In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by liver function tests, and if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof by finding that the patient exhibits an increase or decrease in a parameter in a liver function test (e.g., aspartate aminotransferase level or alanine aminotransferase level) relative to a reference level. I. Serum Bile Acid Test

In some embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia. In some embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by a serum bile acid test. In some embodiments, a patient is monitored for the development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof. In some embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by serum bile acid testing, and an anti-cholestatic agent is administered to the patient if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof.

In some embodiments, a patient is monitored for cholestasis or hyperbilirubinemia by the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) level as measured by a serum bile acid test.

In some embodiments, when one or more of the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels are greater than the norm as measured by a serum bile acid test, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's bile acid level as measured by a serum bile acid test is greater than 5 nmol/mL (e.g., 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's bile acid level as measured by a serum bile acid test is greater than 5 nmol/mL (e.g., 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's ursodeoxycholic acid level as measured by a serum bile acid test is greater than 2 nmol/mL (e.g., 2 nmol/mL, 3 nmol/mL, 4 nmol/mL, 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's ursodeoxycholic acid level as measured by a serum bile acid test is greater than 5 nmol/mL (e.g., 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. II. Liver Function Tests

In some embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by LFTs. In some embodiments, a patient is monitored for the development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof. In some embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by LFTs, and an anti-cholestatic agent is administered to the patient if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof.

In some embodiments, when a patient's LFT parameter (e.g., ASP level or AST level) is greater than an age-adjusted norm as described herein, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient. IIa. Aspartate aminotransferase

In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST level in LFTs. In some embodiments, the patient is monitored for the development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and if the patient exhibits cholestasis or hyperbilirubinemia or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST level in LFTs, and if the patient exhibits cholestasis or hyperbilirubinemia or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST level in LFTs, and when the patient's AST level is greater than the norm, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's AST level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient was determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof.

In some embodiments, when the patient's AST level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. IIb. Alanine aminotransferase

In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT level in LFTs. In some embodiments, the patient is monitored for the development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof. In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT level in LFTs, and an anti-cholestatic agent is administered to the patient if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof.

In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT level in LFTs, and when the patient's ALT level is greater than the norm, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's ALT level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is considered to be presenting with cholestasis or one or more of its symptoms.

In some embodiments, when the patient's ALT level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. Recommended Clinical Parameters for Monitoring the Development of Cholestasis in Patients I. Serum Bile Acid Tests

In some embodiments, a patient is monitored for the development of cholestasis. In some embodiments, a patient is monitored for the development of cholestasis by serum bile acid testing. In some embodiments, a patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, a patient is monitored for the development of cholestasis by serum bile acid testing, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's total bile acid level as measured by a serum bile acid test is greater than normal, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's total bile acid level is greater than 14 μmol/L (e.g., 15 μmol/L, 16 μmol/L, 17 μmol/L, 18 μmol/L, 19 μmol/L, 20 μmol/L, 21 μmol/L, 22 μmol/L, 23 μmol/L, 24 μmol/L, 25 μmol/L, 26 μmol/L, 27 μmol/L, 28 μmol/L, 29 μmol/L, 30 μmol/L, 31 μmol/L, 32 μmol/L, 33 μmol/L, 34 μmol/L, 35 μmol/L, 36 μmol/L, 37 μmol/L, 38 μmol/L, 39 μmol/L, 40 μmol/L, 41 μmol/L, 42 μmol/L, 43 μmol/L, 44 μmol/L, 45 μmol/L, 46 μmol/L, 47 μmol/L, 48 μmol/L, 49 μmol/L, 50 μmol/L, 51 μmol/L, 52 μmol/L, 53 μmol/L, 54 μmol/L, 55 μmol/L, 56 μmol/L, 57 μmol/L, 58 μmol/L, 59 μmol/L, 60 μmol/L, 61 μmol/L, 62 μmol/L, 63 μmol/L µmol/L, 44 µmol/L, 45 µmol/L, 46 µmol/L, 47 µmol/L, 48 µmol/L, 49 µmol/L, 50 µmol/L, 51 µmol/L, 52 µmol/L, 53 µmol/L, 54 µmol/L, 55 µmol/L, 56 µmol/L, 57 µmol/L, 58 µmol 7 4 µmol/L, 75 µmol/L, 76 µmol/L, 77 µmol/L, 78 The patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's blood glucose level is 250 μmol/L, 80 μmol/L, 81 μmol/L, 82 μmol/L, 83 μmol/L, 84 μmol/L, 85 μmol/L, 86 μmol/L, 87 μmol/L, 88 μmol/L, 89 μmol/L, 90 μmol/L, 91 μmol/L, 92 μmol/L, 93 μmol/L, 94 μmol/L, 95 μmol/L, 96 μmol/L, 97 μmol/L, 98 μmol/L, 99 μmol/L, and 100 μmol/L) and an anticholestatic agent is administered to the patient. II. Blood Tests

In some embodiments, a patient is monitored for the development of cholestasis by a blood test (e.g., LFT or bilirubin test). In some embodiments, a patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, a patient is monitored for the development of cholestasis by LFT, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, when one or more parameters (e.g., GGT level, ASP level, AST level, ALT level, and bilirubin level) of a patient's blood test (e.g., LFT or bilirubin test) are greater than age-adjusted norms as described herein, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient. IIa. Liver Function Tests

In some embodiments, a patient is monitored for the development of cholestasis by LFTs. In some embodiments, a patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, a patient is monitored for the development of cholestasis by LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, when one or more parameters of the patient's LFT (e.g., GGT level, ASP level, AST level, and ALT level) are greater than the age-adjusted norms as described herein, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient. IIai. γ- Glucosyltransferase

In some embodiments, the patient is monitored for the development of cholestasis by measuring the patient's GGT level in LFTs. In some embodiments, the patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, the patient is monitored for the development of cholestasis by measuring the patient's GGT level in LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient exhibits GGT levels greater than age-adjusted norms, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a newborn (e.g., 0-6 months), a toddler (e.g., 6-12 months), or a child 1-5 years old. In some embodiments, the patient is a newborn 0-6 months old. In some embodiments, the patient is a toddler 6-12 months old. In some embodiments, the patient is a child 1-5 years old.

In some embodiments, the patient is a newborn (e.g., 0-6 months), and when the patient's GGT level is within the normal range of about 12-122 U/L (e.g., 12-122 U/L, 13-122 U/L, 14-122 U/L, 15-122 U/L, 16-122 U/L, 17-122 U/L, 18-122 U/L, 19-122 U/L, 20-122 U/L, 25-122 U/L, 30-122 U/L, 40-122 U/L, 50-122 U/L, 60-122 U/L, 70-122 U/L, 80-122 U/L, 90-122 U/L, 100-122 U/L, 110-122 U/L, 120-122 U/L, or 121-122 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male newborn (e.g., 0-6 months), and the patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's GGT level is less than 12 U/L (e.g., 11 U/L, 10 U/L, 9 U/L, 8 U/L, 7 U/L, 6 U/L, 5 U/L, 4 U/L, 3 U/L, 2 U/L, or 1 U/L), and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a male newborn (e.g., 0-6 months), and when the patient's GGT level is greater than 122 U/L (e.g., 123 U/L, 124 U/L, 125 U/L, 126 U/L, 127 U/L, 128 U/L, 129 U/L, 130 U/L, 135 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a male infant (e.g., 6-12 months), and when the patient's GGT level is within the normal range of 1-39 U/L (e.g., 2-39 U/L, 3-39 U/L, 4-39 U/L, 5-39 U/L, 6-39 U/L, 7-39 U/L, 8-39 U/L, 9-39 U/L, 10-39 U/L, 11-39 U/L, 12-39 U/L, 13-39 U/L, 14-39 U/L, 15-39 U/L, 16-39 U/L, 17-39 U/L, 18-39 U/L, 19-39 U/L, 20-39 U/L, 21-39 U/L, 22-39 U/L, 23-39 U/L, 24-39 The patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's blood pressure is above 200 U/L, 25-39 U/L, 26-39 U/L, 27-39 U/L, 28-39 U/L, 29-39 U/L, 30-39 U/L, 31-39 U/L, 32-39 U/L, 33-39 U/L, 34-39 U/L, 35-39 U/L, 36-39 U/L, 37-39 U/L, or 38-39 U/L, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male infant (e.g., 6-12 months), and when the patient's GGT level is greater than 39 U/L (e.g., 40 U/L, 41 U/L, 42 U/L, 43 U/L, 44 U/L, 45 U/L, 46 U/L, 47 U/L, 48 U/L, 49 U/L, 50 U/L, 55 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male child aged 1-5 years, and when the patient's GGT level is within the normal range of about 3-22 U/L (e.g., about 3-22 U/L, 4-22 U/L, 5-22 U/L, 6-22 U/L, 7-22 U/L, 8-22 U/L, 9-22 U/L, 10-22 U/L, 11-22 U/L, 12-22 U/L, 13-22 U/L, 14-22 U/L, 15-22 U/L, 16-22 U/L, 17-22 U/L, 18-22 U/L, 19-22 U/L, 20-22 U/L, and 21-22 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male child aged 1-5 years, and when the patient's GGT level is less than 3 U/L (e.g., 2 U/L and 1 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a male child aged 1-5 years, and when the patient's GGT level is greater than 22 U/L (e.g., 23 U/L, 24 U/L, 25 U/L, 26 U/L, 27 U/L, 28 U/L, 29 U/L, 30 U/L, 35 U/L, 40 U/L, 50 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female newborn (e.g., 0-6 months), and when the patient's GGT level is within the normal range of about 15-132 U/L (e.g., 15-132 U/L, 16-132 U/L, 17-132 U/L, 18-132 U/L, 19-132 U/L, 20-132 U/L, 25-132 U/L, 30-132 U/L, 40-132 U/L, 50-132 U/L, 60-132 U/L, 70-132 U/L, 80-132 U/L, 90-132 U/L, 100-132 U/L, 110-132 U/L, 120-132 U/L, 130-132 U/L, and 131-132 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female newborn (e.g., 0-6 months), and the patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's GGT level is less than 15 U/L (e.g., 14 U/L, 13 U/L, 12 U/L, 11 U/L, 10 U/L, 9 U/L, 8 U/L, 7 U/L, 6 U/L, 5 U/L, 4 U/L, 3 U/L, 2 U/L, or 1 U/L), and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a female newborn (e.g., 0-6 months), and when the patient's GGT level is greater than 132 U/L (e.g., 133 U/L, 134 U/L, 135 U/L, 136 U/L, 137 U/L, 138 U/L, 139 U/L, 140 U/L, 145 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a female infant (e.g., 6-12 months old), when the patient's GGT level is within the normal range of 1-39 U/L (e.g., 2-39 U/L, 3-39 U/L, 4-39 U/L, 5-39 U/L, 6-39 U/L, 7-39 U/L, 8-39 U/L, 9-39 U/L, 10-39 U/L, 11-39 U/L, 12-39 U/L, 13-39 U/L, 14-39 U/L, 15-39 U/L, 16-39 U/L, 17-39 U/L, 18-39 U/L, 19-39 U/L, 20-39 U/L, 21-39 U/L, 22-39 U/L, 23-39 U/L, 24-39 The patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's blood pressure is above 200 U/L, 25-39 U/L, 26-39 U/L, 27-39 U/L, 28-39 U/L, 29-39 U/L, 30-39 U/L, 31-39 U/L, 32-39 U/L, 33-39 U/L, 34-39 U/L, 35-39 U/L, 36-39 U/L, 37-39 U/L, or 38-39 U/L, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female infant (e.g., 6-12 months), and when the patient's GGT level is greater than 39 U/L (e.g., 40 U/L, 41 U/L, 42 U/L, 43 U/L, 44 U/L, 45 U/L, 46 U/L, 47 U/L, 48 U/L, 49 U/L, 50 U/L, 55 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female child aged 1-5 years, when the patient's GGT level is within the normal range of about 3-22 U/L (e.g., about 3-22 U/L, 4-22 U/L, 5-22 U/L, 6-22 U/L, 7-22 U/L, 8-22 U/L, 9-22 U/L, 10-22 U/L, 11-22 U/L, 12-22 U/L, 13-22 U/L, 14-22 U/L, 15-22 U/L, 16-22 U/L, 17-22 U/L, 18-22 U/L, 19-22 U/L, 20-22 U/L, and 21-22 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female child aged 1-5 years, and when the patient's GGT level is less than 3 U/L (e.g., 2 U/L and 1 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a female child aged 1-5 years, and when the patient's GGT level is greater than 22 U/L (e.g., 23 U/L, 24 U/L, 25 U/L, 26 U/L, 27 U/L, 28 U/L, 29 U/L, 30 U/L, 35 U/L, 40 U/L, 50 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof , and an anticholestatic agent is administered to the patient.

