Compositions and methods for the treatment of neuromuscular disorders

Abstract

The present disclosure relates to adeno-associated virus (AAV)-mediated delivery of nucleic acids to treat neuromuscular disorders, such as X-linked myotubular myopathy (XLM™), in patients in need thereof. The AAV vectors of the present disclosure may include, for example, a transgene encoding a myotubularin protein operably linked to one or more transcriptional regulators. In certain embodiments, the one or more transcriptional regulators are specifically active in muscle and / or liver tissue.

Description

[Technical Field]

[0001] SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. Said XML copy, created on September 14, 2023, is named "51037-075WO2_Sequence_Listing_9_14_23" and is 27,146 bytes in size. [Background technology]

[0002] X-linked myotubular myopathy (XLMTM) is a fatal monogenic disorder of the skeletal musculature resulting from mutations in the myotubularin 1 (MTM1) gene. Approximately 1 in 50,000 newborns has XLMTM and primarily presents with marked hypotonia and respiratory failure. In extremely rare cases, females may develop a severe form of XLMTM. To survive beyond the postnatal period, intensive support is required, including respiratory support (i.e., mechanical ventilation) at birth in 85–90% of patients, ongoing 24-hour mechanical ventilation in almost 50% of patients, and tracheostomy in approximately 60% of patients. Until recently, only supportive treatment options, such as mechanical ventilation or feeding tubes, were available. Summary of the Invention [Problem to be solved by the invention]

[0003] Recently, gene therapy approaches involving delivery of MTM1 have been developed for the treatment of XLMTM. However, there is a need in the art for improved methods of administering gene therapy to patients with XLMTM. [Means for solving the problem]

[0004] The present disclosure relates to compositions and methods useful for treating neuromuscular disorders, particularly X-linked myotubular myopathy (XLMTM). Using the compositions and methods described herein, a patient (e.g., a human patient) suffering from XLMTM can be administered a viral vector (e.g., an adeno-associated viral (AAV) vector) carrying a transgene encoding myotubularin 1 (MTM1). Exemplary compositions and methods of the present disclosure are described below.

[0005] 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.

[0006] In some embodiments, the promoter is selectively active in liver tissue. In some embodiments, when the vector is contacted separately with one or more liver cells and one or more non-liver cells under comparable conditions (e.g., in vitro or in vivo), the vector causes a level of transgene expression in one or more liver cells that is higher than the level of transgene expression in the one or more non-liver 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 transgene expression in the one or more non-liver cells). In some embodiments, when the vector is separately contacted with one or more hepatocytes and one or more non-hepatocytes, the vector results in a level of transgene expression in the one or more hepatocytes that is 10-fold to 1,000-fold higher than the level of transgene expression in the one or more non-hepatocytes. In some embodiments, when the vector is separately contacted with one or more hepatocytes and one or more non-hepatocytes, the vector results in a level of transgene expression in the one or more hepatocytes that is 50-fold to 1,000-fold higher than the level of transgene expression in the one or more non-hepatocytes. In some embodiments, when the vector is separately contacted with one or more hepatocytes and one or more non-hepatocytes, the vector results in a level of transgene expression in the one or more hepatocytes that is 100-fold to 1,000-fold higher than the level of transgene expression in the one or more non-hepatocytes. In one embodiment, when the vector is separately contacted with one or more hepatocytes and one or more non-hepatocytes, the vector results in an expression level of the transgene in the one or more hepatocytes 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 transgene in the one or more non-hepatocytes.

[0007] In some embodiments, the non-hepatic cells are muscle cells or nerve cells. In some embodiments, the non-hepatic cells are cardiac cells. In certain embodiments, upon contacting the vector with one or more hepatocytes (e.g., from a patient suffering from a neuromuscular disorder), the vector increases the level of transgene expression in the one or more hepatocytes to more closely match the expression of MTM1 in wild-type / healthy hepatocytes. For example, transgene expression can be 0.2- to 10-fold (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-fold) the level of MTM1 expression in wild-type / healthy hepatocytes. In one embodiment, when the vector is contacted with one or more hepatocytes (e.g., of a patient with a neuromuscular disorder), the vector results in an expression level of the transgene in the one or more hepatocytes that is at least 0.2-fold, at least 0.5-fold, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold the wild-type MTM1 expression level in healthy hepatocytes.

[0008] In one embodiment, the promoter comprises the LP1 promoter. In one embodiment, the LP1 promoter comprises the following SEQ ID NO:3:

[0009] [ka] There is a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence represented by: In one embodiment, the LP1 promoter comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 3 (e.g., 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 set forth in SEQ ID NO: 3). In one embodiment, the LP1 promoter comprises a nucleic acid sequence set forth in SEQ ID NO: 3.

[0010] In one embodiment, the promoter comprises an apolipoprotein E (ApoE) promoter. In one embodiment, the ApoE promoter comprises the following sequence: SEQ ID NO: 8:

[0011] [ka] There is a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence represented by: In one embodiment, the ApoE promoter has a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 8 (e.g., 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 set forth in SEQ ID NO: 8). In one embodiment, the ApoE promoter has a nucleic acid sequence set forth in SEQ ID NO:8.

[0012] In one embodiment, the promoter comprises an alpha-1-antitrypsin (A1AT) promoter. In one embodiment, the A1AT promoter comprises the following sequence: SEQ ID NO: 9:

[0013] [ka] There is a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence represented by: In one embodiment, the A1AT promoter has a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:9 (e.g., 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 set forth in SEQ ID NO:9). In one embodiment, the A1AT promoter has a nucleic acid sequence set forth in SEQ ID NO:9.

[0014] In one embodiment, the promoter is a chimeric promoter comprising an ApoE promoter and an A1AT promoter. In one embodiment, the chimeric promoter comprises the following sequence: SEQ ID NO:2:

[0015] [ka] There is a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence represented by: In some embodiments, the chimeric promoter comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in 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 comprises a nucleic acid sequence set forth in SEQ ID NO:2.

[0016] In some embodiments, the promoter comprises a constitutive promoter, ie, a phosphoglycerate kinase (PGK) promoter, an elongation factor-1α (EF 1α) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a cytomegalovirus (CMV) promoter, or a chicken β-actin (CBA) promoter.

[0017] 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.

[0018] In some embodiments, a promoter selectively active in muscle tissue achieves an expression level of the MTM1 transgene in muscle cells (e.g., in vitro or in vivo) that is higher than the expression level 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 expression level 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 the MTM1 transgene in muscle cells that is 10-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 results in expression levels of the MTM1 transgene in muscle cells that are 50- to 1,000-fold higher than the expression levels of the MTM1 transgene driven by the same promoter in non-muscle cells. In some embodiments, a promoter that is selectively active in muscle tissue results in expression levels of the MTM1 transgene in muscle cells that are 100- to 1,000-fold higher than the expression levels of the MTM1 transgene driven by the same promoter in non-muscle cells. In some embodiments, a promoter that is selectively active in muscle tissue results in expression levels of the MTM1 transgene in muscle cells that are 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 levels of the MTM1 transgene driven by the same promoter in non-muscle cells.

[0019] In certain embodiments, upon contacting the vector with one or more hepatocytes (e.g., of a patient suffering from a neuromuscular disorder), the vector results in transgene expression levels in the one or more hepatocytes that more closely resemble MTM1 expression in wild-type / healthy hepatocytes, e.g., transgene expression can be 0.2-fold to 10-fold (e.g., 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold) the MTM1 expression level in wild-type / healthy hepatocytes. In one embodiment, when the vector is contacted with one or more hepatocytes (e.g., of a patient with a neuromuscular disorder), the vector results in an expression level of the transgene in the one or more hepatocytes that is at least 0.2-fold, at least 0.5-fold, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold the wild-type MTM1 expression level in healthy hepatocytes.

[0020] In one embodiment, the second transgene encoding MTM1 is operably linked to a muscle creatine kinase (MCK) promoter or a desmin (DES) promoter. In some embodiments, the transgenes encoding MTM1 each independently have a nucleic acid sequence at least 75% identical to the nucleic acid sequence represented by residues 4927-6748 in SEQ ID NO:1. In some embodiments, the transgenes encoding MTM1 each independently have a nucleic acid sequence at least 80% identical to the nucleic acid sequence represented by residues 4927-6748 in SEQ ID NO:1. (e.g., 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 represented by residues 4927-6748 in SEQ ID NO:1.) In some embodiments, the transgenes encoding MTM1 each independently have a nucleic acid sequence represented by residues 4927-6748 in SEQ ID NO:1.

[0021] In some embodiments, the transgenes encoding MTM1 each independently have a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments, the transgenes encoding MTM1 each independently have a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in 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 set forth in SEQ ID NO: 6). In some embodiments, the transgenes encoding MTM1 each have a nucleic acid sequence set forth in SEQ ID NO: 6.

[0022] In some embodiments, the transgenes encoding MTM1 each independently have a nucleic acid sequence that is at least 75% identical to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the transgenes encoding MTM1 each independently have a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in 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 set forth in SEQ ID NO: 7). In some embodiments, the transgenes encoding MTM1 each have a nucleic acid sequence set forth in SEQ ID NO: 7. In one embodiment, the MTM1 sequence is codon optimized.

[0023] In some embodiments, the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh 10, or AAVrh74 serotype. In some embodiments, the AAV vector is a pseudotyped AAV. In some embodiments, the pseudotyped AAV is AAV2 / 8 or AAV2 / 9.

[0024] In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of an AAV vector of any of the above aspects or embodiments of the disclosure.

[0025] In some embodiments, the patient is 5 years old or younger at the time of administration of the AAV vector. In some embodiments, the patient is 4 years old or younger at the time of administration of the AAV vector, and optionally the patient is 3 years old or younger, 2 years old or younger, 1 year old or younger, or 6 months old or younger.

[0026] In one embodiment, the AAV vector is 3×10 14 In one embodiment, the AAV vector is administered to a patient in an amount of less than 2.5 x 10 vg / kg. 14 vg / kg, and in some cases, the AAV vector is administered to the patient in an amount of less than 2 x 10 14 vg / kg, less than 1.5 × 10 14 vg / kg or less than 1.4 × 10 14 In one embodiment, the AAV vector is administered to a patient in an amount of less than 3×10 vg / kg. 13 vg / kg~2.3×10 14 vg / kg, and in some cases, the AAV vector is administered to the patient in an amount of 8×10 13 vg / kg~1.8×10 14 vg / kg, 1 × 10 14 vg / kg~1.6×10 14 vg / kg, 1.1 × 10 14 vg / kg~1.5×10 14 vg / kg, or 1.2 × 10 14 vg / kg~1.4×10 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.3 x 10 vg / kg. 14 It is administered to patients in an amount of vg / kg. In one embodiment, the AAV vector is administered to the patient by intravenous, intramuscular, intrahepatic, intradermal, or subcutaneous administration.

[0027] In some embodiments, the patient is further administered an anti-choleretic agent selected from the group consisting of bile acids, 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 agents, anti-fibrotic therapies, and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitors. In certain 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 PPAR-α and PPAR-δ dual agonist is elafibranor; (viii) the ASBT inhibitor is odevixibat, maralixibat, or linerixibat; (ix) the immunomodulatory agent is rituximab, abatacept, ustekinumab, infliximab, baricitinib, or FFP-104; (x) the anti-fibrotic therapy is a vitamin D receptor agonist or sintuzumab; and / or (xi) the NOX inhibitor is setanaxib.

[0028] In certain 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, hi some embodiments, the patient has no history of any underlying liver disease.

[0029] In one embodiment, the method includes 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 or selectively active in liver tissue, and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active or selectively active in muscle tissue. In one embodiment, the promoter active in liver tissue comprises the LP1 promoter. In one embodiment, the LP1 promoter has a nucleic acid sequence at least 75% identical to the nucleic acid sequence set forth in SEQ ID NO:3. In one embodiment, the LP1 promoter has a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:3 (e.g., 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 set forth in SEQ ID NO:3). In one embodiment, the LP1 promoter has the nucleic acid sequence set forth in SEQ ID NO:3.

[0030] In some embodiments, the promoter active in liver tissue comprises an ApoE promoter. In some embodiments, the ApoE promoter has a nucleic acid sequence at least 75% identical to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the ApoE promoter has a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 8 (e.g., 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 set forth in SEQ ID NO: 8). In some embodiments, the ApoE promoter has a nucleic acid sequence set forth in SEQ ID NO: 8.

[0031] In some embodiments, the promoter active in liver tissue comprises the A1AT promoter. In some embodiments, the A1AT promoter has a nucleic acid sequence at least 75% identical to the nucleic acid sequence set forth in SEQ ID NO:9. In some embodiments, the A1AT promoter has a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:9 (e.g., 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 set forth in SEQ ID NO:9). In some embodiments, the A1AT promoter has the nucleic acid sequence set forth in SEQ ID NO:9.

[0032] In one embodiment, the promoter active in liver tissue is a chimeric promoter comprising an ApoE promoter and an A1AT promoter. In one embodiment, the chimeric promoter comprises a nucleic acid sequence at least 75% identical to the nucleic acid sequence set forth in SEQ ID NO:2. In one embodiment, the chimeric promoter comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:2 (e.g., 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 set forth in SEQ ID NO:2). In one embodiment, the chimeric promoter comprises the nucleic acid sequence set forth in SEQ ID NO:2.

[0033] In a further aspect, the present invention relates to a method of treating XLMTM in a human patient in need thereof, comprising administering to the patient therapeutically effective amounts of (i) a non-viral composition comprising a nucleic acid encoding MTM1 operably linked to a promoter that is active or selectively active in liver tissue, and (ii) an AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active or 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 capable of delivering a DNA construct similar in composition and size to that delivered via an AAV capsid.

[0034] In some embodiments, the promoter is a DES promoter. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence at least 75% identical to the nucleic acid sequence represented by residues 4927-6748 in SEQ ID NO:1. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have a nucleic acid sequence at least 80% identical to the nucleic acid sequence represented by residues 4927-6748 in SEQ ID NO:1. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently have 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 represented by residues 4927-6748 in SEQ ID NO:1. In one embodiment, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each independently comprise the nucleic acid sequence represented by residues 4927-6748 in SEQ ID NO:1.

[0035] 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 set forth in 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 set forth in 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 set forth in SEQ ID NO: 6. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each have a nucleic acid sequence set forth in SEQ ID NO: 6.

[0036] 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 set forth in 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 set forth in 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 set forth in SEQ ID NO: 7. In some embodiments, the nucleic acid encoding MTM1 and the transgene encoding MTM1 each have a nucleic acid sequence set forth in SEQ ID NO: 7.

[0037] In one embodiment, the AAV vector is resamirigene bilparvovec.

[0038] In a further aspect, the present disclosure provides a kit comprising the AAV vector of any of the above aspects or embodiments of the present disclosure, and optionally further comprising a package insert instructing a user to administer the non-viral composition and the AAV vector to a patient diagnosed with XLMTM.

[0039] In a further aspect, the disclosure provides a kit comprising (i) a non-viral composition comprising a nucleic acid encoding MTM1 (e.g., a non-viral composition described above), and (ii) an AAV vector (e.g., an AAV vector described above) comprising a transgene encoding MTM1 under the control of a promoter active (e.g., selectively active) in muscle tissue. The kit may further include a package insert instructing a user to administer the non-viral composition and the AAV vector to a patient diagnosed with XLMTM. [Brief explanation of the drawings]