In some embodiments, the development of cholestasis in a patient is monitored by measuring the patient's ASP level in LFTs. In some embodiments, the development of cholestasis in a patient is monitored, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, the development of cholestasis in a patient is monitored by measuring the patient's ASP level in LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, the development of cholestasis in a patient is monitored by measuring the patient's ASP level in LFTs, and when the patient's ASP level is greater than the norm, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's ASP level is within the normal range of about 50 to 300 U/L (e.g., about 51 to 300 U/L, about 52 to 300 U/L, about 53 to 300 U/L, about 54 to 300 U/L, about 55 to 300 U/L, about 56 to 300 U/L, about 57 to 300 U/L, about 58 to 300 U/L, about 59 to 300 U/L, about 60 to 300 U/L, about 65 to 300 U/L, about 70 to 300 U/L, about 80 to 300 U/L, about 90 to 300 U/L, about 100 to 300 U/L, about 125 to 300 U/L, about 150 to 300 U/L, about 175 to 300 U/L, about 200 to 300 U/L U/L, about 225 to 300 U/L, about 250 to 300 U/L, or about 275 to 300 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's ASP level is less than 50 U/L (e.g., 50 U/L, 49 U/L, 48 U/L, 47 U/L, 46 U/L, 45 U/L, 44 U/L, 43 U/L, 42 U/L, 41 U/L, 40 U/L, 39 U/L, 38 U/L, 37 U/L, 36 U/L, 35 U/L, 34 U/L, 33 U/L, 32 U/L, 31 U/L, 30 U/L, 29 U/L, 28 U/L, 27 U/L, 26 U/L, 25 U/L, 24 U/L, 23 U/L, 22 U/L, 21 U/L, 20 U/L, 19 U/L, 18 U/L, 17 U/L, 16 U/L, 15 U/L, 14 U/L, 13 U/L, 12 U/L, 11 U/L, 10 U/L, 9 U/L, 8 U/L, 7 U/L, 6 U/L, 5 U/L, 4 U/L, 3 U/L, 2 U/L, and 1 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's ASP level is greater than 300 U/L (e.g., 300 U/L, 301 U/L, 302 U/L, 303 U/L, 304 U/L, 305 U/L, 306 U/L, 307 U/L, 308 U/L, 309 U/L, 310 U/L, 311 U/L, 312 U/L, 313 U/L, 314 U/L, 315 U/L, 316 U/L, 317 U/L, 318 U/L, 319 U/L, 320 U/L, 321 U/L, 322 U/L, 323 U/L, 324 U/L, 325 U/L, 330 U/L, 340 U/L, 350 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. IIaiii. Aspartate aminotransferase

In some embodiments, the patient is monitored for the development of cholestasis by measuring the patient's AST level in LFTs. In some embodiments, the patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, the patient is monitored for the development of cholestasis by measuring the patient's AST level in LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, the development of cholestasis in a patient is monitored by measuring the patient's AST level in LFTs, and when the patient's AST level is greater than the norm, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's AST level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. IIaiv. Alanine aminotransferase

In some embodiments, the patient is monitored for the development of cholestasis by measuring the patient's ALT level in LFTs. In some embodiments, the patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, the patient is monitored for the development of cholestasis by measuring the patient's ALT level in LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, the development of cholestasis in a patient is monitored by measuring the patient's ALT level in LFTs, and when the patient's ALT level is greater than the norm, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's ALT level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient. Recommended Clinical Parameters for Monitoring the Development of Hyperbilirubinemia in Patients Bilerubin Testing

In some embodiments, a patient is monitored for the development of hyperbilirubinemia. In some embodiments, a patient is monitored for the development of hyperbilirubinemia by a bilirubin test. In some embodiments, a patient is monitored for the development of hyperbilirubinemia, and if the patient exhibits hyperbilirubinemia or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient. In some embodiments, a patient is monitored for the development of hyperbilirubinemia by a bilirubin test, and if the patient exhibits hyperbilirubinemia or one or more symptoms thereof, an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof when the patient exhibits bilirubin levels greater than normal, and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's total bilirubin level is greater than 1.2 mg/dL (e.g., 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3 mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL, 3.9 dL), the patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's direct bilirubin level is greater than 0.2 mg/dL (e.g., 0.2 mg/dL, 0.3 mg/dL, 0.4 mg/dL, 0.5 mg/dL, 0.6 mg/dL, 0.7 mg/dL, 0.8 mg/dL, 0.9 mg/dL, 1 mg/dL, 1.1 mg/dL, 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 4 .5 mg/dL, 4.6 mg/dL, 4.7 mg/dL, 4.8 mg/dL, 4.9 mg/dL, 5 mg/dL, 10 mg/dL, 15 mg/dL, 20 mg/dL, 30 mg/dL, 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL, and 100 mg/dL), the patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, when a patient exhibits a bilirubin level greater than 1 mg/dL (e.g., greater than 1 mg/dL, 1.1 mg/dL, 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3 mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 dL), the patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof and an anticholestatic agent is administered to the patient. Recommended clinical parameters for identifying patients who present with cholestasis or hyperbilirubinemia or symptoms

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof by determining that one or more parameters of a patient's serum bile acid test and/or blood test (e.g., LFT) (e.g., total bile acid level, GGT level, ASP level, AST level, and ALT level) are greater than or less than age-adjusted norms as described herein.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof by determining that one or more parameters (e.g., bilirubin levels) of a patient's blood test (e.g., a bilirubin test) are greater than the norms as described herein, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when one or more of the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels are greater than the norm as measured by a serum bile acid test, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when a patient's LFT parameter (e.g., ASP level or AST level) is greater than an age-adjusted norm as described herein, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. I. Serum Bile Acid Test

In some embodiments, when one or more of the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels are greater than the norm as measured by a serum bile acid test, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's bile acid level as measured by a serum bile acid test is greater than 5 nmol/mL (e.g., 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's bile acid level as measured by a serum bile acid test is greater than 5 nmol/mL (e.g., 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's deoxycholic acid level as measured by a serum bile acid test is greater than 6 nmol/mL (e.g., 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof when the patient's ursodeoxycholic acid level as measured by a serum bile acid test is greater than 2 nmol/mL (e.g., 2 nmol/mL, 3 nmol/mL, 4 nmol/mL, 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL).

In some embodiments, when the patient's ursodeoxycholic acid level as measured by a serum bile acid test is greater than 5 nmol/mL (e.g., 2 nmol/mL, 3 nmol/mL, 4 nmol/mL, 5 nmol/mL, 6 nmol/mL, 7 nmol/mL, 8 nmol/mL, 9 nmol/mL, 10 nmol/mL, 15 nmol/mL, 20 nmol/mL, 30 nmol/mL, 40 nmol/mL, 50 nmol/mL, 60 nmol/mL, 70 nmol/mL, 80 nmol/mL, 90 nmol/mL, and 100 nmol/mL), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. II. Liver Function Tests

In some embodiments, when a patient's LFT parameter (e.g., ASP level or AST level) is greater than an age-adjusted norm as described herein, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient. IIa. Aspartate aminotransferase

In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST level in LFTs, and when the patient's AST level is greater than the norm, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's AST level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient was determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof.

In some embodiments, when the patient's AST level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. IIb. Alanine aminotransferase

In some embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT level in LFTs, and when the patient's ALT level is greater than the norm, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's ALT level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is considered to be presenting with cholestasis or one or more of its symptoms.

In some embodiments, when the patient's ALT level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. Recommended Clinical Parameters for Identifying Patients as Exhibiting Cholestasis or Its Symptoms I. Serum Bile Acid Test

In some embodiments, when the patient exhibits bile acid levels greater than norm as measured by a serum bile acid test, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's total bile acid level is greater than 14 μmol/L (e.g., 15 μmol/L, 16 μmol/L, 17 μmol/L, 18 μmol/L, 19 μmol/L, 20 μmol/L, 21 μmol/L, 22 μmol/L, 23 μmol/L, 24 μmol/L, 25 μmol/L, 26 μmol/L, 27 μmol/L, 28 μmol/L, 29 μmol/L, 30 μmol/L, 31 μmol/L, 32 μmol/L, 33 μmol/L, 34 μmol/L, 35 μmol/L, 36 μmol/L, 37 μmol/L, 38 μmol/L, 39 μmol/L, 40 μmol/L, 41 μmol/L, 42 μmol/L, 43 μmol/L, 44 μmol/L, 45 μmol/L, 46 μmol/L, 47 μmol/L, 48 μmol/L, 49 μmol/L, 50 μmol/L, 51 μmol/L, 52 μmol/L, 53 μmol/L, 54 μmol/L, 55 μmol/L, 56 μmol/L, 57 μmol/L, 58 μmol/L, 59 μmol/L, 60 μmol/L, 61 μmol/L, 62 μmol/L, 63 μmol/L µmol/L, 44 µmol/L, 45 µmol/L, 46 µmol/L, 47 µmol/L, 48 µmol/L, 49 µmol/L, 50 µmol/L, 51 µmol/L, 52 µmol/L, 53 µmol/L, 54 µmol/L, 55 µmol/L, 56 µmol/L, 57 µmol/L, 58 µmol 7 4 µmol/L, 75 µmol/L, 76 µmol/L, 77 µmol/L, 78 The patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's blood glucose level is 250 μmol/L, 80 μmol/L, 81 μmol/L, 82 μmol/L, 83 μmol/L, 84 μmol/L, 85 μmol/L, 86 μmol/L, 87 μmol/L, 88 μmol/L, 89 μmol/L, 90 μmol/L, 91 μmol/L, 92 μmol/L, 93 μmol/L, 94 μmol/L, 95 μmol/L, 96 μmol/L, 97 μmol/L, 98 μmol/L, 99 μmol/L, and 100 μmol/L) and an anticholestatic agent is administered to the patient. II. Blood Tests

In some embodiments, when one or more parameters (e.g., GGT level, ASP level, AST level, ALT level, and bilirubin level) of a patient's blood test (e.g., LFT or bilirubin test) are greater than age-adjusted norms as described herein, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient. IIa. Liver Function Tests

In some embodiments, when one or more parameters of the patient's LFT (e.g., GGT level, ASP level, AST level, and ALT level) are greater than the age-adjusted norms as described herein, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient. IIai. γ- Glucosyltransferase

In some embodiments, when a patient exhibits GGT levels greater than age-adjusted norms as measured by LFTs, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a newborn (e.g., 0-6 months), a toddler (e.g., 6-12 months), or a child 1-5 years old. In some embodiments, the patient is a newborn 0-6 months old. In some embodiments, the patient is a toddler 6-12 months old. In some embodiments, the patient is a child 1-5 years old.

In some embodiments, the patient is a newborn (e.g., 0-6 months), and when the patient's GGT level is within the normal range of about 12-122 U/L (e.g., 12-122 U/L, 13-122 U/L, 14-122 U/L, 15-122 U/L, 16-122 U/L, 17-122 U/L, 18-122 U/L, 19-122 U/L, 20-122 U/L, 25-122 U/L, 30-122 U/L, 40-122 U/L, 50-122 U/L, 60-122 U/L, 70-122 U/L, 80-122 U/L, 90-122 U/L, 100-122 U/L, 110-122 U/L, 120-122 U/L, or 121-122 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male newborn (e.g., 0-6 months), and the patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's GGT level is less than 12 U/L (e.g., 11 U/L, 10 U/L, 9 U/L, 8 U/L, 7 U/L, 6 U/L, 5 U/L, 4 U/L, 3 U/L, 2 U/L, or 1 U/L), and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a male newborn (e.g., 0-6 months), and when the patient's GGT level is greater than 122 U/L (e.g., 123 U/L, 124 U/L, 125 U/L, 126 U/L, 127 U/L, 128 U/L, 129 U/L, 130 U/L, 135 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a male infant (e.g., 6-12 months), and when the patient's GGT level is within the normal range of 1-39 U/L (e.g., 2-39 U/L, 3-39 U/L, 4-39 U/L, 5-39 U/L, 6-39 U/L, 7-39 U/L, 8-39 U/L, 9-39 U/L, 10-39 U/L, 11-39 U/L, 12-39 U/L, 13-39 U/L, 14-39 U/L, 15-39 U/L, 16-39 U/L, 17-39 U/L, 18-39 U/L, 19-39 U/L, 20-39 U/L, 21-39 U/L, 22-39 U/L, 23-39 U/L, 24-39 The patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's blood pressure is above 200 U/L, 25-39 U/L, 26-39 U/L, 27-39 U/L, 28-39 U/L, 29-39 U/L, 30-39 U/L, 31-39 U/L, 32-39 U/L, 33-39 U/L, 34-39 U/L, 35-39 U/L, 36-39 U/L, 37-39 U/L, or 38-39 U/L, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male infant (e.g., 6-12 months), and when the patient's GGT level is greater than 39 U/L (e.g., 40 U/L, 41 U/L, 42 U/L, 43 U/L, 44 U/L, 45 U/L, 46 U/L, 47 U/L, 48 U/L, 49 U/L, 50 U/L, 55 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male child aged 1-5 years, and when the patient's GGT level is within the normal range of about 3-22 U/L (e.g., about 3-22 U/L, 4-22 U/L, 5-22 U/L, 6-22 U/L, 7-22 U/L, 8-22 U/L, 9-22 U/L, 10-22 U/L, 11-22 U/L, 12-22 U/L, 13-22 U/L, 14-22 U/L, 15-22 U/L, 16-22 U/L, 17-22 U/L, 18-22 U/L, 19-22 U/L, 20-22 U/L, and 21-22 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a male child aged 1-5 years, and the patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's GGT level is less than about 3 U/L (e.g., 2 U/L and 1 U/L), and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a male child aged 1-5 years, and when the patient's GGT level is greater than 22 U/L (e.g., 23 U/L, 24 U/L, 25 U/L, 26 U/L, 27 U/L, 28 U/L, 29 U/L, 30 U/L, 35 U/L, 40 U/L, 50 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female newborn (e.g., 0-6 months), and when the patient's GGT level is within the normal range of about 15-132 U/L (e.g., 15-132 U/L, 16-132 U/L, 17-132 U/L, 18-132 U/L, 19-132 U/L, 20-132 U/L, 25-132 U/L, 30-132 U/L, 40-132 U/L, 50-132 U/L, 60-132 U/L, 70-132 U/L, 80-132 U/L, 90-132 U/L, 100-132 U/L, 110-132 U/L, 120-132 U/L, 130-132 U/L, and 131-132 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female newborn (e.g., 0-6 months), and the patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's GGT level is less than about 15 U/L (e.g., 14 U/L, 13 U/L, 12 U/L, 11 U/L, 10 U/L, 9 U/L, 8 U/L, 7 U/L, 6 U/L, 5 U/L, 4 U/L, 3 U/L, 2 U/L, or 1 U/L), and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a female newborn (e.g., 0-6 months), and when the patient's GGT level is greater than 132 U/L (e.g., 133 U/L, 134 U/L, 135 U/L, 136 U/L, 137 U/L, 138 U/L, 139 U/L, 140 U/L, 145 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a female infant (e.g., 6-12 months), and when the patient's GGT level is within the normal range of 1-39 U/L (e.g., 2-39 U/L, 3-39 U/L, 4-39 U/L, 5-39 U/L, 6-39 U/L, 7-39 U/L, 8-39 U/L, 9-39 U/L, 10-39 U/L, 11-39 U/L, 12-39 U/L, 13-39 U/L, 14-39 U/L, 15-39 U/L, 16-39 U/L, 17-39 U/L, 18-39 U/L, 19-39 U/L, 20-39 U/L, 21-39 U/L, 22-39 U/L, 23-39 U/L, 24-39 The patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's blood pressure is above 200 U/L, 25-39 U/L, 26-39 U/L, 27-39 U/L, 28-39 U/L, 29-39 U/L, 30-39 U/L, 31-39 U/L, 32-39 U/L, 33-39 U/L, 34-39 U/L, 35-39 U/L, 36-39 U/L, 37-39 U/L, or 38-39 U/L, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female infant (e.g., 6-12 months), and when the patient's GGT level is greater than 39 U/L (e.g., 40 U/L, 41 U/L, 42 U/L, 43 U/L, 44 U/L, 45 U/L, 46 U/L, 47 U/L, 48 U/L, 49 U/L, 50 U/L, 55 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female child aged 1-5 years, when the patient's GGT level is within the normal range of about 3-22 U/L (e.g., about 3-22 U/L, 4-22 U/L, 5-22 U/L, 6-22 U/L, 7-22 U/L, 8-22 U/L, 9-22 U/L, 10-22 U/L, 11-22 U/L, 12-22 U/L, 13-22 U/L, 14-22 U/L, 15-22 U/L, 16-22 U/L, 17-22 U/L, 18-22 U/L, 19-22 U/L, 20-22 U/L, and 21-22 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, the patient is a female child between 1 and 5 years old, and the patient is determined to exhibit cholestasis or one or more symptoms thereof when the patient's GGT level is less than about 3 U/L (e.g., 2 U/L and 1 U/L), and an anti-cholestatic agent is administered to the patient.

In some embodiments, the patient is a female child aged 1-5 years, and when the patient's GGT level is greater than 22 U/L (e.g., 23 U/L, 24 U/L, 25 U/L, 26 U/L, 27 U/L, 28 U/L, 29 U/L, 30 U/L, 35 U/L, 40 U/L, 50 U/L, 60 U/L, 70 U/L, 80 U/L, 90 U/L, 100 U/L, 110 U/L, 1120 U/L, 130 U/L, 140 U/L, 150 U/L, 160 U/L, 170 U/L, 180 U/L, 190 U/L, and 200 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof , and an anticholestatic agent is administered to the patient.

In some embodiments, when a patient exhibits an ASP level greater than norm as measured by LFT, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's ASP level is within the normal range of about 50 to 300 U/L (e.g., about 51 to 300 U/L, about 52 to 300 U/L, about 53 to 300 U/L, about 54 to 300 U/L, about 55 to 300 U/L, about 56 to 300 U/L, about 57 to 300 U/L, about 58 to 300 U/L, about 59 to 300 U/L, about 60 to 300 U/L, about 65 to 300 U/L, about 70 to 300 U/L, about 80 to 300 U/L, about 90 to 300 U/L, about 100 to 300 U/L, about 125 to 300 U/L, about 150 to 300 U/L, about 175 to 300 U/L, about 200 to 300 U/L U/L, about 225 to 300 U/L, about 250 to 300 U/L, or about 275 to 300 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's ASP level is less than about 50 U/L (e.g., 50 U/L, 49 U/L, 48 U/L, 47 U/L, 46 U/L, 45 U/L, 44 U/L, 43 U/L, 42 U/L, 41 U/L, 40 U/L, 39 U/L, 38 U/L, 37 U/L, 36 U/L, 35 U/L, 34 U/L, 33 U/L, 32 U/L, 31 U/L, 30 U/L, 29 U/L, 28 U/L, 27 U/L, 26 U/L, 25 U/L, 24 U/L, 23 U/L, 22 U/L, 21 U/L, 20 U/L, 19 U/L, 18 U/L, 17 U/L, 16 U/L, 15 U/L, 14 U/L, 13 U/L, 12 U/L, 11 U/L, 10 U/L, 9 U/L, 8 U/L, 7 U/L, 6 U/L, 5 U/L, 4 U/L, 3 U/L, 2 U/L, and 1 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's ASP level is greater than 300 U/L (e.g., 300 U/L, 301 U/L, 302 U/L, 303 U/L, 304 U/L, 305 U/L, 306 U/L, 307 U/L, 308 U/L, 309 U/L, 310 U/L, 311 U/L, 312 U/L, 313 U/L, 314 U/L, 315 U/L, 316 U/L, 317 U/L, 318 U/L, 319 U/L, 320 U/L, 321 U/L, 322 U/L, 323 U/L, 324 U/L, 325 U/L, 330 U/L, 340 U/L, 350 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. IIaiii. Aspartate aminotransferase

In some embodiments, when a patient exhibits AST levels greater than norm as measured by LFT, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's AST level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. IIaiv. Alanine aminotransferase

In some embodiments, when a patient exhibits ALT levels greater than norm as measured by LFT, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-cholestatic agent is administered to the patient.

In some embodiments, when the patient's ALT level is greater than 50 U/L (e.g., 51 U/L, 52 U/L, 53 U/L, 54 U/L, 55 U/L, 56 U/L, 57 U/L U/L, 58 U/L, 59 U/L, 60 U/L, 61 U/L, 62 U/L, 63 U/L, 64 U/L, 65 U/L, 66 U/L, 67 U/L, 68 U/L, 69 U/L, 70 U/L, 75 U/L, 80 U/L, 85 U/L, 90 U/L, 100 U/L, 110 U/L, 120 U/L, 130 U/L, 140 U/L, 150 U/L, 200 U/L, 300 U/L, 400 U/L, and 500 U/L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholestatic agent is administered to the patient. Recommended clinical parameters for determining whether a patient exhibits hyperbilirubinemia or its symptoms Vegetarian Test

In some embodiments, a patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof when the patient exhibits bilirubin levels greater than norm as measured in a blood test (e.g., a bilirubin test), and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's total bilirubin level is greater than 1.2 mg/dL (e.g., 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3 mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL, 3.9 dL), the patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, when the patient's direct bilirubin level is greater than 0.2 mg/dL (e.g., 0.2 mg/dL, 0.3 mg/dL, 0.4 mg/dL, 0.5 mg/dL, 0.6 mg/dL, 0.7 mg/dL, 0.8 mg/dL, 0.9 mg/dL, 1 mg/dL, 1.1 mg/dL, 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 4 .5 mg/dL, 4.6 mg/dL, 4.7 mg/dL, 4.8 mg/dL, 4.9 mg/dL, 5 mg/dL, 10 mg/dL, 15 mg/dL, 20 mg/dL, 30 mg/dL, 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL, and 100 mg/dL), the patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof and an anticholestatic agent is administered to the patient.

In some embodiments, when a patient exhibits a bilirubin level greater than 1 mg/dL (e.g., greater than 1 mg/dL, 1.1 mg/dL, 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3 mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 dL), the patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof and an anticholestatic agent is administered to the patient .

The following examples are presented to provide those skilled in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be exemplary of the invention only and are not intended to limit the scope of what the inventors regard as their invention. Example 1. Evaluation of the safety and efficacy of pseudotyped AAV8 vectors including a nucleic acid sequence encoding the myotubularin 1 gene in a mouse model of X-linked myopathy Objectives and study methods

The purpose of this study was to conduct efficacy and sampling studies using myotubularin 1 (MTM1) hemizygous ("HEMI") knockout (KO) mice and to determine the possible impact of liver-specific MTM1 expression in mice. Mice were dosed with different test articles intravenously at approximately 4 weeks of age. Mice were sampled at approximately 16 weeks of age (approximately 12 weeks after dosing). Tissue samples were used for myotubularin and bile salt transporter expression, bile acid level measurements, and histopathology. Whole blood samples were used for hematology analysis, and serum samples were used for clinical chemistry analysis. In addition, a group of naive MTM1 KO mice were sampled at approximately 5 weeks of age and tissues were used for histopathology analysis.

The research was conducted in a test facility or test site according to appropriate methods and standard operating procedures (SOPs). The research complied with standard animal welfare ethics. Materials and Methods

MTM1 KO mice were used in this study, including 28 male wild-type (WT) mice and 70 male KO mice. Animals were sampled at approximately 5 weeks and approximately 16 weeks. Each animal was permanently identified according to the SOP of the testing facility with a unique permanent identification code. Toe and tail numbers and ear tags were used to identify animals. There was an adaptation period of approximately 3 to 4 days before dosing. Animals were group-housed with a maximum of 3 to 4 mice per cage, and at least one WT mouse was placed in each cage for social purposes. Mice were housed using an individual ventilated cage system (IVC) and type II polycarbonate long cages.

Veterinary care was provided throughout the study, and animals were examined by trained responsible personnel and monitored by veterinarians based on clinical signs or other changes.

Before the start of the life stage, any animal deemed unsuitable for use in the study was replaced with a replacement animal obtained from the same batch and maintained under the same environmental conditions. Animals in poor health or at the extremes of the weight range were not grouped. When setting up the groups for this study, the KO mice were randomly grouped so that the entire litter of mice was not assigned to one test group to avoid "nestling effects." The mice were housed in groups of a maximum of 4 to 5 mice. Each cage contained 3 to 4 KO mice and 1 to 2 WT mice for social purposes. No KO mouse was housed alone.

Four articles were tested: 1) AAV8 vector containing the mouse MTM1 (mMTM1) gene under the control of the desmin promoter (AAV8-Des-mMTM1), 2) AAV8 vector containing mMTM1 under the control of the APoE-A1AT promoter (AAV8-APoE-A1AT-mMTM1), 3) AAV8 vector containing the human MTM1 (hMTM1) gene with a stop codon under the control of the desmin promoter (AAV8-Des-hMTM1-STOP), and 4) AAV8 empty capsids. A vehicle control containing a placebo of 0.01% pluronic in Green's lactate solution was used. Dose levels were selected based on previous studies in mice. The high dose is a multiple of the previous high dose in mice, which demonstrated significant pharmacological effects without toxicity. Animals were dosed by slow intravenous (IV) bolus injection into the tail vein. The dose volume (16.33 mL/kg total) was administered as divided IV bolus doses, with 2 to 3 hours between doses. The site of dose, end time, and dose volume were recorded. Animals were anesthetized with isoflurane when necessary. This route of administration is consistent with the proposed human route of administration and is expected to provide appropriate systemic exposure for the study and relevant pharmacological activity. The frequency of administration is one dose on day 1. The expected dose level determines the relevant pharmacological activity that can be achieved. Necropsies were performed approximately 12 weeks after dosing. Treatment/study groups are summarized in Table 4 below. In addition, a group of 10 naive MTM1 KO mice were sampled at approximately 5 (5.3) weeks of age. Table 4. Mouse Study Groups Group genotype Number of animals Treatment group Dosage (vg/kg) Volume (ml/kg) Sampling age ( weeks ) Medication age ( weeks ) 1 Wild type 14 Medium 0 16.33 ~16.4 ~4.1 2 MTM1 KO (HEMI) 14 AAV8-Des-mMTM1 4e14 vg/kg 16.33 ~16.3 ~4.1 3 MTM1 KO (HEMI) 13 AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP 4e14 vg/kg + 2e14 vg/kg 16.33 ~16.4 ~4.2 4 MTM1 KO (HEMI) 14 AAV8-Des-mMTM1 + AAV8 empty capsid 4e14 vg/kg + equivalent to Group 3 shell/kg 16.33 ~16.6 ~4.1 5 MTM1 KO (HEMI) 14 AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 4e14 vg/kg + 2e14 vg/kg 16.33 ~16.7 ~4.2 6 MTM1 KO (HEMI) 12 AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP 4e14 vg/kg + 6e13 vg/kg + 1.4e14 vg/kg 16.33 ~16.8 ~4.1 7 Wild type 14 AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP 4e14 vg/kg + 2e14 vg/kg 16.33 ~16.4 ~4.1