[0040] [Figure 1] 1 is a schematic diagram of an exemplary adeno-associated virus (AAV) vector (e.g., a pseudotyped AAV vector, e.g., an AAV vector comprising AAV2 inverted terminal repeats housed within an AAV8-derived capsid protein) for expression of the human or mouse myotubularin 1 (MTM1) gene. From left to right, the shaded arrow and rectangle represent a nucleic acid sequence encoding a beta-globin intron (b-globin intron), the human or mouse MTM1 gene, an SV40 polyadenylation signal (SV40-polyA), and the ApoE / A1AT liver-specific promoter (ApoE / A1AT) operably linked to adjacent inverted terminal repeats (ITRs). [Figure 2]Schematic diagram of an exemplary AAV vector (e.g., a pseudotyped AAV vector, such as an AAV vector comprising AAV2 inverted terminal repeats housed within an AAV8-derived capsid protein) for expression of the human or mouse myotubularin 1 (MTM1) gene. From left to right, the shaded arrow and rectangle represent the nucleic acid sequence encoding the LP1 liver-specific promoter (LP1 promoter) operably linked to an SV40 intron, the human or mouse MTM1 gene, an SV40 polyadenylation signal (SV40-polyA), and adjacent inverted terminal repeats (ITRs). [Figure 3] 1 is a schematic diagram of an exemplary AAV vector (e.g., a pseudotyped AAV vector (e.g., an AAV vector comprising AAV2 inverted terminal repeats housed within an AAV8-derived capsid protein)) for expression of the human myotubularin 1 (hMTM1) gene. From left to right, the shaded arrow and rectangle indicate nucleic acid sequences encoding a muscle-specific promoter (e.g., MCK, desmin) operably linked to hMTM1, 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). [Figure 4] Schematic diagram of an exemplary AAV vector (e.g., a pseudotyped AAV vector (e.g., an AAV vector comprising AAV2 inverted terminal repeats housed within an AAV8-derived capsid protein)) for expression of the human myotubularin 1 (hMTM1) gene. From left to right, the shaded arrow and rectangle represent nucleic acid sequences encoding a ubiquitous promoter (e.g., PGK, Ef1a, GAPDH), a polyadenylation signal (polyA), and flanking inverted terminal repeats (ITRs) operably linked to hMTM1. [Figure 5]1 is a graph showing body weight (g), mean±SEM (g), against time (age in weeks). n=10-14 (all data), measured by the method described in Example 1 below. Statistical significance: HEMIOAAV 8-Des-mMTM 1 mice vs. WT vehicle mice: p<0.001 at all time points; WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP mice vs. WT vehicle mice: p<0.05 at weeks 7–16; HEMIOAAV 8-Des-mMTM 1 + AAV8 empty capsid mice vs. HEMIOAAV 8-Des-mMTM 1 mice: p<0.05 at weeks 4–5; HEMIOAAV 8-Des-mMTM 1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice vs. HEMIOAAV 8-Des-mMTM 1 mice: p<0.05 at weeks 5–6 and 8–9. Abbreviations in Figure 5: WT, wild type; HEMi, hemizygous. [Figure 6] Figure 6A is a graph showing alkaline phosphatase (AFOS) (U / L) levels, mean + SEM (all n=10-14), measured as described in Example 1 below. No statistical differences were observed. Abbreviations in Figure 6A: WT, wild-type; HEMi, hemizygous; AFOS, alkaline phosphatase. Figure 6B is a graph showing alkaline phosphatase (AFOS) (U / L) levels (mean ± SEM (n=11)) in untreated mice, measured as described in Example 1 below. Figure 6B Abbreviations: AFOS, alkaline phosphatase. [Figure 7]Figure 7A is a graph showing alanine aminotransferase (ALAT) levels (U / L), mean + SEM (n = 0-7 for in-life samples (4-10 weeks): n = 10-14 for terminal samples (16 weeks): n = 10-14 for all), measured as described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Data are not shown for groups that fell below the limit of detection. Statistical difference: *p<0.05 compared to WT vehicle, #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 7A: WT, wild-type; HEMi, hemizygous; ALAT, alanine aminotransferase. Figure 7B is a graph showing alanine aminotransferase (ALAT) (U / L) levels (mean ± SEM (n=5-11)) in untreated mice, measured as described in Example 1 below. Figure 7B Abbreviations: ALAT, alanine aminotransferase. [Figure 8] Figure 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 difference: **p<0.01 compared to WT vehicle. Abbreviations for Figure 8A: WT, wild-type; HEMi, hemizygous; ASAT, aspartate aminotransferase. Figure 8B is a graph showing aspartate aminotransferase (ASAT) (U / L) levels (mean ± SEM (n=11)) in untreated mice, measured as described in Example 1 below. Figure 8B Abbreviations: ASAT, aspartate aminotransferase. [Figure 9]Figure 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. Abbreviations in Figure 9A: WT, wild-type; HEMi, hemizygous. Figure 9B is a graph showing albumin (g / L) levels, mean ± SEM (n=11), in untreated mice, measured as described in Example 1 below. [Figure 10] FIG. 10A is a graph showing gamma-glutamyltransferase (GGT) (U / L) levels (mean ± SEM (n=10-14 for all)) measured in the manner described in Example 1 below. Statistical difference: #p<0.05, ##p<0.01 compared to HEMO AAV8-Des-mMTM1. Abbreviations for FIG. 10A: WT, wild type; HEMi, hemizygous; GGT, gamma-glutamyltransferase. FIG. 10B is a graph showing gamma-glutamyltransferase (GGT) (U / L) levels (mean ± SEM (n=11)) in untreated mice measured in the manner described in Example 1 below. FIG. 10B Abbreviations: GGT, gamma-glutamyltransferase. [Figure 11] Figure 11A is a graph showing total protein (g / L) levels, mean ± SEM (n=10-14 for all), measured in the manner described in Example 1 below. No statistical differences were observed. Abbreviations for Figure 11A: WT, wild type; HEMi, hemizygous; Prottot, total protein. Figure 11B is a graph showing total protein (g / L) levels, mean ± SEM (n=11), in untreated mice, measured in the manner described in Example 1 below. Abbreviations for Figure 11B: Prottot, total protein. [Figure 12] Figure 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. Abbreviations in Figure 12A: WT, wild-type; HEMi, hemizygous. Figure 12B is a graph showing urea (mmol / L) levels (mean ± SEM (n=11)) in untreated mice, measured as described in Example 1 below. [Figure 13] Figure 13A is a graph showing lactate dehydrogenase (LDH) (U / I) levels, mean ± SEM (n=10-14 for all), measured in the manner described in Example 1 below. No statistical differences were observed. Abbreviations in Figure 13A: WT, wild-type; HEMi, hemizygous; LDH, lactate dehydrogenase. Figure 13B is a graph showing lactate dehydrogenase (LDH) (U / I) levels (mean ± SEM (n=11)) in untreated mice, measured in the manner described in Example 1 below. Figure 13B Abbreviations: LDH, lactate dehydrogenase. [Figure 14] Figure 14A is a graph showing bile acid levels (µmol / L), mean ± SEM (n=0-3 for all), measured as described in Example 1 below. Data are not shown for groups that fell below the limit of detection. Statistical difference: #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 14A: WT, wild-type; HEMi, hemizygous. Figure 14B is a graph showing bile acid levels (µmol / L), mean ± SEM (n=11), in untreated mice, measured as described in Example 1 below. [Figure 15] Figure 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 are not shown for groups that fell below the limit of detection. No statistical differences were observed. Abbreviations in Figure 15: WT, wild type; HEMi, hemizygous; BilTot, total bilirubin. [Figure 16] Figure 16A is a graph showing calculated globulin (g / L) levels, mean ± SEM (n=10-14 for all), measured in the manner described in Example 1 below. Statistical difference: *p<0.05 compared to WT vehicle. Abbreviations in Figure 16A: WT, wild-type; HEMi, hemizygous. Figure 16B is a graph showing calculated globulin (g / L) levels, mean ± SEM (n=11) in untreated mice, measured in the manner described in Example 1 below. [Figure 17]Figure 17A is a graph showing calculated albumin / globulin ratio levels, mean ± SEM (n = 10-14 for all), measured in the manner described in Example 1 below. No statistical differences were observed. Abbreviations in Figure 17A: WT, wild-type; HEMi, hemizygous. Figure 17B is a graph showing calculated albumin / globulin ratio levels, mean ± SEM (n = 11), in untreated mice, measured in the manner described in Example 1 below. [Figure 18] Figure 18A is a graph showing creatine kinase (U / L) levels, mean ± SEM (n=10-14 for all), measured in the manner described in Example 1 below. Statistical difference: *p<0.05 compared to WT vehicle. Abbreviations in Figure 18A: WT, wild type; HEMi, hemizygous; CK, creatine kinase. Figure 18B is a graph showing creatine kinase (U / L) levels (mean ± SEM (n=11)) in untreated mice, measured in the manner described in Example 1 below. Figure 18B Abbreviations: CK, creatine kinase. [Figure 19] Figure 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. Abbreviations in Figure 19A: WT, wild-type; HEMi, hemizygous. Figure 19B is a graph showing creatinine (umol / L) levels, mean ± SEM (n=11), in untreated mice, measured as described in Example 1 below. [Figure 20]FIG. 20A is a graph showing white blood cell counts (WBC) (×10 9 cells / L), mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: *p<0.05 compared to WT vehicle, #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 20A: WT, wild-type; HEMi, hemizygous; WBC, white blood cell count. Figure 20B is a graph showing white blood cell count (WBC) (x109 cells / L) levels, mean ± SEM (n=3 / 9), in untreated mice, measured in the manner described in Example 1 below. Abbreviations in Figure 20B: WBC, white blood cell count. [Figure 21]FIG. 21A is a graph showing red blood cell counts (RBCs) (×10 12 cells / L), mean ± SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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. Abbreviations in Figure 21A: WT, wild-type; HEMi, hemizygous; RBC, red blood cell count. Figure 21B is a graph showing red blood cell (RBC) count (x10 cells / L) levels, mean ± SEM (n=3 / 9), in untreated mice, measured in the manner described below in Example 1. Figure 21B Abbreviations: RBC, red blood cell count. [Figure 22]FIG. 22A is a graph showing hemoglobin (g / L) levels, mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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. Abbreviations in Figure 22A: WT, wild-type; HEMi, hemizygous; HGB, hemoglobin. Figure 22B is a graph showing hemoglobin (g / L) levels, mean ± SEM (n=3 / 9), in untreated mice, measured in the manner described below in Example 1. Figure 22B Abbreviations: HGB, hemoglobin. [Figure 23]FIG. 23A is a graph showing hematocrit (HCT) (%) levels, mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 23A: WT, wild type; HEMi, hemizygous; HCT, hematocrit. Figure 23B is a graph showing hematocrit (HCT) (%) levels in untreated mice, mean ± SEM (n=3 / 9), measured in the manner described in Example 1 below. Figure 23B Abbreviations: HCT, hematocrit. [Figure 24]FIG. 24A is a graph showing mean corpuscular volume (MCV) (fL) levels at mean + SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: *p<0.05 compared to WT vehicle, #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 24A: WT, wild type; HEMi, hemizygous; MCV, mean corpuscular volume. Figure 24B is a graph showing mean corpuscular volume (MCV) (fL) levels in untreated mice, mean ± SEM (n=3 / 9), measured in the manner described in Example 1 below. Abbreviations in Figure 24B: MCV, mean corpuscular volume. [Figure 25]FIG. 25A is a graph showing mean corpuscular hemoglobin (MCH) (pg) levels, mean + SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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. Abbreviations in Figure 25A: WT, wild-type; HEMi, hemizygous; MCH, mean corpuscular hemoglobin. Figure 25B is a graph showing mean corpuscular hemoglobin (MCH) (pg) levels, mean ± SEM (n=3 / 9), in untreated mice, measured in the manner described in Example 1 below. Figure 25B Abbreviations: MCH, mean corpuscular hemoglobin. [Figure 26]FIG. 26A is a graph showing mean corpuscular hemoglobin concentration (MCHC) (g / L) levels, mean + SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 26A: WT, wild-type; HEMi, hemizygous; MCHC, mean corpuscular hemoglobin concentration. Figure 26B is a graph showing mean corpuscular hemoglobin concentration (MCHC) (g / L) levels (mean + SEM (n=3 / 9)) in untreated mice, measured in the manner described in Example 1 below. Abbreviations in Figure 26B: MCHC, mean corpuscular hemoglobin concentration. [Figure 27]FIG. 27A is a graph showing platelet counts (×10 9 cells / L), mean±SEM (n=5-14 for all), measured as described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: *p<0.05 compared to WT vehicle. Abbreviations in Figure 27A: WT, wild type; HEMi, hemizygous; PLT, platelets or platelet count. Figure 27B is a graph showing platelet counts (x109 cells / L), mean ± SEM (n=3 / 9), in untreated mice, measured in the manner described in Example 1 below. Figure 27B Abbreviations: PLT, platelets or platelet count. [Figure 28]FIG. 28A is a graph showing relative neutrophil counts (%) neutrophils, mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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. Abbreviations in Figure 28A: WT, wild-type; HEMi, hemizygous; %NEUT, neutrophils (%). Figure 28B is a graph showing relative neutrophil counts (%) neutrophils in untreated mice, mean ± SEM (n=3 / 9), measured in the manner described in Example 1 below. Figure 28B Abbreviations: %NEUT, % neutrophils. [Figure 29]FIG. 29A is a graph showing absolute neutrophil counts (×10 9 cells / L), mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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. Abbreviations in Figure 29A: WT, wild type; HEMi, hemizygous; Abs / absolute-NEUT, absolute neutrophil count. Figure 29B is a graph showing absolute neutrophil counts (x109 cells / L), mean ± SEM (n=3 / 9), in untreated mice, measured as described in Example 1 below. Abbreviations in Figure 29B: Abs / absolute-NEUT, absolute neutrophil count. [Figure 30]FIG. 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 each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 30A: WT, wild type; HEMi, hemizygous; %LYM, lymphocytes (%). Figure 30B is a graph showing relative lymphocyte counts (% lymphocytes) in untreated mice, mean ± SEM (n=3 / 9), measured as described in Example 1 below. Figure 30B Abbreviations: %LYM, lymphocytes (%). [Figure 31]FIG. 31A is a graph showing absolute lymphocyte counts (×10 9 cells / L), mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: *p<0.05 compared to WT vehicle. Abbreviations for Figure 31A: WT, wild type; HEMi, hemizygous; Abs / AbsoluteLYMpHS, absolute lymphocyte count. Figure 31B is a graph showing absolute lymphocyte counts (x109 cells / L), mean ± SEM (n=3 / 9), in untreated mice, measured in the manner described in Example 1 below. Abbreviations for Figure 31B: Abs / AbsoluteLYMpHS, absolute lymphocyte count. [Figure 32]FIG. 32A is a graph showing relative monocyte counts (% monocytes), mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: *p<0.05 compared to WT vehicle. Abbreviations in Figure 32A: WT, wild type; HEMi, hemizygous; %MONO, monocytes (%). Figure 32B is a graph showing relative monocyte numbers (% monocytes) in untreated mice, mean ± SEM (n=3 / 9), measured as described in Example 1 below. Figure 32B Abbreviations: %MONO, monocytes (%). [Figure 33]FIG. 33A is a graph showing absolute monocyte counts (×10 9 cells / L), mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: *p<0.05 compared to WT vehicle. Abbreviations for Figure 33A: WT, wild type; HEMi, hemizygous; Abs / Absolute MONOS, absolute monocyte count. Figure 33B is a graph showing absolute monocyte counts (x109 cells / L), mean ± SEM (n=3 / 9), in untreated mice, measured in the manner described in Example 1 below. Abbreviations for Figure 33B: Abs / Absolute MONOS, absolute monocyte count. [Figure 34]FIG. 34A is a graph showing relative eosinophil counts (% eosinophils), mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: ##p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 34A: WT, wild type; HEMi, hemizygous; %EOS, eosinophils (%). Figure 34B is a graph showing relative eosinophil counts (% eosinophils) in untreated mice, mean ± SEM (n=3 / 9), measured as described in Example 1 below. Figure 34B Abbreviations: %EOS, eosinophils (%). [Figure 35]FIG. 35A is a graph showing absolute eosinophil counts (×10 9 cells / L), mean±SEM (n=5-14 for all), measured as described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 35A: WT, wild type; HEMi, hemizygous; Abs / absoluteEOS, absolute eosinophil count. Figure 35B is a graph showing absolute eosinophil counts (x109 cells / L) in untreated mice, mean±SEM (n=3 / 9), measured as described in Example 1 below. Abbreviations in Figure 35B: Abs / absoluteEOS, absolute eosinophil count. [Figure 36]FIG. 36A is a graph showing relative basophil counts (% basophils), mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Data are not shown for groups below the detection limit. Statistical difference: #p<0.05 compared to HEMO AAV8-Des-mMTM1. Abbreviations in Figure 36A: WT, wild type; HEMi, hemizygous; %BASO, basophils (%). Figure 36B is a graph showing relative basophil counts (% basophils) in untreated mice, mean ± SEM (n=3 / 9), measured as described in Example 1 below. Figure 36B Abbreviations: %BASO, basophils (%). [Figure 37]FIG. 37A is a graph showing absolute basophil counts (×10 9 cells / L), mean±SEM (n=5-14 for all), measured as described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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 are not shown for groups that fell below the limit of detection. Abbreviations in Figure 37A: WT, wild type; HEMi, hemizygous; Abs / absolute BASOS, absolute basophil count. Figure 37B is a graph showing absolute basophil counts (x109 cells / L), mean ± SEM (n=3 / 9), in untreated mice, measured as described in Example 1 below. Data are not shown for groups that fell below the limit of detection. Figure 37B Abbreviations: Abs / absolute BASOS, absolute basophil count. [Figure 38]Figure 38A is a graph showing the relative number of large unstained cells (%), mean ± SEM (n = 5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Statistical difference: **p<0.01 compared to WT vehicle. Abbreviations in Figure 38A: WT, wild type; HEMi, hemizygous; %LUC, large unstained cells (%). Figure 38B is a graph showing the relative number of large unstained cells (% large unstained cells) in untreated mice, mean ± SEM (n = 3 / 9), measured as described in Example 1 below. Figure 38B Abbreviations: %LUC, large unstained cells (%). [Figure 39]Figure 39A is a graph showing the absolute number, mean + SEM, of large unstained cells (x10 cells / L) (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP, and WT AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP. Data are not shown for groups that fell below the limit of detection. Statistical difference: #p<0.05, ##p<0.01 compared to HEMO AAV8-Des-mMTM1. Abbreviations for Figure 39A: WT, wild type; HEMi, hemizygous; Abs / Absolute LUCS, absolute number of large unstained cells. Figure 39B is a graph showing the absolute number of large unstained cells (x109 cells / L) in untreated mice, mean±SEM (n=3 / 9), measured as described in Example 1 below. Data are not shown for groups that fell below the limit of detection. Abbreviations for Figure 39B: Abs / Absolute LUCS, absolute number of large unstained cells. [Figure 40]FIG. 40A is a graph showing relative reticulocyte counts (%) reticulocytes, mean±SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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. Abbreviations in Figure 40A: WT, wild type; HEMi, hemizygous; %Retic, reticulocytes (%). Figure 40B is a graph showing relative reticulocyte counts (reticulocytes (%)), mean ± SEM (n=3 / 9), in untreated mice, measured as described in Example 1 below. Figure 40B Abbreviations: %Retic, reticulocytes (%). [Figure 41]Figure 41A is a graph showing absolute reticulocyte cell counts (x109 cells / L), mean ± SEM (n=5-14 for all), measured in the manner described in Example 1 below. At each time point, 4, 5, 6, 7, 10, and 16 weeks, the following groups were tested (from left to right): WT vehicle, HEMO AAV8-Des-mMTM1, HEMO AAV8-Des-mMTM1 + AAV8-Des-hMTM1-STOP, HEMO AAV8-Des-mMTM1 + AAV8-empty capsid, HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1, HEMO 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. Abbreviations in Figure 41A: WT, wild type; HEMi, hemizygous; Abs / Retic absolute, absolute number of reticulocyte cells. Figure 41B is a graph showing absolute reticulocyte cell counts (x109 cells / L) in untreated mice, mean ± SEM (n=3 / 9), measured as described in Example 1 below. Figure 41B Abbreviations: Abs / Retic absolute, absolute number of reticulocyte cells. DETAILED DESCRIPTION OF THE INVENTION

[0041] [Definition] As used herein, the term "about" refers to a value within 10% above or below the stated value. For example, "100 pounds" used in the context of masses described herein includes amounts within 10% above or below 100 lbs. Additionally, the term "about," when used in the context of a list of numerical quantities, when preceding a list of numerical quantities, should be understood to apply to each individual amount recited in the list.

[0042] As used herein, the terms "administering," "administration," and the like refer to providing 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) directly to a patient by any effective route. Exemplary administration routes are described herein and include, among others, systemic administration routes, such as intravenous injection, and direct administration routes into the patient's central nervous system, such as by intrathecal or intraventricular injection, and direct administration routes into the patient's liver.

[0043] As used herein, the term "age-adjusted standard" refers to the process of normalizing data by age, a technique used to compare populations of subjects even when the populations have different age profiles. As used herein, the term "standard" refers to data that has not undergone normalization by age, because the populations of subjects across age profiles are similar.

[0044] As used herein, the terms "alanine aminotransferase" and "ALT" refer to proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring wild-type ALT protein (e.g., ALT1 and ALT2), as well as proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring allelic variant of ALT (GPT or GPT2, e.g., a splice variant or allelic variant). The human GPT nucleic acid sequence is provided in NCBI Reference SEQ ID NO: NM_005309.2, and an exemplary wild-type ALT1 amino acid sequence is provided in NCBI Reference SEQ ID NO: NM_NP_005300.1. The human GPT2 nucleic acid sequence is provided in NCBI Reference SEQ ID NO: NM_001142466.2, and an exemplary wild-type ALT2 amino acid sequence is provided in NCBI Reference SEQ ID NO: NP_001135938.1.

[0045] As used herein, the terms "alkaline phosphatase" and "ASP" refer to proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring wild-type ASP protein, as well as proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring allelic variant (e.g., a splice variant or allelic variant) of ASP. A human ASP nucleic acid sequence is provided in NCBI Reference SEQ ID NO: NM_000478.5, and an exemplary wild-type ASP amino acid sequence is provided in NCBI Reference SEQ ID NO: NP_000469.3.