The volumes of test articles used before drug administration are summarized in Table 5 below. Table 5. Volumes of test articles used before drug administration Group Treatment group Dosage (vg/kg) AAV8-Des-mMTM1 (mL) AAV8-Des-hMTM1-STOP (mL) AAV8 empty capsid (mL) AAV8-APoE-A1AT-mMTM1 (mL) Placebo volume (ml) Total volume (mL) Total volume (ml/kg) 1 Medium 0 NA NA NA NA 5 5 16.33 2 AAV8-Des-mMTM1 4e14 vg/kg 2.25 NA NA NA 2.75 5 16.33 3 AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP 4e14 vg/kg + 2e14 vg/kg 2.25 1.05 NA NA 1.7 5 16.33 4 AAV8-Des-mMTM1 + AAV8 empty capsid 4e14 vg/kg + equivalent to Group 3 shell/kg 2.25 NA 2.75 NA NA 5 16.33 5 AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 4e14 vg/kg + 2e14 vg/kg 2.25 NA NA 0.5 2.25 5 16.33 6 AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP 4e14 vg/kg + 6e13 vg/kg + 1.4e14 vg/kg 2.25 0.74 NA 0.15 1.86 5 16.33 7 AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP 4e14 vg/kg + 2e14 vg/kg 2.25 1.05 NA NA 1.7 5 16.33

The following humane endpoint criteria apply to this study. If mice meet the pre-defined humane endpoint criteria, they will undergo planned euthanasia for welfare reasons. If any mice need to be euthanized for welfare reasons, they will be opened and macroscopic observations will be recorded. In cases where the overall health of the animals deteriorates significantly, a shortened tissue isolation protocol should be used, if possible. The shortened tissue isolation protocol involves the collection of skeletal muscle (quadriceps) and 1 liver lobe in liquid nitrogen, and the collection of 1 liver lobe and heart (dorsal) tissue in formalin. Skeletal muscle and liver lobes are stored at -80°C, while the liver lobe and heart (dorsal) in formalin are stored at room temperature. Due to limited staff capacity to sample acute cases requiring immediate action during the evenings and weekends, mice were euthanized and decapitated by overdose of CO _2. In this case, no sampling was performed.

Body weight was measured three times a week and mortality was recorded. If surviving to the end of the study, mice were euthanized by deep anesthesia with sodium pentobarbital (180 mg/kg) at the end point, approximately 16-17 weeks of age (approximately 12-13 weeks after dosing). As much whole blood as possible was collected by cardiac puncture. Blood was first collected for hematology (100 μL EDTA whole blood) and then for clinical chemistry (200 μL whole blood to separate a minimum of 80 μL serum). Any remaining serum was collected. The mice were then perfused transcardially with heparinized saline to remove blood from the tissues. The 10 naive MTM1 KO mice in the group were sampled at approximately 5 weeks of age. Sampling included blood sampling (whole blood and serum), perfusion, and dissection of samples for histology and histopathology. No samples were used for other analyses.

Tissue samples were used for myotubularin and bile salt transporter expression, measurement of bile acid levels, and tissue pathology. Intravital and terminal whole blood samples were used for hematological analysis, while serum samples were used for clinical chemistry analysis. The same portion of the sample from each mouse was used for each analysis. The heart was collected with the left and right atria and then divided into two halves along the frontal plane. Tissue collection and analysis are summarized in Table 6 below. Table 6. Mouse tissue collection and analysis organization Histology and Histopathology Analysis of tubulin and bile salt transporter by IHC Myotubularin (ELISA) , vector copy number (VCN) and / or bile acid analysis (ELISA) Additional samples Quadriceps Left side muscles (½) Left side muscles (½) Right side muscles NA Diaphragm 1 sample 1 sample 1 sample NA Liver Left lateral lobe and a small part of right medial lobe (including gallbladder) Left medial lobe and remaining right medial lobe Remaining lobes (right lobe, right lateral lobe, caudate lobe) NA Heart ½ heart (dorsal side) NA ½ heart (ventral side) divided into 2 samples (for each animal, sample 1, vial 1; sample 2, vial 2; same parts) NA Triceps (right) Gastrocnemius (right) Tibialis anterior (right) NA NA NA Full muscle lung 1 sample NA NA NA Kidney Left + right, 2 samples NA NA NA spleen 1 sample NA NA NA Brain 1 sample NA NA NA Histology and Histopathology

The following tissue samples were dissected and placed in 10% neutral buffered formalin: ● Quadriceps femoris (left muscle, ½) ● Diaphragm (1 sample) ● Liver (left lateral lobe, and a small part of the right medial lobe (including gall bladder)) ● Heart (1/2, dorsal) ● Lung (1 sample) ● Kidney (left + right, 2 samples) ● Spleen (1 sample) ● Brain (1 sample)

Samples were embedded in paraffin, cut into sections, and stained with hematoxylin and eosin (H&E) for microscopic evaluation. Sample collection: tubulin (ELISA) , vector copy number (VCN) , and / or bile acid analysis (ELISA) ; tubulin and bile acid transporter analysis by immunohistochemistry (IHC) ; and tissue storage

Sterile collection for bioanalytical analysis (myotubularin (ELISA), vector copy number (VCN), and/or bile acid analysis (ELISA); myotubularin and bile acid transporter analysis by IHC; and stock tissue): Each tissue was collected in an RNAse-free screw-cap polypropylene tube, flash frozen in liquid nitrogen under sterile conditions, and stored at -80°C per the testing facility SOP. Samples were stored on dry ice until storage. Scalpel blades, weigh boats (small dishes), and dissecting instruments were replaced/cleaned before handling new mice. All instruments were wiped with RNAse Zap (or similar) between collection of each tissue. Precautions were taken to prevent cross contamination during tissue collection. Myotubularin (ELISA) , vector copy number (VCN) and / or bile acid analysis (ELISA)

The following tissue samples were dissected and frozen in liquid nitrogen: ● Quadriceps femoris (right muscle) ● Diaphragm (1 sample) ● Liver (right lobe, right lateral lobe, caudate lobe) ● Heart (1/2, ventral) (split into 2 samples) Myotubularin and bile acid transporter were analyzed by IHC

The following tissue samples were dissected and frozen in liquid nitrogen: ● Quadriceps femoris (left muscle, ½) ● Diaphragm (1 sample) ● Liver (left medial lobe and remaining right medial lobe, without gallbladder) Tissue stock

The following tissue samples were dissected and frozen in liquid nitrogen: ● Triceps (right muscle) ● Gastrocnemius (right muscle) ● Tibialis anterior (right muscle) Clinical Chemistry Panel

A panel of clinical biochemical markers was analyzed in non-hemolyzed serum at sampling during life and at terminal autopsy for the following parameters: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (AFOS), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), albumin, total bilirubin, total protein, creatinine, bile acid, calculated globulin, calculated albumin/globulin ratio, and urea nitrogen.

If blood samples are limited, the following priority analysis is performed: 1. Alanine aminotransferase (ALAT) 2. Total bilirubin/direct bilirubin 3. Bile acid 4. Aspartate aminotransferase (ASAT) 5. Alkaline phosphatase (AFOS) 6. Gamma-glutamyl transferase (GGT) 7. Lactate dehydrogenase (LDH) 8. Creatine kinase (CK) 9. Albumin 10. Total protein 11. Creatinine 12. Urea nitrogen

Due to limited serum samples, only ALAT was analyzed in vital samples.

During life and at terminal necropsy, a hematologic marker panel was analyzed in EDTA whole blood for the following parameters: hemoglobin, red blood cell count, white blood cell count (both relative and absolute differential counts), absolute reticulocyte count, reticulocyte %, and thrombocyte count. Additional parameters were included if the assay volume of whole blood permitted: hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. Genotyping Samples

At the endpoint sampling, tail samples were collected for possible re-genotyping by conventional PCR. Statistical Analysis and Graphical Presentation of Data

Data quality checks and validation are performed prior to further statistical analysis. During this process, potential outliers are identified and evaluated. No outliers are removed from the data without a clear reason for removal (e.g., a measurement error found in the laboratory records).

The comparisons planned for this study are: ● Group 1 versus Group 2 and Group 7 ● Group 2 versus Groups 3-6

The normality assumption for each data set was based primarily on experience (e.g., the data within a known population are approximately Gaussian) and observations from the validation phase. Some biological variables are known to follow a log-normal distribution - in such cases, the data are first transformed to logarithms and then parametric statistical tests can be used. The same normality assumption is used for a series of experiments or a set of similar readings from a test. In addition, the D'Agostino-Pearson omnibus normality test was used on clinical chemistry and hematology data to support the decision of whether to use parametric or nonparametric tests. A summary of the readings used for statistical analysis and visualization is summarized in Table 7 below. Table 7. Summary of readings used for statistical analysis and visualization Test / verification Regularization Visualization Weight without Line chart Hematology without Bar chart Clinical Chemistry without Bar chart

Descriptive statistics including group size, mean, SD, and SEM were provided for each parameter. Statistical Analysis of Single Time Point Data

Simple comparisons between two groups were performed using the unpaired Welch's t test or, when the assumptions of normality or lognormality were not met, the Mann-Whitney U test. Repeated observations on the same subjects ( longitudinal data )

Comparisons between the two groups were performed using a two-way mixed effects model (Mixed Anova) with a Geisser-Greenhouse correction (not assuming sphericity), followed by Fisher's LSD test between the two groups at each time point. Time, group, and time × group interaction were fixed effects, and individual subject was a random effect.

In the case of longitudinal comparisons of three or more groups, a two-way mixed-effects model with Geisser-Greenhouse correction was used, followed by Dunnett's multiple comparison test. One household was set at each time point. Time, group, and time × group interaction were fixed effects, and individual subjects were random effects.

The following section provides detailed results for some of the parameters mentioned in Table 6 above.

The results of this study provide an indication of the safety and efficacy of the above-described compositions and methods.

At all time points, HEMI AAV8-Des-mMTM1 mice had significantly lower body weight compared to WT vehicle mice (two-way ANOVA: group: p < 0.0001) (Fisher's LSD multiple comparisons: p < 0.001 for all) ( Figure 5 ).

At weeks 7-16, the body weight of WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP mice was significantly lower than that of WT vehicle mice (two-way ANOVA: group: p < 0.05) (Fisher's LSD multiple comparisons: p < 0.05 for all) ( Figure 5 ).

Body weight of HEMI mice was significantly altered (two-way ANOVA: group effect: NS, group x time interaction effect: p < 0.0001). Post hoc comparisons revealed that body weight of HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice was significantly reduced at weeks 4 and 5 (Dunnett's multiple comparisons: p < 0.05 for both) and that of HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice was significantly reduced at weeks 5-6 and 8-9 (Dunnett's multiple comparisons: p < 0.05 for all) compared with HEMI AAV8-Des-mMTM1 mice. Mortality

No data are presented for animals that died or were euthanized prior to dosing. No mortality was observed in Groups 1-3, 5, and 7. Two mice each in Groups 4, 6, and the initial group died prior to dosing due to factors unrelated to the test article .

All clinical chemistry evaluations for all groups were performed only from terminal samples (16 weeks or 5 weeks in naive mice), except for ALAT, which was analyzed from both mid-life and terminal samples (4-10 weeks). Data from clinical chemistry analyses are shown in Figures 6A to 19B . Alkaline phosphatase (AFOS)

There were no statistically significant differences in AFOS levels among the groups at 16 weeks of age ( Figure 6A ).

The AFOS levels of naive mice at 5 weeks of age are shown in Figure 6B . Alanine aminotransferase (ALAT)

ALAT levels were significantly increased in HEMI AAV8-Des-mMTM1 mice compared to WT vehicle mice at both the 6- and 16-week time points (two-way ANOVA: group effect: p = 0.0005) (Fisher's LSD multiple comparisons: p < 0.05 for both) ( FIG. 7A ).

ALAT levels were significantly increased in HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 mice at 10 weeks of age compared with HEMI AAV8-Des-mMTM1 mice (one-way ANOVA: group effect: p = 0.0026, Dunnett's post hoc comparison: p < 0.05) ( Figure 7A ).

The ALAT levels of naive mice at 5 weeks of age are shown in Figure 7B . Aspartate aminotransferase (ASAT)

ASAT levels were significantly increased in HEMI AAV8-Des-mMTM1 mice at 16 weeks of age compared with WT vehicle mice (t test: p < 0.01) ( Figure 8A ).

The ASAT levels of naive mice at 5 weeks of age are shown in Figure 8B .

There were no statistically significant differences in albumin levels between the groups at 16 weeks of age ( Fig. 9A ).

The albumin levels of naive mice at 5 weeks of age are shown in Figure 9B . γ- Glutaramidoyltransferase (GGT)

GGT levels were significantly increased in HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice and HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice at 16 weeks of age compared with HEMI AAV8-Des-mMTM1 (one-way ANOVA: group effect: p=0.0013, Dunnett's post hoc comparison: p<0.05 for both) ( Figure 10A ).

The GGT levels of naive mice at 5 weeks of age are shown in Figure 10B .

There were no statistically significant differences in total protein levels among the groups at 16 weeks of age ( Figure 11A ).

The total protein levels of naive mice at 5 weeks of age are shown in Figure 11B . Urea

There were no statistically significant differences in urea levels among the groups at 16 weeks of age ( Figure 12A ).

The urea levels of the naive mice at 5 weeks of age are shown in Figure 12B . Lactate dehydrogenase (LDH)

There was no statistically significant difference in LDH levels among the groups at 16 weeks of age ( Figure 13A ).

The LDH levels of naive mice at 5 weeks of age are shown in Figure 13B .

Compared with HEMI AAV8-Des-mMTM1 mice, bile acid levels were significantly increased in HEMI AAV8-Des-mMTM1 + AAV8 empty coat mice at 16 weeks of age (one-way ANOVA: group effect: p = 0.0258, Dunnett's post hoc comparison: p < 0.05) ( Figure 14A ). However, it is worth noting that the majority of bile acid values in all treatment groups were below detection levels.

The bile acid levels of naive mice at 5 weeks of age are shown in Figure 14B . Total bilirubin

There was no statistically significant difference in total bilirubin levels among the groups at 16 weeks of age ( Figure 15 ).

Total bilirubin data were not available for naive mice at 5 weeks of age (all values were below detection levels).

Compared with WT vehicle mice, calculated globulin levels were significantly increased in HEMI AAV8-Des-mMTM1 mice at 16 weeks of age (t test: p < 0.05) ( Figure 16A ).

The calculated globulin levels of naive mice at 5 weeks of age are shown in Figure 16B . Calculation of Albumin / Globulin Ratio

There were no statistically significant differences in the calculated albumin/globulin ratios between the groups at 16 weeks of age ( Fig. 17A ).

The albumin/globulin ratio levels of naive mice at 5 weeks of age are shown in Figure 17B . Creatine kinase

Compared with WT vehicle mice, CK levels were significantly increased in HEMI AAV8-Des-mMTM1 mice at 16 weeks of age (t test: p < 0.05) ( Figure 18A ).

The CK levels of naive mice at 5 weeks of age are shown in Figure 18B . Creatinine

There were no statistically significant differences in creatinine levels between the groups at 16 weeks of age ( FIG. 19A ).

Creatinine levels in naive mice at 5 weeks of age are shown in Figure 19B . Hematology

Due to the differences in blood sampling techniques between vital sampling (greater vein, no anesthesia) and terminal sampling (cardiocentesis, pentobarbital anesthesia), statistical analysis was performed separately for vital samples (weeks 4-10) and terminal samples (week 16). Different blood sampling techniques have been reported to result in significant differences in blood parameters (e.g., total white blood cell count) (Hoggatt et al. Exp Hematol. 44 (2):132–137.e1. (2016)). Data from hematological analyses are shown in Figures 20A to 41B . White blood cell count (WBC)

Compared with WT vehicle mice, the leukocyte counts of HEMI AAV8-Des-mMTM1 mice at 16 weeks of age were significantly increased (t-test, p<0.05) ( Figure 20A ). Compared with HEMI AAV8-Des-mMTM1 mice, the leukocyte counts of HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice at 7 weeks of age were significantly increased (two-way ANOVA: group effect: NS, group x time interaction effect: p<0.05, Dunnett's post hoc comparison: p<0.05) ( Figure 20A ).

The white blood cell counts of naive mice at 4 and 5 weeks of age are shown in FIG20B . Red blood cell counts (RBC)

There were no statistically significant differences in red blood cell counts among the groups ( FIG. 21A ).

The red blood cell counts of the naive mice at 4 and 5 weeks of age are shown in FIG21B . Hemoglobin (HGB)

There were no statistically significant differences in hemoglobin levels among the groups ( FIG. 22A ).

The hemoglobin levels of naive mice at 4 and 5 weeks of age are shown in FIG22B .

The hematocrit level of HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 mice at 6 weeks of age was significantly increased compared with HEMI AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: p < 0.05, Dunnett's post hoc comparison: p < 0.05) ( Figure 23A ).

The hematocrit levels of naive mice at 4 and 5 weeks of age are shown in Figure 23B . Mean Corpuscular Volume (MCV)

MCV levels were significantly reduced in HEMI AAV8-Des-mMTM1 mice at 4 and 5 weeks of age compared to WT vehicle mice (t test, p < 0.01 for all) ( Figure 24A ).

The MCV levels of naive mice at 4 and 5 weeks of age are shown in Figure 24B . Mean corpuscular hemoglobin (MCH)

There was no statistically significant difference in MCH levels among the groups ( Figure 25A ).

The MCH levels of naive mice at 4 and 5 weeks of age are shown in Figure 25B . Mean Corpuscular Hemoglobin Concentration (MCHC)

Compared with HEMI AAV8-Des-mMTM1 mice, MCHC levels were significantly decreased in HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice at 5 and 16 weeks of age and HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 mice at 6 weeks of age, while MCHC levels were significantly increased in HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice at 7 weeks of age (two-way ANOVA: group effect: NS, group x time interaction effect: p < 0.01, Dunnett's post hoc comparison: p < 0.05 for all) ( Figure 26A )

The MCHC levels of naive mice at 4 and 5 weeks of age are shown in Figure 26B .

The thrombocyte or platelet counts of HEMI AAV8-Des-mMTM1 mice at 6 weeks of age were significantly reduced compared with WT vehicle mice (two-way ANOVA: group effect: p < 0.05, Dunnett's post hoc comparison: p < 0.01) ( Figure 27A ).

The thrombocyte or platelet counts of naive mice at 4 and 5 weeks of age are shown in FIG27B . Neutrophil count ( relative ) (Neut(%))

There were no statistically significant differences in relative neutrophil counts between the groups ( FIG. 28A ).

The relative neutrophil counts of naive mice at 4 and 5 weeks of age are shown in FIG28B . Neutrophil counts ( absolute ) (Neut)

There were no statistically significant differences in absolute neutrophil counts between the groups ( Fig. 29A ).

The absolute neutrophil counts of naive mice at 4 and 5 weeks of age are shown in FIG29B . Lymphocyte count ( relative ) (Lymph (%))

Relative lymphocyte counts were significantly reduced in HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice at 5 and 6 weeks of age compared with HEMI AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: NS, group x time interaction: p < 0.05, Dunnett's post hoc comparison: p < 0.05 for all) ( Figure 30A ).

The relative lymphocyte counts of naive mice are shown in Figure 30B . Lymphocyte counts ( absolute ) (Lymph)

The absolute lymphocyte counts of HEMI AAV8-Des-mMTM1 mice at 16 weeks of age were significantly increased compared with WT vehicle mice (t test, p < 0.01) ( Figure 31A ).

The absolute lymphocyte counts of naive mice at 4 and 5 weeks of age are shown in FIG31B .

The relative monocyte counts in WT AAV8-Des-mMTM1+AAV8-Des-hMTM1-STOP mice at 16 weeks of age were significantly increased compared with WT vehicle mice (t-test, p<0.05) ( FIG. 32A ).

The relative monocyte counts of naive mice at 4 and 5 weeks of age are shown in FIG32B .

The absolute monocyte counts in HEMI AAV8-Des-mMTM1 mice at 16 weeks of age were significantly increased compared with WT vehicle mice (t test: p < 0.01) ( Figure 33A ).

The absolute monocyte counts of naive mice at 4 and 5 weeks of age are shown in FIG33B . Eosinophil count ( relative ) (Eos (%))

Compared with HEMI AAV8-Des-mMTM1 mice, the eosinophil counts in HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice at 4 weeks of age were significantly increased (two-way ANOVA: group effect: p < 0.05, Dunnett's post hoc comparison: p < 0.01) ( Figure 34A ).

The eosinophil counts of naive mice at 4 and 5 weeks of age are shown in FIG34B . Eosinophil count ( absolute ) (Eos)

The absolute eosinophil counts in HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice were significantly increased at 5 weeks of age compared with HEMI AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: p = 0.0001, Dunnett's post hoc comparison: p < 0.05) ( Figure 35A ).

The absolute eosinophil counts of naive mice at 4 and 5 weeks of age are shown in FIG35B .

Compared with HEMI AAV8-Des-mMTM1 mice, the relative phagocytic sphere counts of HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice and HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice at 5 weeks of age were significantly reduced (two-way analysis of variance: group effect: NS, group x time interaction effect: p < 0.01, Dunnett's post hoc comparison: p < 0.05) ( Figure 36A ).

The relative basophil counts of naive mice at 4 and 5 weeks of age are shown in FIG36B . Baso counts ( absolute )

There were no statistically significant differences in absolute phagocytic sphere counts among the groups ( Fig. 37A ).

The absolute basophil counts of naive mice at 4 and 5 weeks of age are shown in Figure 37B . Large unstained cell count ( relative ) (LUC (%)

Relative large unstained cell counts were significantly increased in WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP mice at 7 weeks of age compared with WT vehicle mice (two-way ANOVA: group effect: p < 0.05, Dunnett's post hoc comparison: p < 0.01) ( Figure 38A ).

The relative large unstained cell counts of naive mice at 4 and 5 weeks of age are shown in Figure 38B . Large unstained cell counts ( absolute ) (LUC)

The absolute large unstained cell counts were significantly reduced in HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice at 6 weeks of age and HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 mice at 10 weeks of age compared with HEMI AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: NS, group x time interaction: p < 0.05, Dunnett's post hoc comparison: p < 0.05) ( Figure 39A ).

The absolute large unstained cell counts of naive mice at 4 and 5 weeks of age are shown in FIG39B . Reticulocyte count ( relative ) (Retic (%)

There were no statistically significant differences in relative reticulocyte counts between the groups ( Fig. 40A ).

Relative reticulocyte counts of naive mice at 4 and 5 weeks of age are shown in FIG40B . Reticulocyte count ( absolute ) (Retic)

There were no statistically significant differences in absolute reticulocyte counts among the groups ( Fig. 41A ).

The absolute reticulocyte counts of naive mice at 4 and 5 weeks of age are shown in Figure 41B .

The results of the above experiments support the safety and efficacy of AAV vectors encoding the MTM1 transgene under the control of liver-directed regulatory elements, optionally in combination with AAV vectors encoding the MTM1 transgene under the control of muscle-directed regulatory elements, for the treatment of XLMTM. Example 2. Treatment of X-linked muscle microtubular myopathy in human patients by simultaneous administration of a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the myotubularin 1 gene operably linked to a liver-specific promoter and a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the myotubularin 1 gene operably linked to a desmin promoter

Using the compositions and methods disclosed herein, a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific promoter ( FIGS. 1 and 2 ) and a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a desmin promoter (e.g., birelin) can be simultaneously administered to a patient suffering from XLMTM, each at a dose of less than about 3 x 10 ¹⁴ vg/kg (e.g., an amount of less than about 3 x 10 14 vg/kg, 2.9 x 10 ¹⁴ vg/kg, 2.8 x 10 ¹⁴ vg/kg, 2.7 x 10 14 vg/kg, 2.6 x 10 14 vg/kg, 2.5 x 10 14 vg/kg, 2.4 x 10 ¹⁴ vg/kg, 2.6 x 10 ¹⁴ vg/kg, 2.7 x 10 ¹⁴ vg/kg, 2.8 x 10 14 vg/kg, 2.9 x 10 ¹⁴ vg/kg, ^{2.8 ...} vg/kg, 2.3 x 10 ¹⁴ vg/kg, 2.2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/ ^{kg, 1.3 x 10 14 vg/kg, 1.2 x 10 14 vg/kg, 1.1 x 10 14 vg/kg, 1 x 10 14 vg / kg ,} 1 x ^{10 13 vg} /kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg or less).

After concurrent administration to patients of a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific promoter ( FIGS. 1 and 2 ) and a pseudotyped AAV2/8 vector (e.g., birelubin) comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a desmin promoter, the patients exhibited a change in maximum inspiratory pressure relative to baseline. For example, after concurrent administration of a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific promoter ( FIGS . 1 and 2 ) and a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a desmin promoter (e.g., birelizumab) to the patient, the patient exhibited a change in maximum inspiratory pressure relative to baseline at about 24 weeks (e.g., about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks). Example 3. Treatment of X-linked tubulin myopathy in human patients by using a pseudotyped AAV2/8 vector comprising both a nucleic acid sequence encoding myotubularin 1 gene operably linked to a liver-specific promoter and a nucleic acid sequence encoding myotubularin 1 gene operably linked to a desmin promoter

Using the compositions and methods of the present disclosure, a pseudotyped AAV2/8 vector comprising both a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific promoter and a nucleic acid sequence encoding the MTM1 gene operably linked to a muscle-specific promoter ( FIG. 3 ) can be administered to a patient suffering from XLMTM at a dose of less than about 3 x 10 14 vg/kg (e.g., an amount of less than about 3 x 10 ¹⁴ vg/kg, 2.9 x 10 ¹⁴ vg/kg, 2.8 x 10 14 vg/kg, 2.7 x 10 ¹⁴ vg/kg, 2.6 x 10 ¹⁴ vg/kg, 2.5 x 10 14 vg/kg, 2.4 x 10 14 vg/kg, 2.3 x 10 14 vg/kg, 2.2 x 10 ¹⁴ vg/kg, 2.4 x 10 14 vg/kg, 2.5 x 10 14 vg/kg, 2.6 x 10 ¹⁴ vg/kg, 2.7 x 10 ¹⁴ vg/kg, 2.8 x 10 ¹⁴ vg/kg, 2.9 ...8 x 10 ¹⁴ vg/kg, 2.8 x 10 14 vg/kg, 2.8 x 10 14 vg/kg, ^{14 vg / kg , 2.1 /kg、1.3x1014vg/kg、1.2x1014vg/kg、1.1x1014vg/kg、1x1014vg/kg、1x1014vg/kg、1x1013vg/kg、1x1012vg/kg、1x1011vg/kg、1x1010 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​} vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg or less).