[0046] As used herein, the term "anti-choleretic agent" refers to a substance, such as a small molecule, that acts to enhance bile formation and / or antagonize the effects of hydrophobic bile acids on biological membranes. The term "antagonize," as used herein with respect to a protein, reduces signaling resulting from the interaction of the protein with one or more of its binding partners. An antagonist may reduce binding of a protein to one or more of its binding partners compared to binding of the two proteins in the absence of the antagonist.

[0047] As used herein, the terms "aspartate aminotransferase" and "AST" refer to proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring wild-type AST protein, as well as proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring allelic variant (e.g., a splice variant or allelic variant) of AST. The human AST nucleic acid sequence is provided in NCBI Reference SEQ ID NO: NM_002079.2, and an exemplary wild-type AST amino acid sequence is provided in NCBI Reference SEQ ID NO: NP_002070.1.

[0048] 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 taken for baseline, followed by a meal, and a postprandial (i.e., after eating) blood sample is taken approximately two hours later. Both blood samples are tested for bile acid levels, with the preprandial sample used as a reference. As used herein, the term "bile acid" refers to steroid acids found primarily in the bile of mammals and other vertebrates.

[0049] As used herein, the terms "Children's Hospital of Philadelphia Infant Study of Neuromuscular Disorders" and "CHOP INTENT" refer to a validated motor outcome measure developed for the evaluation of frail infants, such as those with skeletal muscle diseases (e.g., X-linked myotubular myopathy (XLM™)). CHOP INTENT uses a 0.64-point scale, with higher scores indicating better motor function. As used herein, the term "motor function score" refers to a score on the CHOP INTENT 0.64-point scale (e.g., a CHOP INTENT scale of >45).

[0050] The term "cholestasis" as used herein refers to a condition in which bile cannot flow from the liver to the duodenum. Two clinical distinctions are "obstructive" cholestasis, in which there is a mechanical blockage in the duct system, which may result from gallstones or malignant tumors, and "metabolic" cholestasis, which is a disorder in bile formation, which may occur due to genetic defects or may be acquired as a side effect of many medications. The term "bile" as used herein refers to the digestive fluid secreted by the liver to aid in the digestion of fats.

[0051] As used herein, the term "combination therapy" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition (e.g., a neuromuscular disorder). In certain embodiments, "combination therapy" can include procedures. The treatment regimen specifies the dosage and periodicity of administration of each agent so that the effects of the individual agents on the subject overlap. In certain embodiments, delivery of two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In other embodiments, two or more agents are not co-formulated but are administered in a sequential manner as part of a prescribed regimen. In certain embodiments, the combined administration of two or more agents or treatments results in a reduction in symptoms or other parameters associated with a disorder that is greater than that observed with one agent or treatment delivered alone or in the absence of the other. 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 can be effected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, intrahepatic, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same or different routes. For example, a first therapeutic agent in the combination can be administered by intravenous injection, while a second therapeutic agent in the combination can be administered enterally. In another example, therapeutic combination agents can be administered by intravenous injection, and a therapeutic combination treatment (e.g., nasobiliary drainage (NBD)) can be performed.

[0052] As used herein, the term "dose" refers to the amount of a therapeutic agent, such as a viral vector described herein, administered to a subject at a particular time for the treatment of a disorder, such as to treat or ameliorate one or more symptoms of a neuromuscular disorder (e.g., XLMTM) described herein. The therapeutic agents described herein may be administered in a single dose or in multiple doses over the course of a treatment period, as defined herein. In each case, the therapeutic agent may be administered using one or more unit dosage forms of the therapeutic agent, and the term refers to one or more separate compositions comprising the therapeutic agent that collectively constitute a single dose of the therapeutic agent.

[0053] As used herein, the terms "effective amount," "therapeutically effective amount," and the like, when used in reference to a therapeutic composition, such as a vector construct described herein, refer to an amount that, when administered to a subject, including a mammal, e.g., a human, is effective to produce a therapeutically effective amount. "Effective amount" refers to an amount sufficient to produce a beneficial or desired result, such as a clinical result. For example, in the context of treating a neuromuscular disorder such as XLMTM, the term refers to an amount of a composition sufficient to achieve a therapeutic response compared to the response obtained without administration of the composition of interest. An "effective amount," "therapeutically effective amount," etc. of a composition, such as a vector construct of the present disclosure, also includes an amount that produces a beneficial or desired result in a subject compared to a control.

[0054] As used herein, the terms "gamma-glutamyltransferase" and "GGT" refer to proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring wild-type GGT protein, as well as proteins whose amino acid sequence comprises or consists of the amino acid sequence of a naturally occurring allelic variant of GGT (GGT1, GGT2, and GGT3, e.g., a splice variant or allelic variant). The human GGT1 nucleic acid sequence is provided in NCBI Reference SEQ ID NO: NM_001288833.1, and an exemplary wild-type GGT1 amino acid sequence is provided in NCBI Reference SEQ ID NO: NP_001275762.1.

[0055] 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 vertebrates in the normal catabolic pathway that breaks down heme. This catabolism is a process necessary for the body's clearance of waste products resulting from the destruction of aging or abnormal red blood cells. As used herein, the term "bilirubin test" refers to the measurement of the amount of bilirubin in a patient's blood.

[0056] As used herein, the term "level" refers to the level of a protein compared to a reference. The reference can be any useful reference as defined herein. "Decreased levels" and "enhanced (enhanced) levels" of a protein refer to a decrease or increase in protein levels compared to a reference (e.g., 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 decrease or increase); ...300%, about 400%, about 500% or more decrease or increase); a decrease or increase in protein levels compared to a reference (e.g., about 5%, about 10%, about 15%, about 20%, about 300%, about 400%, about 500% or more decrease or increase); a decrease or increase in protein levels compared to a reference (e.g., about 5%, about 10%, about 1 "A decrease or enhancement of greater than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%; a decrease or enhancement of about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase of about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold, about 1000-fold, or more. Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, or ng / mL) or as a percentage of total protein in the sample.

[0057] As used herein, the terms "liver function tests" and "LFTs" refer to a liver panel (e.g., a group of blood tests that provide information about the status of a patient's liver). A liver panel may include measurements of gamma glutamyltransferase levels, alkaline phosphatase levels, aspartate aminotransferase levels, alanine aminotransferase levels, albumin levels, bilirubin levels, prothrombin time, activated partial thromboplastin time, or a combination thereof.

[0058] As used herein, the terms "maximum inspiratory pressure" and "MIP" refer to variables in mechanical ventilation that include the sum of delivered airway pressures, typically used to overcome both respiratory system compliance and airway resistance. In pressure-controlled modes, MIP includes the sum of positive end-expiratory pressure and "delta pressure." As used herein, the term "delta pressure" refers to a variable in mechanical ventilation that includes the difference between MIP and positive end-expiratory pressure.

[0059] As used herein, the term "mechanical ventilatory support" is the medical term for artificial ventilation in which mechanical means assist or replace spontaneous breathing. As used herein, the term "invasive mechanical ventilatory support" is the medical term for artificial ventilation in which air is delivered via a tube inserted into the patient's trachea through the mouth or nose and mechanical means are 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 may be placed over the mouth, nose, or entire face.

[0060] As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, positioned so that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single, contiguous molecule, and may or may not be contiguous. For example, a promoter is operably linked to a transcribable polynucleotide molecule.

[0061] A promoter regulates the transcription of a transcribable polynucleotide molecule of interest in a cell. Furthermore, two parts of a transcriptional regulatory element are operably linked to each other if they are linked in such a way that the transcriptional activation functionality of one part is not adversely affected by the presence of the other part. Two transcriptional regulatory elements may be operably linked to each other by a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without any intervening nucleotides.

[0062] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic compounds that is administered to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition that affects or may affect the subject. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response and other problematic complications, commensurate with a reasonable benefit / risk ratio.

[0063] As used herein, the term "promoter" refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the transgene. Exemplary promoters that may be used in conjunction 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, regarding nucleic acid regulatory elements, is incorporated herein by reference. Additionally, the term "promoter" may refer to a synthetic promoter. A promoter is a regulatory DNA sequence that does not naturally occur in a biological system. Synthetic promoters contain portions of a naturally occurring promoter combined with a non-naturally occurring polynucleotide sequence and can be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types. As used herein, the term "selectively active" in the context of promoters refers to a promoter that preferentially drives gene expression in one cell or tissue compared to other cells or tissues. For example, a promoter that is "selectively active" in hepatocytes may achieve a level of gene expression in hepatocytes that is higher, e.g., 2- to 1,000-fold higher, than the level of gene expression achieved by the same promoter under substantially the same conditions in one or more non-hepatic cells (e.g., 2-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or 1,000-fold higher expression). Exemplary methods for measuring gene expression are known in the art and described herein.

[0064] As used herein, a therapeutic agent is considered to be "provided" to a patient when the patient is undergoing treatment. When administered to a patient, a Therapeutic Agent may be a directly administered Therapeutic Agent, or may be a substance that is processed or metabolized in vivo to endogenously yield the Therapeutic Agent. For example, a patient suffering from a neuromuscular disorder described herein, or the like, can be provided with a nucleic acid molecule encoding a Therapeutic protein (e.g., MTM1) by direct administration of the nucleic acid molecule or by administration of a substance (e.g., a viral vector or cells) that is processed in vivo to yield the desired nucleic acid molecule. As used herein, the terms "patient" and "subject" refer to an organism being treated for a particular disease or condition described herein (such as a neuromuscular disorder, e.g., XLMTM). Examples of subjects and patients include mammals, such as humans, being treated for a disease or condition described herein.

[0065] "Reference" refers to any useful reference used to compare 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 reference standard or level. A "reference sample" can be, for example, a control, e.g., 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 normal cells or normal tissue; a sample (e.g., cells or tissue) from a subject free of 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 treated for cholestasis, hyperbilirubinemia, or one or more symptoms thereof; or a sample of a known normal concentration of purified protein (e.g., any of those described herein). "Reference standard or level" refers to a value or number obtained from a reference sample. A "normal control value" is a predetermined value indicative of a non-disease state, e.g., a value expected in a healthy control subject. Normal control values ​​are usually expressed as a range ("X to Y"), a high threshold ("below X"), or a low threshold ("above X"). A subject whose measured value falls within the normal control value for a particular biomarker is usually "within normal limits" for that biomarker. A normal reference standard or level may be a value or number obtained from a normal subject who is free of 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 overall health. A standard curve of purified protein within the normal reference range, e.g., any of the levels described herein, may also be used as a reference.

[0066] As used herein, the term "term age" refers to the age of a patient (e.g., a newborn) born between 37 weeks of gestation and 42 weeks of gestation. For example, if a patient is born at 35 weeks of gestation, the patient is born full-term at 14 days of age.

[0067] As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA or cDNA) that encodes 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 for the gene product, the transgene may include or be operably linked to one or more elements to facilitate or enhance expression, such as a promoter, enhancer, destabilization domain, response element, reporter element, insulator element, polyadenylation signal, and / or other functional element. Embodiments of the present disclosure may utilize any known suitable promoter, enhancer, destabilization domain, response element, reporter element, insulator element, polyadenylation signal, and / or other functional element.

[0068] As used herein, the terms "treat" and "treatment" refer to therapeutic treatment, the purpose of which is, inter alia, to prevent or slow (alleviate) undesirable physiological changes or disorders, such as the progression of a neuromuscular disorder such as XLMTM. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms (e.g., stiffness and / or joint contracture), reduction in the extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliation of disease state, and remission (either partial or total) (either detectable or undetectable). In the context of neuromuscular disorders such as XLMTM, treatment of a patient may be manifested as one or more detectable changes, such as an increase in the concentration of MTM1 protein or nucleic acid (e.g., DNA or RNA such as mRNA) encoding MTM1, or an increase in MTM1 activity (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or more). MTM1 protein concentration can be determined using protein detection assays known in the art, including the ELISA assays described herein. The concentration of nucleic acid encoding MTM1 can be determined using a nucleic acid detection assay (e.g., RNA Seq assay) described herein. Furthermore, treatment of a patient suffering from a neuromuscular disorder such as XLMTM may result in not only an improvement in the patient's muscle function (e.g., skeletal muscle function), but also in improved muscle coordination. For example, the progression of the diaphragm and / or respiratory muscles may be improved.

[0069] As used herein, the terms "X-linked myotubular myopathy" and "XLMTM" refer to an inherited neuromuscular disorder caused by mutations in the MTM1 gene and characterized by symptoms including mild to severe muscle weakness, hypotonia (low muscle tone), feeding difficulties, and / or severe respiratory complications. Human MTM1 is NCBI gene number 4534. An exemplary wild-type human MTM1 nucleic acid sequence is provided in NCBI Reference SEQ ID NO: NM_000252.3, and an exemplary wild-type myotubularin 1 amino acid sequence is provided in NCBI Reference SEQ ID NO: NP_000243.1.

[0070] As used herein, the term "vector" refers to a nucleic acid, e.g., DNA or RNA, that can function as a vehicle for delivery of a gene of interest into a cell (e.g., a mammalian cell, such as a human cell), such as for purposes 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 used, for example, for protein expression and / or recombination of these polynucleotide sequences into the genome of a mammalian cell. Particular vectors that may be used to express the transgenes described herein include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for expressing transgenes contain polynucleotide sequences that increase the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. Such sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells containing the vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0071] [Detailed Description of the Invention] The present disclosure provides compositions and methods that can be used to treat neuromuscular disorders, particularly X-linked myotubular myopathy (XLMTM). The compositions and methods described herein allow for the administration of a viral vector, such as an adeno-associated viral (AAV) vector containing a transgene encoding myotubularin 1 (MTM1), to a patient (e.g., a human patient) suffering from XLMTM. The AAV vector may be a pseudotyped AAV vector, such as an AAV vector containing AAV2 inverted terminal repeats housed within a capsid protein derived from AAV8 (AAV2 / 8). In some embodiments, the MTM1 transgene is operably linked to a transcriptional regulator, such as a promoter, that drives gene expression in hepatocytes. This aspect of the disclosure is based, at least in part, on the discovery that expressing the MTM1 transgene in liver tissue of XLMTM patients may improve the safety of MTM1 gene therapy. In some embodiments, the promoter includes the LP1 promoter, the apolipoprotein E (ApoE) promoter, and / or the alpha-1-antitrypsin (A1AT) promoter (e.g., the LP1 promoter, the ApoE promoter, and / or the A1AT promoter described herein).

[0072] In some embodiments, the MTM1 transgene is operably linked to a constitutive transcriptional regulator, such as the phosphoglycerate kinase (PGK) promoter, the elongation factor-1α (EF 1α) promoter, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, the cytomegalovirus (CMV) promoter, or the chicken β-actin (CBA) promoter. In some embodiments, the viral vector contains 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.

[0073] Also described herein is a method of treating XLMTM in a patient in need thereof, comprising administering to the patient two compositions: (i) a non-viral composition comprising a nucleic acid encoding MTM1 operably linked to a promoter active (e.g., selectively active) in liver tissue, and (ii) a viral vector comprising an MTM1 transgene operably linked to a muscle-specific promoter. Non-viral compositions useful in conjunction with this aspect of the disclosure include, but are not limited to, liposomes, vesicles, synthetic vesicles, exosomes, synthetic exosomes, dendrimers, or nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles. Viral vectors useful in conjunction with this aspect of the disclosure include, but are not limited to, lesamiridin-bilparvovec.

[0074] In one embodiment, the present disclosure describes a method of reducing stiffness and / or joint contracture in a human patient diagnosed with XLMTM by administering one or more compositions described herein. In one embodiment, the present disclosure describes a method of enhancing the progression of diaphragm and / or respiratory muscles in a human patient diagnosed with XLMTM by administering one or more compositions described herein. In certain embodiments, the present disclosure describes methods of preventing cholestasis or hyperbilirubinemia in a human patient diagnosed with XLMTM by administering one or more compositions described herein. The following sections provide a description of therapeutic agents and parameters for assessing cholestasis, hyperbilirubinemia, or one or more symptoms thereof. The following sections also describe various transduction agents that may be used in conjunction with the compositions and methods of the present disclosure.

[0075] X-linked myotubular myopathy XLMTM is a rare, life-threatening congenital myopathy caused by mutations in the MTM1 gene and characterized by severe muscle weakness and hypotonia at birth that leads to severe respiratory insufficiency, inability to stand, erect, or walk, and early death in most patients. 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 condition often lack the muscle strength to breathe on their own and require assistance with mechanical ventilation. Affected individuals may only require mechanical ventilation periodically, such as during sleep, or they may require mechanical ventilation continuously. Patients with XLMTM also have weakness of the muscles that control eye movement (ophthalmoplegia), weakness of other facial muscles, and absence of reflexes (areflexia). In XLMTM, muscle weakness often interferes with normal bone development, resulting in weak bones, an abnormal curvature of the spine (scoliosis), and deformed hip and knee joints (contractures). People with XLMTM may have a narrow, elongated face, a high, arched palate (arched palate), and a large head. Patients may also have liver disease, recurrent ear and respiratory infections, or seizures. Patients with XLMTM typically survive only into childhood due to their severe respiratory distress, although some patients with this condition can survive into adulthood. The compositions and methods of the present disclosure provide an important medical benefit by restoring functional MTM1 expression, thereby extending the patient's lifespan. Furthermore, the present disclosure provides a set of guidelines that can be used to determine a patient's eligibility to be weaned from mechanical ventilation, and the compositions and methods described herein can be used to improve a patient's quality of life after treatment (e.g., reducing joint stiffness and / or contractures or enhancing the progression of the diaphragm and / or respiratory muscles).

[0076] Treatment method In one embodiment, the patient is a newborn (e.g., 0-4 months of age), an infant (e.g., 0-5 months of age), a toddler (e.g., 6-12 months of age), a child aged 1 to about 3 years, or a child aged 3 to about 5 years at the time of administration of the viral vector. In some embodiments, the patient is a newborn (e.g., 0 to 4 months of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is a newborn about 0 to about 4 months of age (e.g., 0 to about 4 months of age, 1 to about 4 months of age, 2 to about 4 months of age, or 3 to about 4 months of age). In some embodiments, the patient is 0 months of age. In some embodiments, the patient is 1 month of age. In some embodiments, the patient is 2 months of age. In some embodiments, the patient is 3 months of age. In some embodiments, the patient is 4 months of age. In some embodiments, the patient is a neonate (e.g., less than about 4 months of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is a neonate less than about 4 months of age. In some embodiments, the patient is less than about 4 months of age. In some embodiments, the patient is less than about 3 months of age. In some embodiments, the patient is less than about 2 months of age. In some embodiments, the patient is less than about 1 month of age.