After patients were administered a pseudotyped AAV2/8 vector comprising both a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific promoter and a nucleic acid sequence encoding the MTM1 gene operably linked to a muscle-specific promoter ( FIG. 3 ), the patients showed changes in the number of hours of mechanical ventilation support over time relative to baseline. For example, after administering to a patient an AAV2/8 vector comprising both a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific promoter and a nucleic acid sequence encoding the MTM1 gene operably linked to a muscle-specific promoter ( FIG. 3 ), the patient exhibits a change in the number of hours of mechanical ventilation support over time relative to a baseline of about 24 weeks (e.g., about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks). Example 4. Treatment of X-linked muscle microtubular myopathy in a human patient by a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the myotubularin 1 gene operably linked to a ubiquitous promoter

Using the compositions and methods of the present disclosure, a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a ubiquitous promoter ( FIG. 4 ) can be administered to a patient suffering from XLMTM at a dose of less than about 3 x 10 ¹⁴ vg/kg (e.g., an amount of less than about 3 x 10 ¹⁴ vg/kg, 2.9 x 10 ¹⁴ vg/kg, 2.8 x 10 ¹⁴ vg/kg, 2.7 x 10 ¹⁴ vg/kg, 2.6 x 10 ¹⁴ vg/kg, 2.5 x 10 ¹⁴ vg/kg, 2.4 x 10 ¹⁴ vg/kg, 2.3 x 10 14 vg/kg, 2.2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2 x ^{10 14} vg/ ^kg , vg/kg, 1.9 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/ kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg or smaller).

After administering to the patient a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a ubiquitous promoter ( FIG. 4 ), the patient achieves functional independent sitting for at least 30 seconds. For example, after administering to the patient a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the MTM1 gene operably linked to a ubiquitous promoter ( FIG. 4 ), the patient achieves functional independent sitting at about 24 weeks (e.g., about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks). Example 5. Treatment of X-linked microtubular myopathy in a human patient by combining a non-viral vector comprising a liver expression construct with a pseudotyped AAV2/8 vector comprising a nucleic acid sequence encoding the myotubularin 1 gene operably linked to a desmin promoter

Using the compositions and methods disclosed herein, a non-viral vector (e.g., lipid nanoparticles) comprising a liver expression construct can be administered to a patient suffering from XLMTM in combination with a pseudotyped AAV2/8 vector (e.g., birelin) comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a desmin promoter, wherein the AAV2/8 vector is administered in an amount of less than about 3 x 10 ¹⁴ vg/kg (e.g., an amount of less than about 3 x 10 ¹⁴ vg/kg, 2.9 x 10 14 vg/kg, 2.8 x 10 ¹⁴ vg/kg, 2.7 x 10 ¹⁴ vg/kg, 2.6 x 10 14 vg/kg, 2.5 x 10 ¹⁴ vg/kg, 2.4 x 10 ¹⁴ vg/kg, 2.3 x 10 14 vg/kg, 2.6 x 10 ¹⁴ vg/kg, 2.7 x 10 14 vg/kg, 2.8 x 10 ¹⁴ vg/kg, 2.9 ^... vg/kg, 2.2 x 10 ¹⁴ vg/kg, 2.1 x 10 ¹⁴ vg/kg, 2 x 10 ¹⁴ vg/kg, 1.9 x 10 ¹⁴ vg/kg, 1.8 x 10 ¹⁴ vg/kg, 1.7 x 10 ¹⁴ vg/kg, 1.6 x 10 ¹⁴ vg/kg, 1.5 x 10 ¹⁴ vg/kg, 1.4 x 10 ¹⁴ vg/kg, 1.3 x 10 ¹⁴ vg/kg, 1.2 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg, 1 x 10 ¹³ vg/kg, 1 x 10 ¹² vg/kg, 1 x 10 ¹¹ vg/kg, 1 x 10 ¹⁰ vg/kg, 1 x 10 ⁹ vg/kg, 1 x 10 ⁸ vg/kg or less).

After administering to the patient a combination of a non-viral vector (e.g., lipid nanoparticles) comprising a liver expression construct and a pseudotyped AAV2/8 vector (e.g., birelugen) comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a desmin promoter, the patient achieves functional independence sitting for at least 30 seconds. For example, after administering to the patient a combination of a non-viral vector (e.g., lipid nanoparticles) comprising a liver expression construct and a pseudotyped AAV2/8 vector (e.g., birelugen) comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a desmin promoter, the patient achieves functional independence sitting for at least 30 seconds at about 24 weeks (e.g., about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks).