[0077] In some embodiments, the patient is an infant (e.g., 0 to 5 months of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is an infant about 0 to about 5 months of age (e.g., 0 to about 5 months of age, 1 to about 5 months of age, 2 to about 5 months of age, 3 to about 5 months of age, or 4 to about 5 months of age). In some embodiments, the patient is 0 months of age. In some embodiments, the patient is 1 month of age. In some embodiments, the patient is 2 months of age. In some embodiments, the patient is 3 months of age. In some embodiments, the patient is 4 months of age. In some embodiments, the patient is 3 months of age. In some embodiments, the patient is 5 months of age.

[0078] In some embodiments, the patient is an infant (e.g., less than about 5 months of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is an infant less than about 5 months of age. In some embodiments, the patient is less than about 5 months of age. In some embodiments, the patient is less than about 4 months of age. In some embodiments, the patient is less than about 3 months of age. In some embodiments, the patient is less than about 2 months of age. In some embodiments, the patient is less than about 1 month of age.

[0079] In some embodiments, the patient is an infant (e.g., 6 to 12 months of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is an infant between about 6 months and about 12 months of age (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 old. In some embodiments, the patient is 7 months old. In some embodiments, the patient is 8 months old. In some embodiments, the patient is 9 months old. In some embodiments, the patient is 10 months old. In some embodiments, the patient is 11 months old. In some embodiments, the patient is 12 months old.

[0080] In some embodiments, the patient is an infant (e.g., less than about 12 months of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is an infant less than about 12 months of age. In some embodiments, the patient is less than about 12 months of age. In some embodiments, the patient is less than about 11 months of age. In some embodiments, the patient is less than about 10 months of age. In some embodiments, the patient is less than about 9 months of age. In some embodiments, the patient is less than about 8 months of age. In some embodiments, the patient is less than about 7 months of age. In some embodiments, the patient is less than about 6 months of age. In some embodiments, the patient is less than about 5 months of age. In some embodiments, the patient is less than about 4 months of age. In some embodiments, the patient is less than about 3 months of age. In some embodiments, the patient is less than about 2 months of age. In some embodiments, the patient is less than about 1 month of age.

[0081] In some embodiments, the patient is a child aged 1 to about 3 years at the time of administration of the viral vector. For example, in some embodiments, the patient is a child aged about 1 to about 3 years (e.g., 1 to about 3 years or 2 to about 3 years). 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.

[0082] In some embodiments, the patient is a child (e.g., under about 3 years of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is a child under about 3 years of age. In some embodiments, the patient is under about 3 years of age. In some embodiments, the patient is under about 2 years of age. In some embodiments, the patient is under about 1 year of age. In some embodiments, the patient is under about 12 months of age. In some embodiments, the patient is under about 11 months of age. In some embodiments, the patient is under about 10 months of age. In some embodiments, the patient is under about 9 months of age. In some embodiments, the patient is under about 8 months of age. In some embodiments, the patient is under about 7 months of age. In some embodiments, the patient is under about 6 months of age. In some embodiments, the patient is under about 5 months of age. In some embodiments, the patient is under about 4 months of age. In some embodiments, the patient is under about 3 months of age. In some embodiments, the patient is under about 2 months of age. In some embodiments, the patient is under about 1 month of age.

[0083] In some embodiments, the patient is a child aged 3 to 5 years at the time of administration of the viral vector. For example, in some embodiments, the patient is a child 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.

[0084] In some embodiments, the patient is a child (e.g., under about 5 years of age) at the time of administration of the viral vector. For example, in some embodiments, the patient is a child under about 5 years of age. In some embodiments, the patient is under about 5 years of age. In some embodiments, the patient is under about 4 years of age. In some embodiments, the patient is under about 3 years of age. In some embodiments, the patient is under about 2 years of age. In some embodiments, the patient is under about 1 year of age. In some embodiments, the patient is under about 12 months of age. In some embodiments, the patient is under about 11 months of age. In some embodiments, the patient is under about 10 months of age. In some embodiments, the patient is under about 9 months of age. In some embodiments, the patient is under about 8 months of age. In some embodiments, the patient is under about 7 months of age. In some embodiments, the patient is under about 6 months of age. In some embodiments, the patient is under about 5 months of age. In some embodiments, the patient is under about 4 months of age. In some embodiments, the patient is under about 3 months of age. In some embodiments, the patient is under about 2 months of age. In some embodiments, the patient is under about 1 month of age.

[0085] In one embodiment, the patient is about 1 month to about 5 years old (e.g., about 1 month to about 5 years old, about 2 months to about 5 years old, about 3 months to about 5 years old, about 4 months to about 5 years old, about 5 months to about 5 years old, about 6 months to about 5 years old, about 1 year to about 5 years old, about 2 years to about 5 years old, about 3 years to about 5 years old, or about 4 years to about 5 years old) at the time of administration of the viral vector.

[0086] In certain embodiments, the patient is born at 35 weeks or greater of gestational age (e.g., 35 weeks of gestational age, 36 weeks of gestational age, 37 weeks of gestational age, 38 weeks of gestational age, 39 weeks of gestational age, 40 weeks of gestational age, 41 weeks of gestational age, and 42 weeks of gestational age) and is between an adjusted full-term age (e.g., 37 weeks of gestational age or greater) and about 5 years of age at the time of administration of the viral vector. For example, if the patient is born at 35 weeks of gestational age, the patient is born full-term at 14 days of age.

[0087] In one embodiment, the patient is born at 35 weeks gestation and is an adjusted age between full term and about 5 years old at the time of administration of the viral vector (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, 1 month to about 5 years old, 2 months to about 5 years old, 3 months to about 5 years old, 4 months to about 5 years old, 5 months to about 5 years old, 6 months to about 5 years old, 1 month to about 5 years old, 2 months to about 5 years old, 3 months to about 5 years old, and 4 months to about 5 years old). In one embodiment, the patient is born at 36 weeks gestation and is between an adjusted full-term age and about 5 years old at the time of administration of the viral vector (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, 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, 1 month to about 5 years old, 2 months to about 5 years old, 3 years to about 5 years old, and 4 years to about 5 years old). In one embodiment, the patient is born at 37 weeks gestation and is between an adjusted full-term age of 1 year and about 5 years old at the time of administration of the viral vector (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, 19 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, 5 days to about 5 years old, 6 days to about 5 years old, 1 year to about 5 years old, 2 years to about 5 years old, 3 years to about 5 years old, and 4 years to about 5 years old). In certain embodiments, the patient is male. In certain embodiments, the patient is female.

[0088] Cholestasis and hyperbilirubinemia Cholestasis refers to any condition in which the flow of bile acids from the liver is slowed or blocked, while hyperbilirubinemia refers to a condition in which bilirubin accumulates in the blood but serum bile acids appear normal. In contrast, cholestatic syndrome is characterized by marked bile acidemia 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, and a finding that the parameter is above a reference level identifies the patient as having cholestasis, hyperbilirubinemia, or one or more symptoms thereof.

[0089] In certain embodiments, a patient is determined to exhibit cholestasis or one or more symptoms thereof if a serum bile acid test and / or blood test (e.g., liver function test (LFT)) measures one or more parameters (e.g., total bile acid level, gamma-glutamyltransferase (GGT) level, alkaline phosphatase (ASP) level, aspartate aminotransferase (AST) level, and / or alanine aminotransferase (ALT) level) that are higher or lower than age-adjusted norms. In certain embodiments, a patient is determined to have hyperbilirubinemia or one or more symptoms thereof when a blood test (eg, a bilirubin test) measures a bilirubin level higher than normal. In some embodiments, the patient has no history of cholestasis or hyperbilirubinemia, hi some embodiments, the patient has no history of any underlying liver disease.

[0090] Vectors for delivery of 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 a gene of interest (e.g., a transgene encoding MTM1) into the genome of a target cell (e.g., a mammalian cell, such as a human cell). Recombinant viral genomes are particularly useful vectors for gene delivery because they deliver the gene of interest to the nucleus of the target cell. For selected viruses, polynucleotides contained within such genomes can be integrated into the genome of target cells by generalized or specialized transduction. These processes occur during the natural viral replication cycle and do not require the addition of proteins or reagents. Examples of recombinant viral vectors used to deliver a gene of interest include AAV, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai viruses)), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and and double-stranded DNA viruses (e.g., adenoviruses, herpes viruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses useful for delivering polynucleotides encoding the antibody light and heavy chains or antibody fragments of the invention include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus.Examples of retroviruses include avian leukosis sarcoma viruses, mammalian type C, type B, and type D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third 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 lentiviruses. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which relates to viral vectors for use in gene therapy and is incorporated herein by reference.

[0091] AAV vectors for nucleic acid delivery In certain embodiments, the 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 useful in the present invention are recombinant nucleic acid constructs that contain (1) a transgene to be expressed (e.g., a polynucleotide encoding an MTM1 protein) and (2) viral nucleic acid that facilitates the integration and expression of a heterologous gene. The viral nucleic acid may include AAV sequences required in cis for DNA replication and packaging into virions (e.g., functional inverted terminal repeats (ITRs)). In a typical application, the transgene encodes MTM1, which is useful for correcting MTM1 mutations in patients with neuromuscular disorders such as XLMTM. The rAAV vector may also contain a marker or reporter gene. Useful rAAV vectors contain one or more AAV wild-type genes deleted in whole or in part but retaining functional flanking ITR sequences. The AAV ITRs may be of any serotype (e.g., from serotype 2) appropriate for the particular application. 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), the disclosures of each of which relate to AAV vectors for gene delivery and are incorporated herein by reference.

[0092] The nucleic acids and vectors described herein can be incorporated into rAAV virions to facilitate the introduction of the nucleic acid or vector into cells. The capsid protein of AAV constitutes the external, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virion assembly. For construction of rAAV virions, see, e.g., U.S. Patent Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as Rabinowitz et al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of each of which relate to AAV vectors for gene delivery and are incorporated herein by reference.

[0093] rAAV virions that can be used in conjunction with the compositions and methods described herein include those derived from various AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, and 9. For targeting muscle cells, rAAV virions containing at least one serotype 1 capsid protein may be particularly useful. rAAV virions containing at least one serotype 6 capsid protein may also be particularly useful, since serotype 6 capsid protein is structurally similar to serotype 1 capsid protein and is therefore expected to also result in high expression of MTM1 in muscle cells. rAAV serotype 9 has also been found to be an efficient inducer of muscle cells. The construction and use of different serotypes of AAV vectors and AAV proteins 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), the disclosures of each of which relate to AAV vectors for gene delivery and are incorporated herein by reference.

[0094] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene 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 AAV8(V) encoding a therapeutic protein pseudotyped with a capsid gene from AAV serotype 2. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art; see, e.g., 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).

[0095] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants can contain ligand insertion mutations that facilitate targeting of AAV to specific cell types. The construction and characterization of AAV capsid mutants, including insertion mutants, alanine screening mutants, and epitope tag mutants, are described in Wu et al., J. Virol. 74:8635-45 (2000). Other rAAV virions that may be used in the methods of the invention include capsid hybrids generated by molecular breeding of viruses and by exon shuffling (see, e.g., Soong et al., Nat. Genet., 25:436-439 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001)).

[0096] Resamirigene Bilparvovec The pseudotyped AAV vector described herein, comprising a nucleic acid sequence encoding the MTM1 gene operably linked to the desmin promoter flanked by AAV2 ITRs and housed within a capsid protein derived from AAV8 (AAV2 / 8), as well as the other genetic components listed in Table 1, refers to a known compound under the International Trademark Name (INN) resamiridin-bilparvovec. Resamiridin-virparvovec is a non-replicating recombinant AAV8 vector expressing 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 reference sequence number _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 was flanked by AAV serotype 2 (AAV2) inverted terminal repeats (ITRs). The vector was generated in AAV8 capsids by two-plasmid transfection in HEK293 cells in suspension culture in a bioreactor using a full GMP process.

[0097] In one embodiment, a method for treating a disorder (e.g., XLMTM) or alleviating one or more symptoms of a disorder (e.g., XLMTM) (e.g., stiffness and / or contracture of joints, or the need for progression of the diaphragm and / or respiratory muscles) in a human patient in need thereof includes administering a therapeutically effective amount of resamirigen bilparvovec to the patient during a treatment period. In one embodiment, a method for weaning a human patient from mechanical ventilation includes a patient who has previously been administered a therapeutically effective amount of resamirigen bilparvovec. The components of resamirigen bilparvovec are set forth below in Table 1.

[0098] Table 1. Nucleic acid sequence of resamiridin-bilparvovec (SEQ ID NO: 1)

[0099] [Table 1] As described herein, resamiridin-virparvovec refers to an AAV vector having the nucleic acid sequence represented by SEQ ID NO: 1 shown below.

[0100] [ka] TIFF2025531998000008.tif251170TIFF2025531998000009.tif247170TIFF2025531998000010.tif245170 TIFF2025531998000011.tif251170TIFF2025531998000012.tif246170TIFF2025531998000013.tif179170 transcriptional regulators Transcriptional regulatory factors that can be used in conjunction with the compositions and methods described herein can include various portions operably linked to each other. For example, transcriptional regulatory factors described herein can include the ApoE and / or A1AT promoter (e.g., the chimeric promoter set forth in SEQ ID NO: 2), or a functional portion thereof. Additional nucleic acid regulatory factors that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules with 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.

[0101] Additionally or alternatively, the transcriptional regulators described herein can comprise the LP1 promoter or a functional portion thereof. For example, the regulator can comprise the LP1 promoter set forth in SEQ ID NO: 3 or a functional portion thereof. Additional nucleic acid regulators that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules with 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.

[0102] Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include promoters that stimulate expression of a transgene operably linked to a liver-specific promoter. Examples of such promoters are the ApoE / A1At and LP1 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 that are capable of stimulating transcription of a transgene operably linked thereto upon introduction into a cell), or functional portions thereof.

[0103] Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include promoters that stimulate expression of a transgene 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 that are capable of stimulating transcription of a transgene operably linked thereto upon introduction into a cell), or functional portions thereof.

[0104] Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include promoters that stimulate expression of a transgene operably linked to a ubiquitous promoter. Examples of such promoters are PGK, Eflα, and GAPDH, 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 that are capable of stimulating transcription of a transgene operably linked thereto upon introduction into a cell), or functional portions thereof.

[0105] The transcriptional regulatory elements described herein can include an SV40 enhancer or a functional portion thereof. For example, the regulatory element can include the SV40 enhancer set forth in SEQ ID NO: 4, or a functional portion thereof. Additional nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules with 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.

[0106] The transcriptional regulators described herein can include a β-globin enhancer or a functional portion thereof. For example, the regulator can include a β-globin enhancer set forth in SEQ ID NO: 5 or a functional portion thereof. Additional nucleic acid regulators that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules with 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.

[0107] Transcriptional regulatory elements that can be used in conjunction with the compositions and methods described herein include enhancers that enhance transgene expression. Examples of such promoters include PGK, Ef 1α, and GAPDH, 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 an operably linked transgene upon introduction into a cell), or functional portions thereof.

[0108] The above nucleic acid regulatory elements are summarized in Table 2 below.

[0109] Table 2: Exemplary Nucleic Acid Regulators

[0110] [Table 2] Additional nucleic acid regulatory elements that can be used in conjunction with the compositions and methods described herein include nucleic acid molecules that have 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.

[0111] Transgene The pseudotyped AAV vectors described herein include a nucleic acid sequence encoding an MTM1 gene flanked by AAV2 ITRs and operably linked to a promoter contained within a capsid protein derived from AAV8 (AAV2 / 8). In one embodiment, the nucleic acid sequence encoding the MTM1 gene encodes the human MTM1 gene. In one embodiment, the nucleic acid sequence encoding the MTM1 gene encodes the mouse MTM1 gene. In one embodiment, the MTM1 gene is codon-optimized. In one embodiment, the promoter is a liver-specific promoter. In one embodiment, the promoter is a muscle-specific promoter. In one embodiment, the promoter is a ubiquitously expressed promoter. In one embodiment, two MTM1 genes can be simultaneously expressed using different promoters on the same AAV vector.

[0112] A transgene that can be used in conjunction with the compositions and methods described herein can comprise the human MTM1 transgene set forth 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 that share 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.

[0113] A transgene that can be used in conjunction with the compositions and methods described herein can comprise the mouse MTM1 transgene set forth 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 that share 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.

[0114] In one embodiment, a method for treating a disorder (e.g., XLMTM) or alleviating one or more symptoms of the disorder (e.g., XLMTM) (e.g., stiffness and / or joint contracture, or the need for progression of the diaphragm and / or respiratory muscles) in a human patient in need thereof comprises administering to the patient for a treatment period a therapeutically effective amount of a pseudotyped AAV vector comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a liver-specific promoter or a muscle-specific promoter flanked by AAV2 ITRs and housed within a capsid protein from AAV8 (AAV2 / 8), as well as other genetic components listed in Table 2. In one embodiment, a method for weaning a human patient from mechanical ventilation includes a patient who has previously received a therapeutically effective amount of a pseudotyped AAV vector comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a liver-specific or muscle-specific promoter flanked by AAV2 ITRs and housed within a capsid protein from AAV8 (AAV2 / 8), as well as other genetic components listed in Table 2.

[0115] The above transgenes are summarized in Table 3 below.

[0116] Table 3. Exemplary transgenes

[0117] [Table 3] TIFF2025531998000016.tif237170TIFF2025531998000017.tif56170Additional transgenes that may be used in conjunction with the compositions and methods described herein include nucleic acid molecules that have 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 3.

[0118] Method for delivering exogenous nucleic acids to target cells Transfection technology Techniques that can be used to introduce transgenes, such as the MTM1 transgene described herein, into target cells are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the cells of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are then predisposed to the uptake of exogenous nucleic acids (e.g., nucleic acids that can be expressed in neurons, glial cells, or non-neuronal cells such as colon and kidney cells). Electroporation of mammalian cells is described in detail, for example, in 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 useful for using this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005) and U.S. Patent Application Publication No. 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0119] Another technique useful for transfection of target cells is squeeze-perforation. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied stress. This technique has the advantage that no vector is required to deliver nucleic acid to cells, such as human target cells. Squeeze-perforation is described in detail, for example, in Sharei et al., J. Vis. Exp. 81:e50980 (2013) (the disclosure of which is incorporated herein by reference).

[0120] Lipofection represents another technique useful for transfecting target cells. This method involves loading nucleic acids into liposomes, which often present cationic functional groups, such as quaternary amines or protonated amines, facing the outside of the liposome. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, ultimately leading to the uptake of the exogenous nucleic acid, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids is to contact cells with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for 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-dextran, 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.

[0121] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laser transfection, also known as optical transfection, which involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique has been found to be similar to, and in some cases superior to, electroporation.

[0122] Impalefection is another technique that can be used to deliver genetic material into 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 intended for intracellular delivery is attached to the nanostructure surface. The chip with an array of these needles is then pressed against cells or tissue. Cells penetrated by the nanostructures 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.

[0123] MAGNETOFECTION™ can also be used to deliver nucleic acids to target cells. The principle of MAGNETOFECTION™ is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made from fully biodegradable iron oxide and coated with specific cationic molecules, which vary depending on the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on target cells by the influence of an external magnetic field generated by a magnet. This technique is described 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, as this methodology utilizes an applied magnetic field to direct the uptake of nucleic acids. This technique is described in detail, for example, in U.S. Patent Application Publication No. 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0124] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves using sound (typically ultrasonic frequencies) to alter the permeability of the plasma membrane of a cell, permeabilizing the cell and allowing polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods Cell Biol. 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0125] Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins to cells, which then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the cell's genome for covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also known as 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], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122. Incorporation of Target Genes by Gene Editing Techniques.

[0126] Targeted gene integration using gene editing technology In addition to the above, various tools have been developed that can be used to integrate a gene of interest into target cells, such as human cells. One method that can be used to integrate a polynucleotide encoding a target gene into a target cell is the use of a transposon. A transposon is a polynucleotide that encodes a transposase enzyme and contains a polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered into a cell, expression of the transposase gene is initiated, resulting in an active enzyme that excises the gene of interest from the transposon. This activity is mediated by site-specific recognition of the transposon excision site by the transposase. In some instances, these excision sites may be terminal repeats or inverted terminal repeats. 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 cell's nuclear genome. This allows the gene of interest to be inserted into the nuclear DNA cleaved at the complementary excision site, and the integration process is completed by subsequent covalent ligation of a phosphodiester bond that links the gene of interest to the DNA of the mammalian cell genome. In some instances, the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA before integration into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail, e.g., in WO 2010 / 085699) and the sleeping beauty transposon (described in detail, e.g., in U.S. Patent Application Publication No. 2005 / 0112764), the disclosures of each of which are incorporated herein by reference with respect to transposons used to deliver genes to cells of interest.

[0127] Another tool for integrating target genes into the genome of target cells is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. CRISPR / Cas systems contain palindromic repeat sequences within plasmid DNA and the associated Cas9 nuclease. This DNA and protein ensemble first integrates foreign DNA into the CRISPR locus, thereby directing site-specific DNA cleavage of the target sequence. Polynucleotides containing these foreign sequences and the repetitive spacer elements of the CRISPR locus are then transcribed in the host cell to generate guide RNAs, which then anneal to the target sequence and localize the Cas9 nuclease to this site. In this way, the interaction that brings Cas9 within the vicinity of the target DNA molecule is governed by RNA:DNA hybridization, allowing highly site-specific Cas9-mediated DNA cleavage to occur in the foreign polynucleotide. As a result, CRISPR / Cas systems can be designed to cleave any target DNA molecule of interest. This technology has been utilized 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 before integrating a gene encoding a target gene. The use of CRISPR / Cas to regulate gene expression is described, for example, in U.S. Patent No. 8,697,359, the disclosure of which is incorporated herein by reference with respect to the use of CRISPR / Cas systems for genome editing. Alternative methods for site-specific cleavage of genomic DNA before integrating a gene of interest into a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike CRISPR / Cas systems, these enzymes do not contain guide polynucleotides for localization to specific target sequences.

[0128] target The specificity is controlled by the DNA binding domain within these 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), the disclosures of each of which are incorporated herein by reference for their disclosures regarding compositions and methods for genome editing. Another genome editing technique that can be used to integrate a polynucleotide encoding a target gene into a target genome is the use of ARCUS™ meganucleases, which can be rationally designed to site-specifically cleave genomic DNA. The use of these enzymes to integrate a gene encoding a target gene into the genome of a mammalian cell is advantageous, given the specific structure-activity relationship established for these enzymes. Single-stranded meganucleases can be modified at specific amino acid positions to create nucleases that selectively cleave DNA at desired positions, allowing target cells to site-specifically integrate the target gene into nuclear DNA. These single-stranded nucleases are extensively described, for example, in U.S. Patent No. 8,021,867 and U.S. Patent No. 8,445,251, the disclosures of which relate to compositions and methods for genome editing and are incorporated herein by reference.

[0129] Pharmaceutical Compositions and Routes of Administration The gene therapy agents described herein can include a transgene, such as a transgene encoding MTM1, and can be incorporated into a vehicle for administration to a patient, such as a human patient with a neuromuscular disorder (e.g., XLMTM). Pharmaceutical compositions containing a vector, e.g., a viral vector, comprising a transcriptional regulator described herein (e.g., an ApoE / A1AT promoter) operably linked to a therapeutic transgene can be prepared using methods known in the art. For example, such compositions can be prepared in a desired form, e.g., a lyophilized formulation or an aqueous solution, using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference).

[0130] Viral vectors, e.g., AAV vectors and others described herein, comprising a transcriptional regulatory element 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 routes of administration can vary, for example, depending on the onset and severity of the disease, and include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intrahepatic, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. Intravascular administration involves delivery to the patient's vasculature. In some embodiments, administration is into a blood vessel considered a vein (intravenous), while in some administrations, administration is into a blood vessel considered an artery (intraarterial). Veins include, but are not limited to, the internal jugular vein, peripheral veins, coronary veins, hepatic veins, portal vein, saphenous vein, pulmonary veins, superior vena cava, inferior vena cava, gastric vein, splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and / or femoral vein. Arteries include, but are not limited to, coronary arteries, pulmonary arteries, hepatic arteries, brachial arteries, internal carotid arteries, aortic arches, femoral arteries, peripheral arteries, and / or ciliary arteries. It is contemplated that delivery may be through or directed to arterioles or capillaries.

[0131] Mixtures of the nucleic acids and viral vectors described herein can be prepared in water, suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. These preparations may contain a preservative to prevent the growth of microorganisms under ordinary conditions of storage and use. 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 U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In all cases, the formulation may be sterile and fluid to the extent that easy syringability exists. The formulation may be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0132] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered, if necessary, and liquid diluents first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, intrahepatic, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous vehicles that can be used will be known to those skilled in the art from the present disclosure. For example, a single dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion fluid or injected at a designated infusion site. Dosage amounts will necessarily vary to some extent depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations will meet sterility, pyrogenicity, general safety, and purity standards to the extent required by FDA Office of Biologics standards.

[0133] kit The compositions described herein may be provided in kits for use in treating neuromuscular disorders (e.g., XLM™). In some embodiments, the kits may include one or more viral vectors described herein. The kits may include a package insert instructing a user of the kit, e.g., a physician skilled in the art, to perform any one of the methods described herein. The kits may optionally include a syringe or other device for administering the composition. In some embodiments, the kits may include one or more additional therapeutic agents. The kit may include a package insert instructing a user of the kit, e.g., a physician skilled in the art, to perform any one of the methods described herein. The kit may optionally include a syringe or other device for administering the composition. In certain embodiments, the kit may include one or more additional therapeutic agents.

[0134] Dosage regimen Dosage regimen containing AAV-MTM1 vector Using the compositions and methods of the present disclosure, a patient suffering from a neuromuscular disorder (e.g., XLMTM) can be injected with an AAV vector containing a transgene encoding MTM1 at a dose of approximately 1.3 x 1014 vg / kg, which may be administered to a patient. Administration of the vector to a patient in such amounts may achieve the beneficial effect of increasing MTM1 expression in the patient to, for example, within 50% or 200% of wild-type levels without inducing toxic side effects.

[0135] In one embodiment, the AAV vector is about 3 x 10 14 Amounts less than 3 × 10 vg / kg (e.g., about 3 × 10 14 vg / kg, 2.9 × 10 14 vg / kg, 2.8 × 10 14 vg / kg, 2.7 × 10 14 vg / kg, 2.6 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.4 × 10 14 vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The patient will be administered a dose of 100 mg / kg or less. For example, AAV vectors can be expressed as 3 x 10 14 vg / kg, 2.9 × 10 14 vg / kg, 2.8 × 10 14vg / kg, 2.7 × 10 14 vg / kg, 2.6 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.4 × 10 14 vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 vg / kg may be administered to the patient.

[0136] In one embodiment, the AAV vector is about 2.5 x 10 14 vg / kg (2.5 × 10 14 vg / kg, 2.4 × 10 14 vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 1014 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The patient will be administered a dose of 100 mg / kg or less. For example, AAV vectors can be expressed at 2.5 × 10 14 vg / kg, 2.4 × 10 14 vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 vg / kg may be administered to the patient.

[0137] In one embodiment, the AAV vector is about 2 x 10 14 vg / kg (2 × 10 14vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The patient will be administered a dose of 100 mg / kg or less. For example, AAV vectors can be expressed at 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 vg / kg may be administered to the patient.

[0138] In one embodiment, the AAV vector is about 1.5 x 10 14 vg / kg (1.5 × 1014 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The patient will be administered a dose of 100 mg / kg or less. For example, AAV vectors can be expressed at 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 vg / kg may be administered to the patient.

[0139] In one embodiment, the AAV vector is about 1 x 10 14 Amounts less than vg / kg (1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The patient will be administered a dose of 100 mg / kg or less. For example, AAV vectors can be expressed at 1 × 10 14vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 vg / kg may be administered to the patient.

[0140] In one embodiment, the AAV vector is about 3 x 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg (e.g., approximately 3 × 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg) is administered to the patient. For example, AAV vectors are approximately 3 × 10 13 vg / kg, 3.1 × 10 13 vg / kg, 3.2 × 10 13 vg / kg, 3.3 × 10 13 vg / kg, 3.4 × 10 13 vg / kg, 3.5 × 10 13 vg / kg, 3.6 × 10 13 vg / kg, 3.7 × 10 13 vg / kg, 3.8 × 10 13 vg / kg, 3.9 × 10 13 vg / kg, 4 × 10 13 vg / kg, 4.1 × 10 13 vg / kg, 4.2 × 10 13 vg / kg, 4.3 × 10 13 vg / kg, 4.4 × 10 13 vg / kg, 4.5 × 10 13 vg / kg, 4.7 × 10 13 vg / kg, 4.8 × 10 13 vg / kg, 4.9 × 10 13 vg / kg, 5 × 10 13 vg / kg, 5.1 × 10 13 vg / kg, 5.2 × 10 13 vg / kg, 5.3 × 10 13 vg / kg, 5.4 × 10 13 vg / kg, 5.5 × 10 13vg / kg、5.6×10 13 vg / kg、5.7×10 13 vg / kg、5.8×10 13 vg / kg、5.9×10 13 vg / kg、6×10 13 vg / kg、6.1×10 13 vg / kg、6.2×10 13 vg / kg、6.3×10 13 vg / kg、6.4×10 13 vg / kg、6.5×10 13 vg / kg、6.6×10 13 vg / kg、6.7×10 13 vg / kg、6.8×10 13 vg / kg、6.9×10 13 vg / kg、7×10 13 vg / kg、7.1×10 13 vg / kg、7.2×10 13 vg / kg、7.3×10 13 vg / kg、7.4×10 13 vg / kg、7.5×10 13 vg / kg、7.6×10 13 vg / kg、7.7×10 13 vg / kg、7.8×10 13 vg / kg、7.9×10 13 vg / kg、8×10 13 vg / kg、8.1×10 13 vg / kg、8.2×10 13 vg / kg、8.3×10 13 vg / kg、8.4×10 13 vg / kg、8.5×10 13 vg / kg、8.6×10 13 vg / kg、8.7×10 13 vg / kg、8.8×10 13 vg / kg、8.9×10 13 vg / kg、9×10 13 vg / kg、9.1×10 13 vg / kg、9.2×10 13 vg / kg、9.3×10 13 vg / kg、9.4×10 13 vg / kg、9.5×10 13vg / kg, 9.6 × 10 13 vg / kg, 9.7 × 10 13 vg / kg, 9.8 × 10 13 vg / kg, 9.9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14 The amount may be administered to the patient in an amount of vg / kg.

[0141] In one embodiment, the AAV vector is about 4 x 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg, e.g., 4 x 10 13 vg / kg, 4.1 × 10 13 vg / kg, 4.2 × 10 13 vg / kg, 4.3 × 10 13 vg / kg, 4.4 × 10 13 vg / kg, 4.5 × 10 13 vg / kg, 4.7 × 10 13 vg / kg, 4.8 × 10 13 vg / kg, 4.9 × 10 13 vg / kg, 5 × 10 13 vg / kg, 5.1 × 10 13 vg / kg, 5.2 × 10 13 vg / kg, 5.3 × 10 13 vg / kg, 5.4 × 10 13 vg / kg, 5.5 × 10 13 vg / kg, 5.6 × 10 13 vg / kg, 5.7 × 10 13vg / kg、5.8×10 13 vg / kg、5.9×10 13 vg / kg、6×10 13 vg / kg、6.1×10 13 vg / kg、6.2×10 13 vg / kg、6.3×10 13 vg / kg、6.4×10 13 vg / kg、6.5×10 13 vg / kg、6.6×10 13 vg / kg、6.7×10 13 vg / kg、6.8×10 13 vg / kg、6.9×10 13 vg / kg、7×10 13 vg / kg、7.1×10 13 vg / kg、7.2×10 13 vg / kg、7.3×10 13 vg / kg、7.4×10 13 vg / kg、7.5×10 13 vg / kg、7.6×10 13 vg / kg、7.7×10 13 vg / kg、7.8×10 13 vg / kg、7.9×10 13 vg / kg、8×10 13 vg / kg、8.1×10 13 vg / kg、8.2×10 13 vg / kg、8.3×10 13 vg / kg、8.4×10 13 vg / kg、8.5×10 13 vg / kg、8.6×10 13 vg / kg、8.7×10 13 vg / kg、8.8×10 13 vg / kg、8.9×10 13 vg / kg、9×10 13 vg / kg、9.1×10 13 vg / kg、9.2×10 13 vg / kg、9.3×10 13 vg / kg、9.4×10 13 vg / kg、9.5×10 13 vg / kg、9.6×10 13 vg / kg、9.7×10 13vg / kg, 9.8 × 10 13 vg / kg, 9.9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14 The patient is administered a dose of 0.05 mg / kg of the active ingredient.

[0142] In one embodiment, the AAV vector is about 5 x 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg, e.g., 5 x 10 13 vg / kg, 5.1 × 10 13 vg / kg, 5.2 × 10 13 vg / kg, 5.3 × 10 13 vg / kg, 5.4 × 10 13 vg / kg, 5.5 × 10 13 vg / kg, 5.6 × 10 13 vg / kg, 5.7 × 10 13 vg / kg, 5.8 × 10 13 vg / kg, 5.9 × 10 13 vg / kg, 6 × 10 13 vg / kg, 6.1 × 10 13 vg / kg, 6.2 × 10 13 vg / kg, 6.3 × 10 13 vg / kg, 6.4 × 10 13 vg / kg, 6.5 × 10 13 vg / kg, 6.6 × 10 13 vg / kg, 6.7 × 10 13 vg / kg, 6.8 × 10 13vg / kg、6.9×10 13 vg / kg、7×10 13 vg / kg、7.1×10 13 vg / kg、7.2×10 13 vg / kg、7.3×10 13 vg / kg、7.4×10 13 vg / kg、7.5×10 13 vg / kg、7.6×10 13 vg / kg、7.7×10 13 vg / kg、7.8×10 13 vg / kg、7.9×10 13 vg / kg、8×10 13 vg / kg、8.1×10 13 vg / kg、8.2×10 13 vg / kg、8.3×10 13 vg / kg、8.4×10 13 vg / kg、8.5×10 13 vg / kg、8.6×10 13 vg / kg、8.7×10 13 vg / kg、8.8×10 13 vg / kg、8.9×10 13 vg / kg、9×10 13 vg / kg、9.1×10 13 vg / kg、9.2×10 13 vg / kg、9.3×10 13 vg / kg、9.4×10 13 vg / kg、9.5×10 13 vg / kg、9.6×10 13 vg / kg、9.7×10 13 vg / kg、9.8×10 13 vg / kg、9.9×10 13 vg / kg、1×10 14 vg / kg、1.1×10 14 vg / kg、1.2×10 14 vg / kg、1.3×10 14 vg / kg、1.4×10 14 vg / kg、1.5×10 14 vg / kg、1.6×10 14 vg / kg、1.7×10 14 vg / kg、1.8×10 14vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14 The patient is administered a dose of 0.05 mg / kg of the active ingredient.

[0143] In one embodiment, the AAV vector is about 6 x 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg, e.g., 6 x 10 13 vg / kg, 6.1 × 10 13 vg / kg, 6.2 × 10 13 vg / kg, 6.3 × 10 13 vg / kg, 6.4 × 10 13 vg / kg, 6.5 × 10 13 vg / kg, 6.6 × 10 13 vg / kg, 6.7 × 10 13 vg / kg, 6.8 × 10 13 vg / kg, 6.9 × 10 13 vg / kg, 7 × 10 13 vg / kg, 7.1 × 10 13 vg / kg, 7.2 × 10 13 vg / kg, 7.3 × 10 13 vg / kg, 7.4 × 10 13 vg / kg, 7.5 × 10 13 vg / kg, 7.6 × 10 13 vg / kg, 7.7 × 10 13 vg / kg, 7.8 × 10 13 vg / kg, 7.9 × 10 13 vg / kg, 8 × 10 13 vg / kg, 8.1 × 10 13 vg / kg, 8.2 × 10 13 vg / kg, 8.3 × 10 13 vg / kg, 8.4 × 10 13 vg / kg, 8.5 × 10 13 vg / kg, 8.6 × 10 13 vg / kg, 8.7 × 10 13 vg / kg, 8.8 × 10 13 vg / kg, 8.9 × 10 13vg / kg, 9 × 10 13 vg / kg, 9.1 × 10 13 vg / kg, 9.2 × 10 13 vg / kg, 9.3 × 10 13 vg / kg, 9.4 × 10 13 vg / kg, 9.5 × 10 13 vg / kg, 9.6 × 10 13 vg / kg, 9.7 × 10 13 vg / kg, 9.8 × 10 13 vg / kg, 9.9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14 The patient is administered a dose of 0.05 mg / kg of the active ingredient.

[0144] In one embodiment, the AAV vector is about 7 x 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg, e.g., 7 x 10 13 vg / kg, 7.1 × 10 13 vg / kg, 7.2 × 10 13 vg / kg, 7.3 × 10 13 vg / kg, 7.4 × 10 13 vg / kg, 7.5 × 10 13 vg / kg, 7.6 × 10 13 vg / kg, 7.7 × 10 13 vg / kg, 7.8 × 10 13 vg / kg, 7.9 × 10 13 vg / kg, 8 × 10 13vg / kg, 8.1 × 10 13 vg / kg, 8.2 × 10 13 vg / kg, 8.3 × 10 13 vg / kg, 8.4 × 10 13 vg / kg, 8.5 × 10 13 vg / kg, 8.6 × 10 13 vg / kg, 8.7 × 10 13 vg / kg, 8.8 × 10 13 vg / kg, 8.9 × 10 13 vg / kg, 9 × 10 13 vg / kg, 9.1 × 10 13 vg / kg, 9.2 × 10 13 vg / kg, 9.3 × 10 13 vg / kg, 9.4 × 10 13 vg / kg, 9.5 × 10 13 vg / kg, 9.6 × 10 13 vg / kg, 9.7 × 10 13 vg / kg, 9.8 × 10 13 vg / kg, 9.9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14 The patient is administered a dose of 0.05 mg / kg of the active ingredient.