FIG. 1 is a schematic diagram of an exemplary adeno-associated virus (AAV) vector for expressing the human or mouse myotubularin 1 (MTM1) gene (e.g., a pseudotyped AAV vector, such as an AAV vector containing AAV2 inverted terminal repeats packaged in capsid proteins from AAV8). From left to right, the shaded arrows and rectangles represent nucleic acid sequences encoding: the ApoE/A1AT liver-specific promoter (ApoE/A1AT) operably linked to the b-globin intron, the human or mouse MTM1 gene, the SV40 polyadenylation signal (SV40-polyA), and flanking inverted terminal repeats (ITRs). FIG. 2 is a schematic diagram of an exemplary AAV vector for expressing the human or mouse myotubularin 1 (MTM1) gene (e.g., a pseudotyped AAV vector, such as an AAV vector containing AAV2 inverted terminal repeats packaged in capsid proteins from AAV8). From left to right, the shaded arrows and rectangles represent nucleic acid sequences encoding: the LP1 liver-specific promoter (LP1 promoter) operably linked to an SV40 intron, the human or mouse MTM1 gene, the SV40 polyadenylation signal (SV40-polyA), and flanking inverted terminal repeats (ITRs). FIG3 is a schematic diagram of an exemplary AAV vector for expressing the human myotubularin 1 (hMTM1) gene (e.g., a pseudotyped AAV vector, such as an AAV vector containing AAV2 inverted terminal repeats packaged in capsid proteins from AAV8). From left to right, shaded arrows and rectangles represent nucleic acid sequences encoding: a muscle-specific promoter (e.g., MCK, desmin) operably linked to hMTM1 and a polyadenylation signal (polyA), a transcriptional pause site, a liver-specific promoter (e.g., ApoE/A1AT, LP1) operably linked to hMTM1, a polyadenylation signal (polyA), and flanking inverted terminal repeats (ITRs). FIG. 4 is a schematic diagram of an exemplary AAV vector for expressing the human myotubularin 1 (hMTM1) gene (e.g., a pseudotyped AAV vector, such as an AAV vector containing AAV2 inverted terminal repeats packaged in capsid proteins from AAV8). From left to right, shaded arrows and rectangles represent nucleic acid sequences encoding: a ubiquitous promoter (e.g., PGK, Ef1a, GAPDH) operably linked to hMTM1, a polyadenylation signal (polyA), and flanking inverted terminal repeats (ITRs). FIG. 5 is a graph showing body weight (g) over time (age in weeks), mean + SEM (n = 10-14 for all), measured as described in Example 1 below. Statistical significance: HEMI AAV8-Des-mMTM1 mice vs. WT vehicle mice: p < 0.001 for all time points; WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP mice vs. WT vehicle mice: p < 0.05 for weeks 7-16; HEMI AAV8-Des-mMTM1 + AAV8 empty shell mice vs. HEMI AAV8-Des-mMTM1 mice: p < 0.05 for weeks 4 and 5; HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice vs. HEMI AAV8-Des-mMTM1 mice: p < 0.05 for weeks 5-6 and 8-9 < 0.05. FIG5 Abbreviations: WT, wild type; HEMI, hemizygous. FIG6A is a graph showing the levels of alkaline phosphatase (AFOS) (U/L), mean + SEM (n = 10-14, for all), measured as described in Example 1 below. No statistical differences were observed. FIG6A Abbreviations: WT, wild type; HEMI, hemizygous; AFOS, alkaline phosphatase. FIG6B is a graph showing the levels of alkaline phosphatase (AFOS) (U/L) in naïve mice, mean + SEM (n = 11),. FIG6B Abbreviations: AFOS, alkaline phosphatase. FIG. 7A is a graph showing alanine aminotransferase (ALAT) (U/L) levels, mean + SEM (mid-life samples (weeks 4-10): n=0-7; terminal samples (week 16): n=10-14, for all), measured as described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Data for groups below the limit of detection are not shown. Statistical differences: *p<0.05, compared to WT vehicle; ^# p<0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 7A Abbreviations: WT, wild type; HEMI, hemizygous; ALAT, alanine aminotransferase. FIG. 7B is a graph showing alanine aminotransferase (ALAT) (U/L) levels in naive mice, mean + SEM (n=5-11), measured as described in Example 1 below. FIG. 7B Abbreviations: ALAT, alanine aminotransferase. FIG. 8A is a graph showing aspartate aminotransferase (ASAT) (U/L) levels, mean + SEM (n=10-14, for all), measured as described in Example 1 below. Statistical differences: **p<0.01, compared to WT vehicle. FIG. 8A abbreviations: WT, wild type; HEMI, hemizygous; ASAT, aspartate aminotransferase. FIG. 8B is a graph showing aspartate aminotransferase (ASAT) (U/L) levels in naive mice, mean + SEM (n=11), measured as described in Example 1 below. FIG. 8B abbreviations: ASAT, aspartate aminotransferase. FIG. 9A is a graph showing albumin (g/L) levels, mean + SEM (n=10-14, for all), measured as described in Example 1 below. No statistical differences were observed. FIG. 9A abbreviations: WT, wild type; HEMI, hemizygous. FIG. 9B is a graph showing albumin (g/L) levels in naive mice, mean + SEM (n=11), measured as described in Example 1 below. FIG. 10A is a graph showing the levels of γ-glutamidyltransferase (GGT) (U/L), mean + SEM (n=10-14 for all), measured as described in Example 1 below. Statistical differences: ^# p<0.05, ^## p<0.01, compared to HEMI AAV8-Des-mMTM1. FIG. 10A abbreviations: WT, wild type; HEMI, hemizygous; GGT, γ-glutamidyltransferase. FIG. 10B is a graph showing the levels of γ-glutamidyltransferase (GGT) (U/L) in naive mice, mean + SEM (n=11), measured as described in the Example below. FIG. 10B abbreviations: GGT, γ-glutamidyltransferase. FIG . 11A is a graph showing total protein (g/L) levels, mean + SEM (n = 10-14 for all), measured as described in Example 1 below. No statistical differences were observed. FIG. 11A abbreviations: WT, wild type; HEMI, hemizygous, Prot tot, total protein. FIG. 11B is a graph showing total protein (g/L) levels in naive mice, mean + SEM (n = 11), measured as described in Example 1 below. FIG. 11B abbreviations: Prot tot, total protein. FIG. 12A is a graph showing urea (mmol/L) levels, mean + SEM (n = 10-14 for all), measured as described in Example 1 below. No statistical differences were observed. FIG. 12A abbreviations: WT, wild type; HEMI, hemizygous. FIG . 12B is a graph showing urea (mmol/L) levels in naive mice, mean + SEM (n = 11), measured as described in Example 1 below. FIG. 13A is a graph showing lactate dehydrogenase (LDH) (U/I) levels, mean + SEM (n = 10-14, for all), measured as described in Example 1 below. No statistical differences were observed. FIG. 13A Abbreviations: WT, wild type; HEMI, hemizygous; LDH, lactate dehydrogenase. FIG. 13B is a graph showing lactate dehydrogenase (LDH) (U/I) levels in naive mice, mean + SEM (n = 11), measured as described in Example 1 below. FIG. 13B Abbreviations: LDH, lactate dehydrogenase. FIG. 14A is a graph showing bile acid (µmol/L) levels, mean + SEM (n=0-3 for all), measured as described in Example 1 below. Data from groups below the limit of detection are not shown. Statistical difference: ^# p<0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 14A abbreviations: WT, wild type; HEMI, hemizygous. FIG. 14B is a graph showing bile acid (µmol/L) levels in naive mice, mean + SEM (n=11), measured as described in Example 1 below. FIG. 15 is a graph showing total bilirubin (µmol/L) levels, mean + SEM (n=0-8 for all), measured as described in Example 1 below. Data from groups below the limit of detection are not shown. No statistical differences were observed. FIG. 15 Abbreviations: WT, wild type; HEMI, hemizygous; BilTot, total bilirubin. FIG. 16A is a graph showing calculated globulin (g/L) levels, mean + SEM (n = 10-14 for all), measured as described in Example 1 below. Statistical differences: *p < 0.05, compared to WT vehicle. FIG. 16A Abbreviations: WT, wild type; HEMI, hemizygous. FIG. 16B is a graph showing calculated globulin (g/L) levels in naive mice, mean + SEM (n = 11), measured as described in Example 1 below. FIG. 17A is a graph showing calculated albumin/globulin ratio levels, mean + SEM (n = 10-14 for all), measured as described in Example 1 below. No statistical differences were observed. FIG. 17A Abbreviations: WT, wild type; HEMI, hemizygous. FIG. 17B is a graph showing calculated albumin/globulin ratio levels in naive mice, mean + SEM (n = 11), measured as described in Example 1 below. FIG. 18A is a graph showing creatine kinase (U/L) levels, mean + SEM (n = 10-14, for all), measured as described in Example 1 below. Statistical differences: *p < 0.05, compared to WT vehicle. FIG. 18A Abbreviations: WT, wild type; HEMI, hemizygous; CK, creatine kinase. FIG . 18B is a graph showing creatine kinase (U/L) levels in naive mice, mean + SEM (n=11), measured as described in Example 1 below. FIG. 18B abbreviations: CK, creatine kinase. FIG. 19A is a graph showing creatinine (umol/L) levels, mean + SEM (n=10-14, for all), measured as described in Example 1 below. No statistical differences were observed. FIG. 19A abbreviations: WT, wild type; HEMI, hemizygous. FIG. 19B is a graph showing creatinine (umol/L) levels in naive mice, mean + SEM (n=11), measured as described in Example 1 below. Figure 20A is a graph showing white blood cell counts (WBC) ( ^x109 cells/L), mean + SEM (n=5-14 for all), measured as described in Example 1 below. At time points of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: *p<0.05, compared to WT vehicle; ^# p<0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 20A Abbreviations: WT, wild type; HEMI, hemizygous; WBC, white blood cell count. FIG. 20B is a graph showing white blood cell count (WBC) (x10 ⁹ cells/L) levels in naive mice, mean + SEM (n=3/9), measured as described in Example 1 below. FIG. 20B Abbreviations: WBC, white blood cell count. FIG. 21A is a graph showing red blood cell count (RBC) (x10 ¹² cells/L), mean + SEM (n=5-14, for all), measured as described in Example 1 below. At each time point of weeks 4, 5, 6, 7, 10, and 16, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. FIG. 21A Abbreviations: WT, wild type; HEMI, hemizygous; RBC, red blood cell count. FIG. 21B is a graph showing red blood cell count (RBC) (x10 ¹² cells/L) levels in naive mice, mean + SEM (n=3/9), measured as described in Example 1 below. FIG. 21B Abbreviations: RBC, red blood cell count. FIG. 22A is a graph showing hemoglobin (g/L) levels, mean + SEM (n=5-14, for all), measured as described in Example 1 below. At each time point of weeks 4, 5, 6, 7, 10, and 16, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. FIG. 22A Abbreviations: WT, wild type; HEMI, hemizygous; HGB, hemoglobin. FIG. 22B is a graph showing hemoglobin (g/L) levels in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 22B Abbreviations: HGB, hemoglobin. FIG. 23A is a graph showing hematocrit (HCT) (%) levels, mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At time points of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: ^# p < 0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 23A Abbreviations: WT, wild type; HEMI, hemizygous; HCT, hematocrit. FIG. 23B is a graph showing hematocrit (HCT) (%) levels in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 23B Abbreviations: HCT, hematocrit. FIG. 24A is a graph showing mean corpuscular volume (MCV) (fL) levels, mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At time points of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: *p<0.05, compared to WT vehicle; ^# p<0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 24A Abbreviations: WT, wild type; HEMI, hemizygous; MCV, mean corpuscular volume. FIG. 24B is a graph showing mean corpuscular volume (MCV) (fL) levels in naive mice, mean + SEM (n=3/9), measured as described in the examples below. FIG. 24B Abbreviations: MCV, mean corpuscular volume. FIG. 25A is a graph showing mean corpuscular hemoglobin (MCH) (pg) levels, mean + SEM (n=5-14, for all), measured as described in the examples below. At each time point of weeks 4, 5, 6, 7, 10, and 16, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. FIG. 25A Abbreviations: WT, wild type; HEMI, hemizygous; MCH, mean corpuscular hemoglobin. FIG. 25B is a graph showing mean corpuscular hemoglobin (MCH) (pg) levels in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 25B Abbreviations: MCH, mean corpuscular hemoglobin. FIG. 26A is a graph showing mean corpuscular hemoglobin concentration (MCHC) (g/L) levels, mean + SEM (n = 5-14 for all), measured as described in Example 1 below. At time points of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: ^# p < 0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 26A Abbreviations: WT, wild type; HEMI, hemizygous; MCHC, mean corpuscular hemoglobin concentration. FIG. 26B is a graph showing mean corpuscular hemoglobin concentration (MCHC) (g/L) levels in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 26B Abbreviations: MCHC, mean corpuscular hemoglobin concentration. FIG. 27A is a graph showing thrombocyte counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: *p<0.05 compared to WT vehicle. FIG. 27A abbreviations: WT, wild type; HEMI, hemizygous; PLT, platelet or thrombocyte count. FIG. 27B is a graph showing thrombocyte counts (x10 ⁹ cells/L) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 27B abbreviations: PLT, platelet or thrombocyte count. FIG. 28A is a graph showing relative neutrophil counts (neutrophils (%)), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of weeks 4, 5, 6, 7, 10, and 16, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. FIG. 28A abbreviations: WT, wild type; HEMI, hemizygous; %NEUT, neutrophil (%). FIG. 28B is a graph showing relative neutrophil counts (neutrophil (%)) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 28B abbreviations: %NEUT, neutrophil (%). FIG. 29A is a graph showing absolute neutrophil counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of weeks 4, 5, 6, 7, 10, and 16, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. Figure 29A abbreviations: WT, wild type; HEMI, hemizygous; Abs/absolute NEUT, absolute neutrophil count. Figure 29B is a graph showing absolute neutrophil counts (x10 ⁹ cells/L) in naive mice, mean + SEM (n = 3/9), measured in the manner described in Example 1 below. Figure 29B abbreviations: Abs/absolute NEUT, absolute neutrophil count. Figure 30A is a graph showing relative lymphocyte counts (lymphocytes (%)), mean + SEM (n = 5-14, for all), measured in the manner described in Example 1 below. At time points of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: ^# p < 0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 30A Abbreviations: WT, wild type; HEMI, hemizygous; %LYM, lymphocyte (%). FIG. 30B is a graph showing relative lymphocyte counts (lymphocytes (%)) in naive mice, mean + SEM (n = 3/9), measured as described in the Example below. FIG. 30B Abbreviations: %LYM, lymphocytes (%). FIG. 31A is a graph showing absolute lymphocyte counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: *p<0.05 compared to WT vehicle. FIG. 31A abbreviations: WT, wild type; HEMI, hemizygous; Abs/absolute LYMPHS, absolute lymphocyte count. FIG. 31B is a graph showing absolute lymphocyte counts (x10 ⁹ cells/L) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 31B abbreviations: Abs/absolute LYMPHS, absolute lymphocyte count. FIG. 32A is a graph showing relative monocyte counts (monocytes (%)), mean + SEM (n = 5-14, for all), measured as described in the Example below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: *p<0.05 compared to WT vehicle. FIG. 32A abbreviations: WT, wild type; HEMI, hemizygous; %MONO, monocyte (%). FIG. 32B is a graph showing relative monocyte counts (monocytes (%)) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 32B abbreviations: %MONO, monocyte (%). FIG. 33A is a graph showing absolute monocyte counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: *p<0.05 compared to WT vehicle. FIG. 33A abbreviations: WT, wild type; HEMI, hemizygous; Abs/absolute MONOS, absolute monocyte count. FIG. 33B is a graph showing absolute monocyte counts (x10 ⁹ cells/L) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 33B abbreviations: Abs/absolute MONOS, absolute monocyte count. FIG. 34A is a graph showing relative eosinophil counts (eosinophils (%)), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At time points of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: ^## p<0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 34A Abbreviations: WT, wild type; HEMI, hemizygous; %EOS, eosinophils (%). FIG. 34B is a graph showing relative eosinophil counts (eosinophils (%)) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 34B Abbreviations: %EOS, eosinophils (%). FIG. 35A is a graph showing absolute eosinophil counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At time points of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: ^# p < 0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 35A Abbreviations: WT, wild type; HEMI, hemizygous; Abs/absolute EOS, absolute eosinophil count. FIG. 35B is a graph showing absolute eosinophil counts (x10 ⁹ cells/L) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 35B Abbreviations: Abs/absolute EOS, absolute eosinophil count. FIG. 36A is a graph showing relative basophil counts (basophils (%)), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Data for groups below the limit of detection are not shown. Statistical differences: ^# p < 0.05, compared to HEMI AAV8-Des-mMTM1. FIG. 36A Abbreviations: WT, wild type; HEMI, hemizygous; % BASO, basophils (%). FIG. 36B is a graph showing relative basophil counts (basophils (%)) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 36B Abbreviations: % BASO, basophils (%). FIG. 37A is a graph showing absolute basophil counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. Data for groups below the detection limit are not shown. Figure 37A abbreviations: WT, wild type; HEMI, hemizygous; Abs/absolute BASOS, absolute basophil count. Figure 37B is a graph showing absolute basophil counts (x10 ⁹ cells/L) in naive mice, mean + SEM (n = 3/9), measured in the manner described in Example 1 below. Data for groups below the detection limit are not shown. Figure 37B abbreviations: Abs/absolute BASOS, absolute basophil count. Figure 38A is a graph showing relative large unstained cell counts (large unstained cells (%)), mean + SEM (n = 5-14, for all), measured in the manner described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical differences: **p<0.01 compared to WT vehicle. FIG. 38A abbreviations: WT, wild type; HEMI, hemizygous; %LUC, large unstained cells (%). FIG . 38B is a graph showing relative large unstained cell counts (large unstained cells (%)) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 38B abbreviations: %LUC, large unstained cells (%). FIG. 39A is a graph showing absolute large unstained cell counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Data for groups below the limit of detection are not shown. Statistical differences: ^# p < 0.05, ^## p < 0.01, compared to HEMI AAV8-Des-mMTM1. FIG. 39A Abbreviations: WT, wild type; HEMI, hemizygous; Abs/absolute LUCS, absolute large unstained cell count. FIG. 39B is a graph showing absolute large unstained cell counts (x10 ⁹ cells/L) in naive mice, mean + SEM (n=3/9), measured as described in Example 1 below. Data for groups below the limit of detection are not shown. FIG. 39B Abbreviations: Abs/absolute LUCS, absolute large unstained cell count. Figure 40A is a graph showing relative reticulocyte count (Reticulocyte (%)), mean + SEM (n = 5-14 for all), measured as described in the Examples below. At each time point of weeks 4, 5, 6, 7, 10, and 16, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. FIG. 40A Abbreviations: WT, wild type; HEMI, hemizygous; %Retic, reticulocyte (%). FIG. 40B is a graph showing relative reticulocyte counts (Reticulocyte (%)) in naive mice, mean + SEM (n = 3/9), measured as described in Example 1 below. FIG. 40B Abbreviations: %Retic, reticulocyte (%). FIG. 41A is a graph showing absolute reticulocyte counts (x10 ⁹ cells/L), mean + SEM (n = 5-14, for all), measured as described in Example 1 below. At each time point of weeks 4, 5, 6, 7, 10, and 16, the following groups were tested from left to right: WT vehicle, HEMI AAV8-Des-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMI AAV8-Des-mMTM1 + AAV8 empty capsid, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMI AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. No statistical differences were observed. Figure 41A abbreviations: WT, wild type; HEMI, hemizygous; Abs/Absolute Retic, absolute reticulocyte count. Figure 41B is a graph showing absolute reticulocyte count (x10 ⁹ cells/L) in naive mice, mean + SEM (n=3/9), measured as described in Example 1 below. Figure 41B abbreviations: Abs/Absolute Retic, absolute reticulocyte count.