[0145] In one embodiment, the AAV vector is about 8 x 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg, e.g., 8 x 10 13 vg / kg, 8.1 × 10 13vg / kg, 8.2 × 10 13 vg / kg, 8.3 × 10 13 vg / kg, 8.4 × 10 13 vg / kg, 8.5 × 10 13 vg / kg, 8.6 × 10 13 vg / kg, 8.7 × 10 13 vg / kg, 8.8 × 10 13 vg / kg, 8.9 × 10 13 vg / kg, 9 × 10 13 vg / kg, 9.1 × 10 13 vg / kg, 9.2 × 10 13 vg / kg, 9.3 × 10 13 vg / kg, 9.4 × 10 13 vg / kg, 9.5 × 10 13 vg / kg, 9.6 × 10 13 vg / kg, 9.7 × 10 13 vg / kg, 9.8 × 10 13 vg / kg, 9.9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14 The patient is administered a dose of 0.05 mg / kg of the active ingredient.

[0146] In one embodiment, the AAV vector is about 9 x 10 13 vg / kg ~ approx. 2.3×10 14 vg / kg, e.g., 9 x 10 13 vg / kg, 9.1 × 10 13 vg / kg, 9.2 × 10 13vg / kg, 9.3 × 10 13 vg / kg, 9.4 × 10 13 vg / kg, 9.5 × 10 13 vg / kg, 9.6 × 10 13 vg / kg, 9.7 × 10 13 vg / kg, 9.8 × 10 13 vg / kg, 9.9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14 The patient is administered a dose of 0.05 mg / kg of the active ingredient.

[0147] In one embodiment, the AAV vector is about 1 x 10 14 vg / kg ~ approx. 2.3×10 14 vg / kg, e.g., 1 x 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2.2 × 10 14 vg / kg or 2.3 × 10 14The patient is administered a dose of 0.05 mg / kg of the active ingredient.

[0148] In one embodiment, the AAV vector is about 3 x 10 13 In one embodiment, the AAV vector is administered to a patient in an amount of about 4×10 vg / kg. 13 In one embodiment, the AAV vector is administered to a patient in an amount of about 5×10 vg / kg. 13 In one embodiment, the AAV vector is administered to a patient in an amount of about 6×10 vg / kg. 13 In one embodiment, the AAV vector is administered to a patient in an amount of about 7×10 vg / kg. 13 In one embodiment, the AAV vector is administered to a patient in an amount of about 8×10 vg / kg. 13 In one embodiment, the AAV vector is administered to a patient in an amount of about 9×10 vg / kg. 13 In one embodiment, the AAV vector is administered to a patient in an amount of about 1 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.1 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.2 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.3 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.4 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.5 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.6 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.7 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.8 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 1.9 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2×10 vg / kg.14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.1 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.2 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.3 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.4 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.5 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.6 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.7 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.8 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 2.9 x 10 vg / kg. 14 In one embodiment, the AAV vector is administered to a patient in an amount of about 3×10 vg / kg. 14 It is administered to patients in an amount of vg / kg.

[0149] In one embodiment, the AAV vector is administered in an amount (e.g., about 3×10 14 The patient is given a single dose containing less than 100 mg / kg of vasopressin. In one embodiment, the AAV vectors are combined in an amount (e.g., about 3×10 14 The patient is administered two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) doses containing less than 100 mg / kg of the active ingredient. In one embodiment, the AAV vectors are each individually administered in an amount (e.g., about 3×10 14 The patient is administered two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) doses containing less than 100 mg / kg of the active ingredient. In one embodiment, the AAV vector is administered to the patient in two or more doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) spaced apart by a period of one year or more (e.g., 1 year, 1 year and 1 day, 1 year and 1 month, 1 year and 6 months, 2 years, 3 years, 4 years, or 5 years). In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) doses are administered to the patient within about 12 months of each other (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).

[0150] Combination therapy An AAV vector comprising a transgene encoding MTM1 as described herein can be administered in combination with one or more additional AAV vectors comprising a transgene encoding MTM1 as described herein, or in combination with one or more therapeutic procedures (e.g., nasobiliary drainage (NBD)) and / or agents (e.g., anti-choleretic agents) for treating a neuromuscular disorder (e.g., XLMTM).

[0151] Treatment Procedure In some embodiments, the one or more additional therapeutic treatments is NBD. NBD is a therapeutic treatment performed to assist bile drainage (e.g., when the bile duct is blocked, bile drainage helps bile flow from the liver to the intestine). In some embodiments, NBD is performed using a bile drainage tube (also known as a bile stent), which is a thin, hollow, flexible tube with several small holes along the side. A bile drainage tube can be inserted into a patient's bile duct to induce drainage.

[0152] therapeutic agent In certain embodiments, the one or more additional therapeutic agents are anti-choleretic agents (e.g., bile acids, 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, anti-fibrotic therapies, and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitors), or combinations thereof. In some embodiments, the anti-choleretic agent is administered to the patient in one or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, and 70 doses) beginning within about six weeks before or after (e.g., about six weeks, about five weeks, about four weeks, about three weeks, about two weeks, or about one week before or after) administration of the viral vector to the patient. In some embodiments, the anti-choleretic agent is administered to the patient in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, and 70) doses beginning within about 5 weeks before or after (e.g., about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week before or after) administration of the viral vector to the patient. In some embodiments, the anti-choleretic agent is administered to the patient in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, and 70) doses beginning within about one week before or after (e.g., about one week, about six days, about five days, about four days, about three days, about two days, or about one day before or after) administration of the viral vector to the patient.

[0153] In one embodiment, the anti-choleretic agent is administered to the patient in one or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, and 70 doses) on the same day (at 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) of administration of the viral vector to the patient.

[0154] In one embodiment, the anti-choleretic agent is a bile acid. In one embodiment, the bile acid is ursodeoxycholic acid or a derivative thereof or nor-ursodeoxycholic acid. In one embodiment, the bile acid is ursodiol. In one embodiment, the anti-choleretic agent is an FXR ligand, hi one embodiment, the FXR ligand is obeticholic acid, cilofexor, tropifexor, tretinoin, or EDP-305. In certain embodiments, the one or more anti-choleretic agents is an FGF-19 mimetic. In certain embodiments, the FGF-19 mimetic is aldafermin. In one embodiment, the anti-choleretic agent is a TGR5 agonist, hi one embodiment, the TGR5 agonist is INT-777 or INT-767. In one embodiment, the anti-choleretic agent is a PPAR agonist, hi one embodiment, the PPAR agonist is bezafibrate, seladelper, or elafibrinor. In certain embodiments, the anti-choleretic agent is a PPAR-α agonist. In certain embodiments, the PPAR-α agonist is fenofibrate. In one embodiment, the anti-choleretic agent is a PPAR-δ agonist. In one embodiment, the PPAR-δ agonist is seladelpar. In one embodiment, the anti-choleretic agent is a dual PPAR-α and PPAR-δ agonist, hi one embodiment, the dual PPAR-α-δ agonist is elafibranor. In certain embodiments, the one or more anti-choleretic agents is an ASBT inhibitor, hi certain embodiments, the ASBT inhibitor is odevixibat, maralixibat, or linelixibat. In one embodiment, the anti-choleretic agent is an immunomodulatory agent, hi one embodiment, the immunomodulatory agent is rituximab, abatacept, ustekinumab, infliximab, baricitinib, or FFP104. In one embodiment, the anti-choleretic agent is an anti-fibrotic therapy. In one embodiment, the anti-fibrotic therapy is a vitamin D receptor (VDR) agonist or sintuzumab. In one embodiment, the anti-choleretic agent is a NOX inhibitor. In one embodiment, the NOX inhibitor is setanaxib.

[0155] Recommended clinical parameters for monitoring patients for the development of cholestasis or hyperbilirubinemia In certain embodiments, patients are monitored for the development of cholestasis by serum bile acid testing and / or blood tests (eg, LFTs) described herein. In certain embodiments, patients are monitored for the development of hyperbilirubinemia by a blood test (eg, a bilirubin test) described herein. In certain embodiments, the patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic agent. In one embodiment, the patient is monitored for the development of hyperbilirubinemia, and if the patient exhibits hyperbilirubinemia or one or more symptoms thereof, the patient is administered an anti-choleretic agent. In certain embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by blood tests (e.g., serum acid bile tests or liver function tests). In certain embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia by blood tests (e.g., serum acid bile tests or liver function tests), and if the patient exhibits cholestasis or hyperbilirubinemia or one or more symptoms thereof, the patient is administered an anti-choleretic agent.

[0156] In one embodiment, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, by finding that the patient exhibits a parameter (e.g., serum bile acid level) in a blood test (e.g., serum bile acid test) that is elevated compared to a reference level. In some embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, by finding that a blood test (e.g., a serum bile acid test) shows elevated serum bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels compared to a reference level.

[0157] In some embodiments, the blood test is a liver function test. In one embodiment, 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, the patient is administered an anti-choleretic agent. In one embodiment, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, by the finding that a parameter in a liver function test (e.g., aspartate aminotransferase level or alanine aminotransferase level) is elevated or decreased compared to a reference level.

[0158] I. Serum bile acid test In certain embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia. In certain embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia using a serum bile acid test. In certain 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, the patient is administered an anti-choleretic. In certain embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia using a serum bile acid test, and if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof, the patient is administered an anti-choleretic.

[0159] In certain embodiments, the patient is monitored for cholestasis or hyperbilirubinemia by the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels as measured by a serum bile acid test. In one embodiment, if the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels, as measured by a serum bile acid test, are higher than normal, the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and should be administered an anti-choleretic agent. In one embodiment, if a patient's cholic acid level as measured by serum bile acid testing 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 have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In one embodiment, if a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and the patient's cholic acid level as measured by serum bile acid testing 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), an anti-choleretic agent is administered.

[0160] In one embodiment, if the patient's chenodeoxycholic acid level as measured by serum bile acid testing 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 be experiencing cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In one embodiment, if a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and the patient's chenodeoxycholic acid level as measured by serum bile acid testing 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), an anti-choleretic agent is administered.

[0161] In one embodiment, if the patient's deoxycholic acid level as measured by serum bile acid testing 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 have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In one embodiment, if a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and the patient's deoxycholic acid level as measured by serum bile acid testing 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), an anti-choleretic agent is administered.

[0162] In one embodiment, if the patient's ursodeoxycholic acid level as measured by serum bile acid testing 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), the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In one embodiment, if a patient's ursodeoxycholic acid level as measured by serum bile acid testing 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 be suffering from cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and should be administered an anti-choleretic agent.

[0163] II. Liver function tests In certain embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia associated with LFTs. In certain 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, the patient is administered an anti-choleretic. In certain embodiments, the patient is monitored for the development of cholestasis or hyperbilirubinemia associated with LFTs, and if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, if the patient's LFT parameters (e.g., ASP level or AST level) described herein are greater than the age-adjusted norm, the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is to be administered an anti-choleretic agent.

[0164] IIa. Aspartate aminotransferase In certain embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST levels on LFTs. In certain embodiments, a 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, the patient is administered an anti-choleretic. In certain embodiments, a patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST levels on LFTs, and if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST level in LFTs, and if the patient's AST level is higher than normal, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0165] In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U U / L, 70U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is administered an anticholinergic.

[0166] IIb. Alanine aminotransferase In one embodiment, a patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT levels in LFTs. In one embodiment, a 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, the patient is administered an anti-choleretic. In one embodiment, a patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT levels in LFTs, and if the patient exhibits cholestasis or hyperbilirubinemia, or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT level in LFTs, and if the patient's ALT level is higher than normal, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0167] In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U U / L, 70U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is administered an anticholinergic.

[0168] Recommended clinical parameters for monitoring patients for the development of cholestasis I. Serum bile acid test In some embodiments, the patient is monitored for the development of cholestasis. In some embodiments, the patient is monitored for the development of cholestasis using a serum bile acid test. In some embodiments, the patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In some embodiments, the patient is monitored for the development of cholestasis with a serum bile acid test, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, if the patient's total bile acid level as measured by a serum bile acid test is higher than normal, it is determined that the patient exhibits cholestasis or one or more symptoms thereof and should be administered an anti-choleretic agent.

[0169] In certain embodiments, if the patient's total bile acid level as measured by serum bile acid testing 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, 64 μmol / L, 65 μmol / L, 66 μmol / L, 67 μmol / L, 68 μmol / L, 69 μmol / L, 70 μmol / L, 72 μmol / L, 73 μmol / L, 74 μmol / L, 3 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, 64 μmol / L, 65 μmol / L, 66 μmol / L, 67 μmol / L, 68 μmol / L, 69 μmol / L, 70 μmol / L, 71 μmol / L, 72 μmol / L, 73 μmol / L, 74 μmol / L, 75 μmol / L, 76 μmol / L, 77 μmol / L, 78 μmol / L, 79 μmol / L, 80 μmol / L, 81 μmol / L, 8 2 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, 100 μmol / L), if the patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic agent.

[0170] II. Blood Test In some embodiments, the patient is monitored for the development of cholestasis using blood tests (e.g., LFT or bilirubin tests). In some embodiments, the patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In some embodiments, the patient is monitored for the development of cholestasis involving LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In some embodiments, if 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 have cholestasis or one or more symptoms thereof and will be administered an anti-choleretic agent.

[0171] IIa. Liver function tests In one embodiment, the patient is monitored for the development of cholestasis associated with LFTs. In one embodiment, the patient is monitored for the development of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, the patient is monitored for the development of cholestasis associated with LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, if one or more parameters of the patient's LFTs (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 have cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent.

[0172] IIai. γ-glutamyltransferase In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's GGT levels in LFTs. In one embodiment, a patient is monitored for the onset of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's GGT levels in LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, a patient is determined to have cholestasis or one or more symptoms thereof and will be administered an anti-choleretic agent if the patient exhibits a GGT level higher than the age-adjusted norm. In some embodiments, the patient is a newborn (e.g., 0-6 months 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.

[0173] In certain embodiments, the patient is a newborn (e.g., 0-6 months of age) and the patient's GGT level is outside 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, 26-122 U / L, 27-122 U / L, 28-122 U / L, 29-122 U / L, 30-122 U / L, 31-122 U / L, 32-122 U / L, 33-122 U / L, 34-122 U / L, 35-122 U / L, 36-122 U / L, 37-122 U / L, 38-122 U / L, 39-122 U / L, 40-122 U / L, 41-122 U / L, 42-122 U / L, 43-122 U / L, 44-122 U / L, 45-122 U / L, 46-122 U / L, 47-122 U / L, 48-122 U / L, 49-122 U / L, 50-122 U / L, 51-122 U / L, 52-122 U / L, 53-122 U / L, 54-122 U / L, 55-122 U / L, 56-122 U / L, 57-1 U / L, 30-122U / L, 40-122U / L, 50-122U / L, 60-122U / L, 70-122U / L, 80-122U / L, 90-122U / L, 100-122U / L, 110-122U / L, 120-122U / L, or 121-122U / L), the patient is determined to have cholestasis or one or more of its symptoms, and an anticholinergic is administered. In one embodiment, the patient is a male newborn (e.g., 0-6 months old), and if 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), the patient is determined to have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a male newborn (e.g., 0-6 months of age), and if 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0174] In certain embodiments, the patient is a male infant (e.g., 6-12 months of age) and the patient's GGT level is outside the normal range of about 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 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, 38-39 U / L, 39-39 U / L, 39-39 U / L, 39-39 U / L, 40-40 U / L, 41-41 U / L, 42-41 U / L, 43-41 U / L, 44-41 U / L, 45-41 U / L, 46-4 L, 20-39U / L, 21-39U / L, 22-39U / L, 23-39U / L, 24-39U / L, 25-39U / L, 26-39U / L, 27-39U / L, 28-39U / L, 29-39U / L, 30-39U / L, 31-39U / L, 32-39U / L, 33-39U / L, 34-39U / L, 35-39U / L, 36-39U / L, 37-39U / L, or 38-39U / L), the patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic agent. In one embodiment, the patient is a male infant (e.g., 6-12 months of age), and if 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, 120 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0175] In one embodiment, the patient is a male infant between 1 and about 5 years of age, and if the patient's GGT level is outside the normal range of about 3 to 22 U / L (e.g., about 3 to 22 U / L, 4 to 22 U / L, 5 to 22 U / L, 6 to 22 U / L, 7 to 22 U / L, 8 to 22 U / L, 9 to 22 U / L, 10 to 22 U / L, 11 to 22 U / L, 12 to 22 U / L, 13 to 22 U / L, 14 to 22 U / L, 15 to 22 U / L, 16 to 22 U / L, 17 to 22 U / L, 18 to 22 U / L, 19 to 22 U / L, 20 to 22 U / L, and 21 to 22 U / L), the patient is determined to be exhibiting cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a male infant between 1 and about 5 years of age, and the patient is determined to have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered when the patient's GGT level is less than 3 U / L (e.g., between 2 U / L and 1 U / L). In one embodiment, the patient is a male infant between 1 and about 5 years of age, and if 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, 120 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0176] In certain embodiments, the patient is a female newborn (e.g., 0-6 months of age) and the patient's GGT level is outside 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, 140-132 U / L, 150-132 U / L, 160-132 U / L, 170-132 U / L, 180-132 U / L, 190-132 U / L, 200-132 U / L, 250-132 U / L, 300-132 U / L, 400-132 U / L, 500-132 U / L, 500-132 U / L, 600-132 U / L, 600-132 U / L, 700-132 U / L, 700-132 U / L, 800-132 U / L, 800-132 U / L, 800-132 U / L, 900-132 U / L, 900-132 U / L, U / L, 50–132U / L, 60–132U / L, 70–132U / L, 80–132U / L, 90–132U / L, 100–132U / L, 110–132U / L, 120–132U / L, 130–132U / L, and 131–132U / L), the patient is determined to have cholestasis or one or more of its symptoms, and an anticholinergic is administered. In one embodiment, the patient is a female newborn (e.g., 0-6 months old), and if 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), the patient is determined to have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a female newborn (e.g., 0-6 months of age), and if 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0177] In certain embodiments, the patient is a female infant (e.g., 6-12 months of age) and the patient's GGT level is outside the normal range of about 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 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, 38-39 U / L, 39-39 U / L, 39-39 U / L, 39-39 U / L, 40-40 U / L, 41-41 U / L, 42-41 U / L, 43-41 U / L, 44-41 U / L, 45-41 U / L, 46-4 / L, 20-39U / L, 21-39U / L, 22-39U / L, 23-39U / L, 24-39U / L, 25-39U / L, 26-39U / L, 27-39U / L, 28-39U / L, 29-39U / L, 30-39U / L, 31-39U / L, 32-39U / L, 33-39U / L, 34-39U / L, 35-39U / L, 36-39U / L, 37-39U / L, or 38-39U / L), the patient is determined to have cholestasis or one or more of its symptoms, and an anticholinergic is administered.

[0178] In one embodiment, the patient is a female (e.g., 6-12 months of age) and if 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, 120 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 have cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent. In one embodiment, the patient is a female child between 1 and about 5 years of age, and if the patient's GGT level is outside the normal range of about 3 to 22 U / L (e.g., about 3 to 22 U / L, 4 to 22 U / L, 5 to 22 U / L, 6 to 22 U / L, 7 to 22 U / L, 8 to 22 U / L, 9 to 22 U / L, 10 to 22 U / L, 11 to 22 U / L, 12 to 22 U / L, 13 to 22 U / L, 14 to 22 U / L, 15 to 22 U / L, 16 to 22 U / L, 17 to 22 U / L, 18 to 22 U / L, 19 to 22 U / L, 20 to 22 U / L, and 21 to 22 U / L), the patient is determined to be exhibiting cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a female child between 1 and about 5 years of age, the patient's GGT level is less than 3 U / L (e.g., between 2 U / L and 1 U / L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholeretic is administered. In one embodiment, the patient is a female child between 1 and about 5 years of age, and if 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, 120 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 have cholestasis or one or more symptoms thereof, and an anticholeretic is administered.

[0179] IIaii. Alkaline phosphatase In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's ASP levels in LFTs. In one embodiment, a patient is monitored for the onset of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's ASP levels in LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, the patient is monitored for the development of cholestasis by measuring the patient's ASP level in LFTs, and if the patient's ASP level is higher than normal, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0180] In one embodiment, if the patient's ASP level is outside the normal range of about 50-300 U / L (e.g., about 51-300 U / L, about 52-300 U / L, about 53-300 U / L, about 54-300 U / L, about 55-300 U / L, about 56-300 U / L, about 57-300 U / L, about 58-300 U / L, about 59-300 U / L, about 60-300 U / L, about 65-300 U / L, about 70-300 U / L, about 71-300 U / L, about 72-300 U / L, about 73-300 U / L, about 74-300 U / L, about 75-300 U / L, about 76-300 U / L, about 77-300 U / L, about 78-300 U / L, about 79-300 U / L, about 80-300 U / L, about 81-300 U / L, about 82-300 U / L, about 83-300 U / L, about 84-300 U / L, about 85-300 U / L, about 86-300 U / L, about 87-300 U / L, about 88-300 U / L, about 89-300 U / L, about 90-300 U / L, about 91-300 U / L, about 92-300 U / L, about 93-300 U / L, about 94-300 U / L, about 95-300 U / L, about 96-300 U / L, about 97-300 U / L, about 98-300 0 U / L, about 80-300 U / L, about 90-300 U / L, about 100-300 U / L, about 125-300 U / L, about 150-300 U / L, about 175-300 U / L, about 200-300 U / L, about 225-300 U / L, about 250-300 U / L, or about 275-300 U / L), the patient is determined to have cholestasis or one or more symptoms thereof and is to be administered an anticholinergic agent. In certain embodiments, if 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, 28 U / L, 29 ... 5U / L, 24U / L, 23U / L, 22U / L, 21U / L, 20U / L, 19U / L, 18U / L, 17U / L, 16U / L, 15U / L, 14U / L, 13U / L, 12U / L, 11U / L, 10U / L, 9U / L, 8U / L, 7U / L, 6U / L, 5U / L, 4U / L, 3U / L, 2U / L, and 1U / L), when a patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic. In certain embodiments, if 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, 326 U / L, 327 U / L, 328 U / L, 329 U / L, 330 U / L, 331 U / L, 332 U / L, 333 U / L, 334 U / L, 335 U / L, 336 U / L, 337 U / L, 338 U / L, 339 U / L, 340 U / L, 341 U / L, 342 U / L, 343 U / L, 344 U / L, 345 U / L, 346 U / L, 347 U / L, 348 U / L, 349 U / L, 350 U / L, 351 U / L, 352 U / L, 353 U / L, 354 U / L, 355 U / L, 356 U / L, 357 U / L, 358 U / L, 359 U / L, 36 6U / L, 317U / L, 318U / L, 319U / L, 320U / L, 321U / L, 322U / L, 323U / L, 324U / L, 325U / L, 330U / L, 340U / L, 350U / L, 400U / L, and 500U / L), the patient is determined to have cholestasis or one or more of its symptoms and is to be administered an anticholinergic agent.

[0181] IIaiii. Aspartate aminotransferase In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's AST levels on LFTs. In one embodiment, a patient is monitored for the onset of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's AST levels on LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, the patient is monitored for the development of cholestasis by measuring the patient's AST level in LFTs, and if the patient's AST level is higher than normal, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In certain embodiments, if 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, 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, , 70U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if a patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic.

[0182] IIaiv. Alanine aminotransferase In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's ALT levels in LFTs. In one embodiment, a patient is monitored for the onset of cholestasis, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, a patient is monitored for the onset of cholestasis by measuring the patient's ALT levels in LFTs, and if the patient exhibits cholestasis or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, the patient is monitored for the development of cholestasis by measuring the patient's ALT level in LFTs, and if the patient's ALT level is higher than normal, the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In certain embodiments, if 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, 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, , 70U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if a patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic.

[0183] Recommended clinical parameters for monitoring patients for the development of hyperbilirubinemia Bilirubin test In some embodiments, the patient is monitored for the development of hyperbilirubinemia. In some embodiments, the patient is monitored for the development of hyperbilirubinemia using a bilirubin test. In some embodiments, the patient is monitored for the development of hyperbilirubinemia, and if the patient exhibits hyperbilirubinemia or one or more symptoms thereof, the patient is administered an anti-choleretic. In some embodiments, the patient is monitored for the development of hyperbilirubinemia with a bilirubin test, and if the patient exhibits hyperbilirubinemia or one or more symptoms thereof, the patient is administered an anti-choleretic. In one embodiment, a patient exhibits hyperbilirubinemia or one or more symptoms thereof, and it is determined that an anti-choleretic agent is to be administered when the patient exhibits bilirubin levels higher than normal.

[0184] In certain embodiments, if the patient's TB level is above 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 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 mg / dL, 3.9 mg / dL, 3.1 ... 7mg / dL, 3.8mg / dL, 3.9mg / dL, 4mg / dL, 4.1mg / dL, 4.2mg / dL, 4.3mg / dL, 4.4mg / dL, 4.5mg / dL, 4.6mg / dL, 4.7mg / dL, 4.8mg / dL, 4.9mg / dL, 5mg / dL, 10mg / dL, 15mg / dL, 20mg / dL, 30mg / dL, 40mg / dL, 50mg / dL, 60mg / dL, 70mg / dL, 80mg / dL, 90mg / dL and 100mg / dL), the patient is determined to have hyperbilirubinemia or one or more symptoms thereof and should be administered an anticholeretic. In certain embodiments, if 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, / dL, 1.5mg / dL, 1.6mg / dL, 1.7mg / dL, 1.8mg / dL, 1.9mg / dL, 2mg / dL, 2.1mg / dL, 2.2mg / dL, 2.3mg / dL, 2.4mg / dL, 2.5mg / dL, 2.6mg / dL, 2.7mg / dL, 2.8mg / dL, 2.9mg / dL, 3mg / dL, 3.1mg / dL, 3.2mg / dL, 3.3mg / dL, 3.4mg / dL, 3.5mg / dL, 3.6mg / dL, 3.7mg / dL, 3.8mg / dL, 3.9mg / dL, 4mg / dL, 4.1mg / dL, 4.2mg / dL, 4.3mg / dL, 4.4mg / dL, 4.5mg / dL, 4.6mg / dL, 4.7mg / dL, 4.8mg / 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 have hyperbilirubinemia or one or more symptoms thereof and should be administered an anticholeretic.

[0185] In certain embodiments, a patient is determined to have hyperbilirubinemia or one or more symptoms thereof, and the patient is determined to have hyperbilirubinemia if the patient has a blood bilirubin level above 1 mg / dL (e.g., 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 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, 3.10 mg / dL, 3.11 mg / dL, 3.12 mg / dL, 3.13 mg / dL, 3.14 mg / dL, 3.15 mg / dL, 3.16 mg / dL, 3.17 mg / dL, 3.18 mg / dL, 3.19 mg / dL, 3.20 mg / dL, 3.21 mg / dL, 3.22 mg / dL, 3.23 mg / dL, 3.24 mg / dL, 3.25 mg / dL, 3.26 mg / dL, 3.27 mg / dL, 3.28 mg / dL, 3.2 5mg / dL, 3.6mg / dL, 3.7mg / dL, 3.8mg / dL, 3.9mg / dL, 4mg / dL, 4.1mg / dL, 4.2mg / dL, 4.3mg / dL, 4.4mg / dL, 4.5mg / dL, 4.6mg / dL, 4.7mg / dL, 4.8mg / dL, 4.9mg / dL, 5mg / dL, 10mg / dL, 15mg / dL, 20mg / dL, 30mg / dL, 40mg / dL, 50mg / dL, 60mg / dL, 70mg / dL, 80mg / dL, 90mg / dL and 100mg / dL), the patient is determined to have hyperbilirubinemia or one or more symptoms thereof and should be administered an anticholeretic.

[0186] Recommended Clinical Parameters for Determining if a Patient Exhibits Cholestasis or Hyperbilirubinemia or Symptoms In certain embodiments, a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, by determining that one or more parameters (e.g., total bile acid level, GGT level, ASP level, AST level, and ALT level) of the patient's serum bile acid test and / or blood test (e.g., LFT) are higher or lower than age-adjusted standards as described herein, and the patient is administered an anti-choleretic agent. In one embodiment, a patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, by determining that one or more parameters (e.g., bilirubin level) of a patient's blood test (e.g., bilirubin test) are greater than a standard as described herein, and the patient is administered an anti-choleretic agent. In one embodiment, if the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels, as measured by a serum bile acid test, are higher than normal, the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and should be administered an anti-choleretic agent. In one embodiment, if the patient's LFT parameters (e.g., ASP level or AST level) described herein are greater than the age-adjusted norm, the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is to be administered an anti-choleretic agent.

[0187] I. Serum bile acid test In one embodiment, if the patient's bile acid (e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid) levels, as measured by a serum bile acid test, are higher than normal, the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and should be administered an anti-choleretic agent. In one embodiment, if a patient's cholic acid level as measured by serum bile acid testing 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 have cholestasis, hyperbilirubinemia, or one or more symptoms thereof.

[0188] In one embodiment, if a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and the patient's cholic acid level as measured by serum bile acid testing 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), an anti-choleretic agent is administered.

[0189] In one embodiment, if the patient's chenodeoxycholic acid level as measured by serum bile acid testing 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 be experiencing cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In one embodiment, if a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and the patient's chenodeoxycholic acid level as measured by serum bile acid testing 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), an anti-choleretic agent is administered.

[0190] In one embodiment, if the patient's deoxycholic acid level as measured by serum bile acid testing 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 be experiencing cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In one embodiment, the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-choleretic agent is administered if 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).

[0191] In one embodiment, if the patient's ursodeoxycholic acid level as measured by serum bile acid testing 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), the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In one embodiment, an anti-choleretic agent is administered if a patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and 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).

[0192] II. Liver function tests In one embodiment, if the patient's LFT parameters (e.g., ASP level or AST level) described herein are greater than the age-adjusted norm, the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is to be administered an anti-choleretic agent.

[0193] IIa. Aspartate aminotransferase In one embodiment, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's AST level in LFTs, and if the patient's AST level is higher than normal, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0194] In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U U / L, 70U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is administered an anticholinergic.

[0195] IIb. Alanine aminotransferase In one embodiment, the patient is monitored for the development of cholestasis or hyperbilirubinemia by measuring the patient's ALT level in LFTs, and if the patient's ALT level is higher than normal, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0196] In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U U / L, 70U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof. In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is administered an anticholinergic.

[0197] Recommended clinical parameters for determining whether a patient has cholestasis or its symptoms I. Serum bile acid test In one embodiment, a patient is determined to exhibit cholestasis or one or more symptoms thereof, and is administered an anti-choleretic agent if the patient exhibits higher than normal levels of acidic bile as measured by a serum bile acid test.

[0198] In certain embodiments, if the patient's total bile acid level as measured by serum bile acid testing 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, 64 μmol / L, 65 μmol / L, 66 μmol / L, 67 μmol / L, 68 μmol / L, 69 μmol / L, 70 μmol / L, 72 μmol / L, 73 μmol / L, 74 μmol / L, 3 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, 64 μmol / L, 65 μmol / L, 66 μmol / L, 67 μmol / L, 68 μmol / L, 69 μmol / L, 70 μmol / L, 71 μmol / L, 72 μmol / L, 73 μmol / L, 74 μmol / L, 75 μmol / L, 76 μmol / L, 77 μmol / L, 78 μmol / L, 79 μmol / L, 80 μmol / L, 81 μmol / L, 8 2 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, 100 μmol / L), if the patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic agent.

[0199] II. Blood Test In one embodiment, a patient is determined to have cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent if one or more parameters of the patient's blood tests (e.g., LFT or bilirubin tests) (e.g., GGT level, ASP level, AST level, ALT level, and bilirubin level) are greater than the age-adjusted norms described herein.

[0200] IIa. Liver function tests In one embodiment, a patient is determined to exhibit cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent if one or more parameters of the patient's LFTs (e.g., GGT level, ASP level, AST level, and ALT level) are greater than age-adjusted norms as described herein.

[0201] IIai. γ-glutamyltransferase In one embodiment, a patient is determined to exhibit cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent if the patient exhibits GGT levels, as measured by LFTs, higher than age-adjusted norms. In some embodiments, the patient is a newborn (e.g., 0-6 months old), a toddler (e.g., 6-12 months old), or a child aged 1 to about 5 years. In some embodiments, the patient is a newborn aged 0-6 months. In some embodiments, the patient is a toddler aged 6-12 months. In some embodiments, the patient is a child aged 1 to about 5 years.

[0202] In certain embodiments, the patient is a newborn (e.g., 0-6 months of age) and the patient's GGT level is outside 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, 26-122 U / L, 27-122 U / L, 28-122 U / L, 29-122 U / L, 30-122 U / L, 31-122 U / L, 32-122 U / L, 33-122 U / L, 34-122 U / L, 35-122 U / L, 36-122 U / L, 37-122 U / L, 38-122 U / L, 39-122 U / L, 40-122 U / L, 41-122 U / L, 42-122 U / L, 43-122 U / L, 44-122 U / L, 45-122 U / L, 46-122 U / L, 47-122 U / L, 48-122 U / L, 49-122 U / L, 50-122 U / L, 51-122 U / L, 52-122 U / L, 53-122 U / L, 54-122 U / L, 55-122 U / L, 56-122 U / L, 57-1 U / L, 30-122U / L, 40-122U / L, 50-122U / L, 60-122U / L, 70-122U / L, 80-122U / L, 90-122U / L, 100-122U / L, 110-122U / L, 120-122U / L, or 121-122U / L), the patient is determined to have cholestasis or one or more of its symptoms, and an anticholinergic is administered. In one embodiment, the patient is a male newborn (e.g., 0-6 months old), and if 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), the patient is determined to have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a male newborn (e.g., 0-6 months of age), and if 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0203] In certain embodiments, the patient is a male infant (e.g., 6-12 months of age) and the patient's GGT level is outside the normal range of about 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 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, 38-39 U / L, 39-39 U / L, 39-39 U / L, 39-39 U / L, 40-40 U / L, 41-41 U / L, 42-41 U / L, 43-41 U / L, 44-41 U / L, 45-41 U / L, 46-4 L, 20-39U / L, 21-39U / L, 22-39U / L, 23-39U / L, 24-39U / L, 25-39U / L, 26-39U / L, 27-39U / L, 28-39U / L, 29-39U / L, 30-39U / L, 31-39U / L, 32-39U / L, 33-39U / L, 34-39U / L, 35-39U / L, 36-39U / L, 37-39U / L, or 38-39U / L), the patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic agent. In one embodiment, the patient is a male infant (e.g., 6-12 months of age), and if 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, 120 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0204] In one embodiment, the patient is a male infant between 1 and about 5 years of age, and if the patient's GGT level is outside the normal range of about 3 to 22 U / L (e.g., about 3 to 22 U / L, 4 to 22 U / L, 5 to 22 U / L, 6 to 22 U / L, 7 to 22 U / L, 8 to 22 U / L, 9 to 22 U / L, 10 to 22 U / L, 11 to 22 U / L, 12 to 22 U / L, 13 to 22 U / L, 14 to 22 U / L, 15 to 22 U / L, 16 to 22 U / L, 17 to 22 U / L, 18 to 22 U / L, 19 to 22 U / L, 20 to 22 U / L, and 21 to 22 U / L), the patient is determined to be exhibiting cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a male infant between 1 and about 5 years of age, and the patient is determined to have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered when the patient's GGT level is less than 3 U / L (e.g., between 2 U / L and 1 U / L). In one embodiment, the patient is a male infant between 1 and about 5 years of age, and if 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, 120 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0205] In certain embodiments, the patient is a female newborn (e.g., 0-6 months of age) and the patient's GGT level is outside 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, 140-132 U / L, 150-132 U / L, 160-132 U / L, 170-132 U / L, 180-132 U / L, 190-132 U / L, 200-132 U / L, 250-132 U / L, 300-132 U / L, 400-132 U / L, 500-132 U / L, 500-132 U / L, 600-132 U / L, 600-132 U / L, 700-132 U / L, 700-132 U / L, 800-132 U / L, 800-132 U / L, 800-132 U / L, 900-132 U / L, 900-132 U / L, U / L, 50–132U / L, 60–132U / L, 70–132U / L, 80–132U / L, 90–132U / L, 100–132U / L, 110–132U / L, 120–132U / L, 130–132U / L, and 131–132U / L), the patient is determined to have cholestasis or one or more of its symptoms, and an anticholinergic is administered. In one embodiment, the patient is a female newborn (e.g., 0-6 months old), and if 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), the patient is determined to have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a female newborn (e.g., 0-6 months of age), and if 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 have cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered.

[0206] In certain embodiments, the patient is a female infant (e.g., 6-12 months of age) and the patient's GGT level is outside the normal range of about 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 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, 38-39 U / L, 39-39 U / L, 39-39 U / L, 39-39 U / L, 40-40 U / L, 41-41 U / L, 42-41 U / L, 43-41 U / L, 44-41 U / L, 45-41 U / L, 46-4 / L, 20-39U / L, 21-39U / L, 22-39U / L, 23-39U / L, 24-39U / L, 25-39U / L, 26-39U / L, 27-39U / L, 28-39U / L, 29-39U / L, 30-39U / L, 31-39U / L, 32-39U / L, 33-39U / L, 34-39U / L, 35-39U / L, 36-39U / L, 37-39U / L, or 38-39U / L), the patient is determined to have cholestasis or one or more of its symptoms, and an anticholinergic is administered.