TW202424194A_112136358_SEQL.xml

Claims (91)

A recombinant adeno-associated virus (AAV) vector comprises a transgene encoding myotubularin 1 (MTM1), wherein the transgene is operably linked to a promoter active in liver tissue. The AAV vector of claim 1, wherein the promoter is selectively active in liver tissue. The AAV vector of claim 2, wherein after the vector is contacted with one or more liver cells and one or more non-liver cells respectively under the same conditions (for example, in vitro or in vivo), the vector causes the expression level of the transgenic gene in the one or more liver cells to be higher than the expression level of the transgenic gene in the one or more non-liver cells. The AAV vector of claim 3, wherein after the vector is contacted with the one or more liver cells and the one or more non-liver cells, the vector causes the expression level of the transgene in the one or more liver cells to be 2 to 1,000 times higher than the expression level of the transgene in the one or more non-liver cells. The AAV vector of claim 4, wherein after the vector is contacted with the one or more liver cells and the one or more non-liver cells, the vector causes the expression level of the transgene in the one or more liver cells to be 50 to 1,000 times higher than the expression level of the transgene in the one or more non-liver cells. The AAV vector of claim 5, wherein after the vector is contacted with the one or more hepatocytes and the one or more non-hepatocytes, the vector causes the expression level of the transgene in the one or more hepatocytes to be 100 to 1,000 times higher than the expression level of the transgene in the one or more non-hepatocytes. The AAV vector of claim 2, wherein after contacting the vector with the one or more liver cells, the vector causes the expression level of the transgene in the one or more liver cells to be at least 0.2 times, at least 0.5 times, at least 1 times, at least 2 times, at least 5 times, or at least 10 times the expression level of wild-type MTM1 in healthy liver cells. The AAV vector of any one of claims 3 to 6, wherein the non-hepatic cells are muscle cells or nerve cells. The AAV vector of any one of claims 3 to 6, wherein the non-hepatic cells are cardiomyocytes. The AAV vector of any one of claims 1 to 9, wherein the promoter comprises the LP1 promoter. The AAV vector of claim 10, wherein the LP1 promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 3. The AAV vector of claim 11, wherein the LP1 promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 3, optionally wherein the LP1 promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 3. The AAV vector of claim 12, wherein the LP1 promoter comprises the nucleic acid sequence of SEQ ID NO: 3. The AAV vector of any one of claims 1 to 13, wherein the promoter comprises an apolipoprotein E (ApoE) promoter. The AAV vector of claim 14, wherein the ApoE promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 8. The AAV vector of claim 15, wherein the ApoE promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 8, optionally wherein the ApoE promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 8. The AAV vector of claim 16, wherein the ApoE promoter comprises the nucleic acid sequence of SEQ ID NO: 8. The AAV vector of any one of claims 1 to 17, wherein the promoter comprises an alpha-1-antitrypsin (A1AT) promoter. The AAV vector of claim 18, wherein the A1AT promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 9. The AAV vector of claim 19, wherein the A1AT promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 9, optionally wherein the A1AT promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 9. The AAV vector of claim 20, wherein the A1AT promoter comprises the nucleic acid sequence of SEQ ID NO: 9. The AAV vector of any one of claims 1 to 21, wherein the promoter is a chimeric promoter comprising an ApoE promoter and an A1AT promoter. The AAV vector of claim 22, wherein the chimeric promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 2. The AAV vector of claim 23, wherein the chimeric promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 2, optionally wherein the chimeric promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 2. The AAV vector of claim 24, wherein the chimeric promoter comprises the nucleic acid sequence of SEQ ID NO: 2. The AAV vector of any one of claims 1 to 25, wherein the promoter comprises a constitutive promoter. The AAV vector of claim 26, wherein the constitutive promoter is a phosphoglycerate kinase (PGK) promoter, an elongation factor-1α (EF1α) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a cytomegalovirus (CMV) promoter, or a chicken-β-actin (CBA) promoter. The AAV vector of any one of claims 1 to 27, wherein the vector further comprises a second transgene encoding MTM1. The AAV vector of claim 28, wherein the second transgene encoding MTM1 is operably linked to a promoter active in muscle tissue. The AAV vector of claim 29, wherein the promoter active in muscle tissue is selectively active in muscle tissue. The AAV vector of claim 30, wherein the promoter that is selectively active in muscle tissue achieves (e.g., in vitro or in vivo) expression of the MTM1 transgene in muscle cells at a level that is 2 to 1,000 times higher than the expression level of the MTM1 transgene achieved by the same promoter in non-muscle cells. The AAV vector of claim 31, wherein the promoter that is selectively active in muscle tissue achieves an expression level of the MTM1 transgene in muscle cells that is 10 to 1,000 times higher than the expression level of the MTM1 transgene achieved by the same promoter in non-muscle cells. The AAV vector of claim 32, wherein the promoter that is selectively active in muscle tissue achieves an expression level of the MTM1 transgene in muscle cells that is 50 to 1,000 times higher than the expression level of the MTM1 transgene achieved by the same promoter in non-muscle cells. The AAV vector of claim 33, wherein the promoter that is selectively active in muscle tissue achieves an expression level of the MTM1 transgene in muscle cells that is 100 to 1,000 times higher than the expression level of the MTM1 transgene achieved by the same promoter in non-muscle cells. The AAV vector of claim 34, wherein the promoter that is selectively active in muscle tissue achieves expression of the MTM1 transgene in muscle cells at a level that is at least 2 times, at least 5 times, at least 10 times, at least 50 times, or at least 100 times higher than the expression level of the MTM1 transgene achieved by the same promoter in non-muscle cells. The AAV vector of any one of claims 28 to 35, wherein the second transgene encoding MTM1 is operably linked to a muscle creatine kinase (MCK) promoter or a desmin (DES) promoter. An AAV vector as claimed in any one of claims 1 to 36, wherein each transgene encoding MTM1 independently comprises: a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 6; or a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 7. The AAV vector of claim 37, wherein each transgene encoding MTM1 independently comprises: a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1, wherein each transgene encoding MTM1 independently comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 6, wherein each transgene encoding MTM1 independently comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; 6; or a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 7, wherein each transgenic gene encoding MTM1 independently comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 7. As in claim 38, the AAV vector, wherein each transgene encoding MTM1 comprises: The nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or The nucleic acid sequence of SEQ ID NO: 6; or The nucleic acid sequence of SEQ ID NO: 7. The AAV vector of any one of claims 1 to 39, wherein the AAV is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 or AAVrh74. The AAV vector of any one of claims 1 to 40, wherein the AAV vector is a pseudotyped AAV. The AAV vector of claim 41, wherein the pseudotyped AAV is AAV2/8 or AAV2/9, depending on the case, wherein the pseudotyped AAV is AAV2/8. A method of treating X-linked microtubular myopathy (XLMTM) in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the AAV vector of any one of claims 1 to 42. The method of claim 43, wherein the patient is five years of age or younger at the time of administration of the AAV vector. The method of claim 44, wherein the patient is four years of age or younger at the time of administration of the AAV vector, optionally wherein the patient is three years of age or younger, two years of age or younger, one year of age or younger, or six months of age or younger. The method of any one of claims 43 to 45, wherein the AAV vector is administered to the patient in an amount of less than 3 x 10 ¹⁴ vg/kg. The method of claim 46, wherein the AAV vector is administered to the patient in an amount of less than 2.5 x 10 ¹⁴ vg/kg, optionally wherein the AAV vector is administered to the patient in an amount of less than 2 x 10 ¹⁴ vg/kg, less than 1.5 x 10 ¹⁴ vg/kg, or less than 1.4 x 10 ¹⁴ vg/kg. The method of any one of claims 43 to 45, wherein the AAV vector is administered to the patient in an amount of 3 x 10 ¹³ vg/kg to 2.3 x 10 ¹⁴ vg/kg, optionally wherein the AAV vector is administered to the patient in an amount of 8 x 10 ¹³ vg/kg to 1.8 x 10 ¹⁴ vg/kg, 1 x 10 ¹⁴ vg/kg to 1.6 x 10 ¹⁴ vg/kg, 1.1 x 10 ¹⁴ vg/kg to 1.5 x 10 ¹⁴ vg/kg, or 1.2 x 10 ¹⁴ vg/kg to 1.4 x 10 ¹⁴ vg/kg. The method of any one of claims 43 to 48, wherein the AAV vector is administered to the patient in an amount of about 1.3 x 10 ¹⁴ vg/kg. The method of any one of claims 43 to 49, wherein the AAV vector is administered to the patient by intravenous, intramuscular, intrahepatic, intradermal, or subcutaneous administration. The method of any one of claims 43 to 50, wherein the patient is further administered an anticholestatic agent. The method of claim 51, wherein the anticholestatic agent is selected from the group consisting of bile acid, farnesoid X receptor (FXR) ligand, fibroblast growth factor 19 (FGF-19) mimetic, Takeda-G protein receptor 5 (TGR5) agonist, peroxisome proliferator-activated receptor (PPAR) agonist, PPAR-α agonist, PPAR-δ agonist, dual PPAR-α and PPAR-δ agonist, apical sodium-dependent bile acid transporter (ASBT) inhibitor, immunomodulatory drug, antifibrotic chemotherapy and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor. The method of claim 52, wherein: (i) the FXR ligand is obeticholic acid, cilofexor, tropifexor, tretinoin, or EDP-305; (ii) the FGF-19 mimetic is aldafermin; (iii) the TGR5 agonist is INT-777 or INT-767; (iv) the PPAR agonist is bezafibrate, seladelpar, or elafibrinor; (v) the PPAR-α agonist is fenofibrate; (vi) the PPAR-δ agonist is seladelpar; (vii) The dual PPAR-α and PPAR-δ agonist is elafibranor; (viii) the ASBT inhibitor is odevixibat, maralixibat, or linerixibat; (ix) the immunomodulatory drug is rituximab, abatacept, ustekinumab, infliximab, baricitinib, or FFP-104; (x) the antifibrotic chemotherapy is a vitamin D receptor agonist or simtuzumab; and/or (xi) the NOX inhibitor is setanaxib. The method of claim 52, wherein the bile acid is ursodeoxycholic acid, norursodeoxycholic acid, or a pharmaceutically acceptable salt thereof. The method of any one of claims 43 to 54, wherein the patient has no history of cholestasis or hyperbilirubinemia. The method of any one of claims 43 to 55, wherein the patient has no history of any underlying liver disease. A method as in any of claims 43 to 56, wherein the method comprises administering to a patient a therapeutically effective amount of: (i) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in liver tissue, and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in muscle tissue. The method of claim 57, wherein the promoter active in liver tissue comprises the LP1 promoter. The method of claim 58, wherein the LP1 promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 3. The method of claim 59, wherein the LP1 promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 3, optionally wherein the LP1 promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 3. The method of claim 60, wherein the LP1 promoter comprises the nucleic acid sequence of SEQ ID NO: 3. The method of any one of claims 57 to 61, wherein the promoter active in liver tissue comprises an ApoE promoter. The method of claim 62, wherein the ApoE promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 8. The method of claim 63, wherein the ApoE promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 8, optionally wherein the ApoE promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 8. The method of claim 64, wherein the ApoE promoter comprises the nucleic acid sequence of SEQ ID NO: 8. The method of any one of claims 57 to 65, wherein the promoter active in liver tissue comprises an A1AT promoter. The method of claim 66, wherein the A1AT promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 9. The method of claim 67, wherein the A1AT promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 9, optionally wherein the A1AT promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 9. The method of claim 68, wherein the A1AT promoter comprises the nucleic acid sequence of SEQ ID NO: 9. The method of any one of claims 57 to 69, wherein the promoter active in liver tissue is a chimeric promoter comprising an ApoE promoter and an A1AT promoter. The method of claim 70, wherein the chimeric promoter comprises a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 2. The method of claim 71, wherein the chimeric promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 2, optionally wherein the chimeric promoter comprises a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 2. The method of claim 72, wherein the chimeric promoter comprises the nucleic acid sequence of SEQ ID NO: 2. The method of any one of claims 57 to 73, wherein the promoter active in muscle tissue comprises the MCK promoter or the DES promoter. A method for treating XLMTM in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of: (i) a non-viral composition comprising a nucleic acid encoding MTM1 operably linked to a promoter that is active (e.g., selectively active) in liver tissue, and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in muscle tissue. The method of claim 75, wherein the non-viral composition is a liposome, a vesicle, a synthetic vesicle, an exosome, a synthetic exosome, a dendrimer, or a nanoparticle. The method of claim 76, wherein the nanoparticles are lipid nanoparticles. A method as in any of claims 75 to 77, wherein the initiator is a DES initiator. A method as claimed in any one of claims 75 to 78, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise: a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 6; or a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 7. The method of claim 79, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise: a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 6, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; ID NO: 6 nucleic acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; or a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO: 7, as the case may be, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise a nucleic acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 7. The method of claim 80, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each comprise: The nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or The nucleic acid sequence of SEQ ID NO: 6; or The nucleic acid sequence of SEQ ID NO: 7. The method of any one of claims 75 to 81, wherein the AAV vector is resamirigene bilparvovec. A kit comprising the AAV vector of any one of claims 1 to 42, wherein the kit further comprises a packaging insert instructing a user to administer the AAV vector to a patient diagnosed with XLMTM. A kit comprising: (i) a non-viral composition comprising a nucleic acid encoding MTM1, and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active (e.g., selectively active) in muscle tissue, wherein the kit further comprises a packaging insert instructing a user to administer the non-viral composition and the AAV vector to a patient diagnosed with XLMTM. A kit as in claim 84, wherein the non-viral composition is a liposome, a vesicle, a synthetic vesicle, an exosome, a synthetic exosome, a dendrimer or a nanoparticle. The kit of claim 85, wherein the nanoparticles are lipid nanoparticles. A set as in any of claims 84 to 86, wherein the initiator is a DES initiator. A kit as claimed in any one of claims 84 to 87, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise: a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 6; or a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence of SEQ ID NO: 1. The kit of claim 88, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise: a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1, optionally wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; 6, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 6; or a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 7, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each independently comprise a nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 6; or 7. A nucleic acid sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of 7. The kit of claim 89, wherein the nucleic acid encoding MTM1 and the transgenic gene encoding MTM1 each comprise: The nucleic acid sequence of residues 4927-6748 of SEQ ID NO: 1; or The nucleic acid sequence of SEQ ID NO: 6; or The nucleic acid sequence of SEQ ID NO: 7. The kit of any one of claims 84 to 90, wherein the AAV vector is birelutin.

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