[0207] In one embodiment, the patient is a female (e.g., 6-12 months of age) and if 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, 120 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 have cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent. In one embodiment, the patient is a female child between 1 and about 5 years of age, and if the patient's GGT level is outside the normal range of about 3 to 22 U / L (e.g., about 3 to 22 U / L, 4 to 22 U / L, 5 to 22 U / L, 6 to 22 U / L, 7 to 22 U / L, 8 to 22 U / L, 9 to 22 U / L, 10 to 22 U / L, 11 to 22 U / L, 12 to 22 U / L, 13 to 22 U / L, 14 to 22 U / L, 15 to 22 U / L, 16 to 22 U / L, 17 to 22 U / L, 18 to 22 U / L, 19 to 22 U / L, 20 to 22 U / L, and 21 to 22 U / L), the patient is determined to be exhibiting cholestasis or one or more symptoms thereof, and an anti-choleretic agent is administered. In one embodiment, the patient is a female child between 1 and about 5 years of age, the patient's GGT level is less than 3 U / L (e.g., between 2 U / L and 1 U / L), the patient is determined to exhibit cholestasis or one or more symptoms thereof, and an anticholeretic is administered. In one embodiment, the patient is a female child between 1 and about 5 years of age, and if 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, 120 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 have cholestasis or one or more symptoms thereof, and an anticholeretic is administered.

[0208] IIaii. Alkaline phosphatase In one embodiment, a patient is determined to exhibit cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent if the patient exhibits higher than normal ASP levels as measured by LFTs.

[0209] In one embodiment, if the patient's ASP level is outside the normal range of about 50-300 U / L (e.g., about 51-300 U / L, about 52-300 U / L, about 53-300 U / L, about 54-300 U / L, about 55-300 U / L, about 56-300 U / L, about 57-300 U / L, about 58-300 U / L, about 59-300 U / L, about 60-300 U / L, about 65-300 U / L, about 70-300 U / L, about 71-300 U / L, about 72-300 U / L, about 73-300 U / L, about 74-300 U / L, about 75-300 U / L, about 76-300 U / L, about 77-300 U / L, about 78-300 U / L, about 79-300 U / L, about 80-300 U / L, about 81-300 U / L, about 82-300 U / L, about 83-300 U / L, about 84-300 U / L, about 85-300 U / L, about 86-300 U / L, about 87-300 U / L, about 88-300 U / L, about 89-300 U / L, about 90-300 U / L, about 91-300 U / L, about 92-300 U / L, about 93-300 U / L, about 94-300 U / L, about 95-300 U / L, about 96-300 U / L, about 97-300 U / L, about 98-300 0 U / L, about 80-300 U / L, about 90-300 U / L, about 100-300 U / L, about 125-300 U / L, about 150-300 U / L, about 175-300 U / L, about 200-300 U / L, about 225-300 U / L, about 250-300 U / L, or about 275-300 U / L), the patient is determined to have cholestasis or one or more symptoms thereof and is to be administered an anticholinergic agent. In certain embodiments, if 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, 28 U / L, 29 ... 5U / L, 24U / L, 23U / L, 22U / L, 21U / L, 20U / L, 19U / L, 18U / L, 17U / L, 16U / L, 15U / L, 14U / L, 13U / L, 12U / L, 11U / L, 10U / L, 9U / L, 8U / L, 7U / L, 6U / L, 5U / L, 4U / L, 3U / L, 2U / L, and 1U / L), when a patient is determined to have cholestasis or one or more symptoms thereof and is administered an anticholinergic. In certain embodiments, if 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, 326 U / L, 327 U / L, 328 U / L, 329 U / L, 330 U / L, 331 U / L, 332 U / L, 333 U / L, 334 U / L, 335 U / L, 336 U / L, 337 U / L, 338 U / L, 339 U / L, 340 U / L, 341 U / L, 342 U / L, 343 U / L, 344 U / L, 345 U / L, 346 U / L, 347 U / L, 348 U / L, 349 U / L, 350 U / L, 351 U / L, 352 U / L, 353 U / L, 354 U / L, 355 U / L, 356 U / L, 357 U / L, 358 U / L, 359 U / L, 36 6U / L, 317U / L, 318U / L, 319U / L, 320U / L, 321U / L, 322U / L, 323U / L, 324U / L, 325U / L, 330U / L, 340U / L, 350U / L, 400U / L, and 500U / L), the patient is determined to have cholestasis or one or more of its symptoms and is to be administered an anticholinergic agent.

[0210] IIaiii. Aspartate aminotransferase In one embodiment, a patient is determined to exhibit cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent if the patient exhibits higher than normal AST levels as measured in LFTs. In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is administered an anticholinergic.

[0211] IIaiv. Alanine aminotransferase In one embodiment, a patient is determined to exhibit cholestasis or one or more symptoms thereof and is administered an anti-choleretic agent if the patient exhibits higher than normal ALT levels as measured by LFTs. In certain embodiments, if 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, 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, 71 U / L, 72 U / L, 73 U / L, 74 U / L, 75 U / L, 76 U / L, 77 U / L, 78 U / L, 79 U / L, 80 U / L, 81 U / L, 82 U / L, 83 U / L, 84 U / L, 85 U / L, 86 U / L, 87 U / L, 88 U / L, 89 U / L, 90 U / L, 91 U / L, 92 U / L, 93 U / L, 94 U / L, 95 U / L, 96 U / L, 97 U / L, 98 U / L, 99 U / L, 100 U / L, 101 U / L, 102 U / L, 103 U / L, 104 U / L, 105 U / L, 106 U / L, 107 U / L, 108 U / L, 109 U / L, 110 U / L, 111 U / L, 112 U / L, 113 U / L, 114 U / L, 115 U / L, 116 U / L, 117 U / L, 75U / L, 80U / L, 85U / L, 90U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 200U / L, 300U / L, 400U / L, and 500U / L), if the patient is determined to have cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and is administered an anticholinergic.

[0212] Recommended Clinical Parameters for Determining if a Patient Exhibits Hyperbilirubinemia or Symptoms of Hyperbilirubinemia Bilirubin test In one embodiment, a patient is determined to have hyperbilirubinemia or one or more symptoms thereof if they exhibit higher than normal bilirubin levels as measured by a blood test (e.g., a bilirubin test), and are administered an anticholeretic agent.

[0213] In certain embodiments, if the patient's TB level is above 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 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 mg / dL, 3.9 mg / dL, 3.1 ... 7mg / dL, 3.8mg / dL, 3.9mg / dL, 4mg / dL, 4.1mg / dL, 4.2mg / dL, 4.3mg / dL, 4.4mg / dL, 4.5mg / dL, 4.6mg / dL, 4.7mg / dL, 4.8mg / dL, 4.9mg / dL, 5mg / dL, 10mg / dL, 15mg / dL, 20mg / dL, 30mg / dL, 40mg / dL, 50mg / dL, 60mg / dL, 70mg / dL, 80mg / dL, 90mg / dL and 100mg / dL), the patient is determined to have hyperbilirubinemia or one or more symptoms thereof and should be administered an anticholeretic. In certain embodiments, if 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, / dL, 1.5mg / dL, 1.6mg / dL, 1.7mg / dL, 1.8mg / dL, 1.9mg / dL, 2mg / dL, 2.1mg / dL, 2.2mg / dL, 2.3mg / dL, 2.4mg / dL, 2.5mg / dL, 2.6mg / dL, 2.7mg / dL, 2.8mg / dL, 2.9mg / dL, 3mg / dL, 3.1mg / dL, 3.2mg / dL, 3.3mg / dL, 3.4mg / dL, 3.5mg / dL, 3.6mg / dL, 3.7mg / dL, 3.8mg / dL, 3.9mg / dL, 4mg / dL, 4.1mg / dL, 4.2mg / dL, 4.3mg / dL, 4.4mg / dL, 4.5mg / dL, 4.6mg / dL, 4.7mg / dL, 4.8mg / 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 have hyperbilirubinemia or one or more symptoms thereof and should be administered an anticholeretic.

[0214] In certain embodiments, a patient is determined to have hyperbilirubinemia or one or more symptoms thereof, and the patient is determined to have hyperbilirubinemia if the patient has a blood bilirubin level above 1 mg / dL (e.g., 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 mg / dL, 3.7 mg / dL, 3.8 mg / dL, 3.9 mg / dL, 3.10 mg / dL, 3.11 mg / dL, 3.12 mg / dL, 3.13 mg / dL, 3.14 mg / dL, 3.15 mg / dL, 3.16 mg / dL, 3.17 mg / dL, 3.18 mg / dL, 3.19 mg / dL, 3.20 mg / dL, 3.21 mg / dL, 3.22 mg / dL, 3.23 mg / dL, 3.24 mg / dL, 3.25 mg / dL, 3.26 mg / dL, 3.27 mg / dL, 3.28 mg / dL, 3.2 5mg / dL, 3.6mg / dL, 3.7mg / dL, 3.8mg / dL, 3.9mg / dL, 4mg / dL, 4.1mg / dL, 4.2mg / dL, 4.3mg / dL, 4.4mg / dL, 4.5mg / dL, 4.6mg / dL, 4.7mg / dL, 4.8mg / dL, 4.9mg / dL, 5mg / dL, 10mg / dL, 15mg / dL, 20mg / dL, 30mg / dL, 40mg / dL, 50mg / dL, 60mg / dL, 70mg / dL, 80mg / dL, 90mg / dL and 100mg / dL), the patient is determined to have hyperbilirubinemia or one or more symptoms thereof and should be administered an anticholeretic.

[0215] [Example] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. [Example]

[0216] Evaluation of safety and efficacy of pseudotyped AAV8 vectors containing nucleic acid sequences encoding the myotubularin 1 gene in a mouse model of X-linked myotubular myopathy Objectives and Test Methodology The purpose of this study was to conduct efficacy and sampling studies using myotubularin 1 (MTM1) hemizygous ("HEMi") knockout (KO) mice to determine the possible effects of liver-specific MTM1 expression in mice. Mice were intravenously administered different test articles at approximately 4 weeks of age. Mice were sampled at approximately 16 weeks of age (approximately 12 weeks post-dose). Tissue samples were used for measuring myotubularin and bile salt transporter expression, bile acid levels, and histopathology. Whole blood samples were used for hematology analysis, and serum samples were used for clinical chemistry analysis. Additionally, samples were collected from a group of naive MTM1 KO mice at approximately 5 weeks of age, and tissues were used for histopathology. The study was conducted in accordance with appropriate methodology and standard operating procedures (SOPs) at the testing facility or testing site. The study complied with standard ethical animal welfare practices.

[0217] Materials and Methods In this study, MTM1 KO mice were used along with 28 male wild-type (WT) mice and 70 male KO mice. Animals were sampled at approximately 5 and 16 weeks. Each animal was permanently identified with a unique permanent identification number in accordance with the laboratory's standard operating procedures. Animals were identified using toe and tail numbering and ear marking. An acclimation period of approximately 3–4 days was allowed before administration. Animals were housed in groups of 3–4 per cage, with at least one WT mouse in each cage for social purposes. Mice were housed using individually ventilated cage systems (IVCs) and polycarbonate type II long cages. Veterinary care was available throughout the course of the study, and animals were examined by responsible trained personnel and supervised by a veterinarian as warranted by clinical signs or other changes. Prior to the start of the survival phase, any animals deemed unsuitable for use in the study were replaced with replacement animals obtained from the same shipment and maintained under the same environmental conditions. Animals in poor health or at the extremes of the weight range were not assigned to groups. When setting up groups for this study, KO mice were randomized into groups so that entire litters of mice were not in a single test group to avoid the "litter effect." Mice were housed in groups of up to 4-5 mice. Each cage contained 3-4 KO mice and 1-2 WT mice for socialization purposes. None of the KO mice were housed individually.

[0218] Four types of AAV8 vectors were tested: 1) AAV8 vectors containing the mouse MTM1 (mMTM1) gene under the control of the desmin promoter (AAV8-Des-mMTM1), 2) AAV8 vectors containing mMTM1 under the control of the APoE-A1AT promoter (AAV8-APoE-A1AT-mMTM1), 3) AAV8 vectors 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 capsid. A vehicle control containing a placebo of 0.01% Pluronic® in Ringer's lactate solution was used. Dose levels were selected based on previous studies in mice. Because previously tested high doses demonstrated significant pharmacological effects without toxicity, the high doses were multiples of the previous high doses in mice. 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 2–3 h between doses. The administration site, completion time, and dose amount were recorded. Animals were anesthetized with isoflurane, as needed. This administration route is consistent with the proposed route of administration in humans and is expected to provide adequate systemic exposure for testing and relevant pharmacological activity. The dosing frequency was once on day 1. The dose level was expected to identify achievable relevant pharmacological activity. Necropsies were performed approximately 12 weeks after dosing. The treatment / test groups are summarized in Table 4 below. Additionally, a group of 10 naive MTM1 KO mice was sampled at approximately 5 (5.3) weeks of age.

[0219] Table 4. Mouse test groups

[0220] [Table 4] The volumes of test article used before dosing are summarized in Table 5 below. Table 5: Test article volume before administration

[0221] [Table 5] The following humane endpoint criteria were applicable to this study. Mice were euthanized for welfare reasons if they met the predetermined humane endpoint criteria. If any of the mice needed to be euthanized for welfare reasons, a laparotomy was performed and gross observations were recorded. If the animals' general health significantly deteriorated, a shortened tissue isolation protocol was applied, if possible. The shortened tissue isolation protocol consisted of collecting skeletal muscle (quadriceps) and one liver lobe in liquid nitrogen, and one liver lobe and heart (dorsal portion) tissue in formalin. Skeletal muscle and liver lobes were stored at -80°C, while the liver lobe and heart (dorsal portion) in formalin were stored at room temperature. Due to limited staff capacity to perform sampling for acute cases requiring immediate action in the evenings and weekends, mice were euthanized by CO2 overdose and decapitated. No sampling was performed in these cases.

[0222] Body weights were measured three times weekly, and mortality was recorded. At the endpoint, mice were euthanized by deep anesthesia with sodium pentobarbital (180 mg / kg) at approximately 16–17 weeks of age (approximately 12–13 weeks post-dose) if they survived to the end of the study. As much whole blood as possible was collected by cardiac puncture. Blood was collected first for hematology (100 μL of EDTA whole blood) and then for clinical chemistry (200 μL of whole blood to separate a minimum of 80 μL of serum). The remaining serum was collected. Mice were then transcardially perfused with heparinized saline to remove blood from tissues. Groups of 10 naive MTM1 KO mice 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 collected for other analyses. Tissue samples were used for the measurement of myotubularin and bile salt transporter expression, bile acid levels, and histopathology. In-life and endpoint whole blood samples were used for hematology analysis, and 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 harvested along with the left and right atria and then halved in the coronal plane. Tissue harvesting and analysis are summarized in Table 6 below.

[0223] Table 6. Collection and analysis of mouse tissues

[0224] [Table 6] Histology and Histopathology The following tissue samples were dissected and placed in 10% neutral buffered formalin: ●Quadriceps (L (left) muscle, 1 / 2) ●Diaphragm (1 sample) Liver (left lobe and small part of the right middle lobe (including the gallbladder)) Heart (1 / 2, dorsal) Lungs (1 sample) ●Kidney (L+R (right), 2 samples) ●Spleen (1 sample) Brain (1 sample) Samples were embedded in paraffin, sectioned onto slides, and stained with hematoxylin and eosin (H&E) for microscopic evaluation. Sample collection for: myotubularin (ELISA), vector copy number (VCN), and / or bile acid analysis (ELISA); myotubularin and bile salt transporter analysis by immunohistochemistry (IHC); and spare tissue Sterile collection for bioanalysis (myotubularin (ELISA), vector copy number (VCN) and / or bile acid analysis (ELISA); myotubularin and bile salt transporter analysis by IHC; and spare tissue): Each tissue was collected into an RNase-free screw-top polypropylene tube, flash-frozen in liquid nitrogen under sterile conditions, and stored at -80°C according to laboratory SOPs. Samples were stored on dry ice before storage. Scalpel blades, weighing boats (small dishes), and dissection instruments were changed / cleaned before handling a new mouse. All equipment was wiped with RNAse Zap (or similar) between each tissue collection. Precautions were taken to prevent cross-contamination during tissue collection.

[0225] 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 (R muscle) ●Diaphragm (1 sample) ● Liver (right lobe, right lateral lobe, caudate lobe) Heart (half, ventral side) (split into two specimens) Myotubularin and bile salt transporter analysis by IHC The following tissue samples were dissected and frozen in liquid nitrogen: ●Quadriceps (L muscle, 1 / 2) ●Diaphragm (1 sample) ●Liver (left middle lobe and remaining right middle lobe without the gallbladder).

[0226] Reserve organization The following tissue samples were dissected and frozen in liquid nitrogen: ●Triceps (R muscle) ●Gastrocnemius (R muscle) ● Tibialis anterior (R muscle) Clinical Chemistry Panels At lifetime sampling and terminal necropsy, a clinical biochemistry panel of markers was analyzed in nonhemolyzed serum for the following parameters: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (AFOS), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), creatinine kinase (CK), albumin, total bilirubin, total protein, creatinine, bile acids, calculated globulin, calculated albumin / globulin ratio, and urea nitrogen. When blood samples were limited, analytical priorities were given in the following order: 1. Alanine aminotransferase (ALAT) 2. Total / direct bilirubin 3. Bile acids 4. Aspartate aminotransferase (ASAT) 5. Alkaline phosphatase (AFOS) 6. γ-glutamyltransferase (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 from in-life samples.

[0227] Hematology Panel At in-life sampling and terminal necropsy, a hematology panel of markers was analyzed in EDTA whole blood for the following parameters: hemoglobin, red blood cell count, white blood cell count with relative and absolute differential count, absolute reticulocyte count, % reticulocytes, and platelet count. When the assay volume of whole blood allowed, additional parameters included hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.

[0228] Genotyping of samples Tail samples were taken at endpoint sampling for possible re-genotyping using conventional PCR.

[0229] Statistical analysis and graphical presentation of data Data quality checks and validation were performed prior to further statistical analysis. During that process, potential outliers were identified and assessed. No outliers were excluded from the data unless there was a clear reason for their exclusion (e.g., measurement errors identified in laboratory records). The planned comparisons in this study were: Group 1 vs. Group 2 and Group 7 ●Group 2 vs. Group 3~6 The normality assumption for each dataset was based primarily on experience (e.g., data within a population are known to be approximately Gaussian) and observation during the validation phase. Some biological variables are known to follow a log-normal distribution; in these cases, the data are first transformed to logarithms, after which parametric statistical tests can be used. The same normality assumption was used for groups of similar readouts from a series of experiments or assays. Furthermore, the D'Agostino-Pearson omnibus normality test was used for clinical chemistry and hematology data to support the decision of whether to use a parametric or nonparametric test. A summary of the readouts for statistical analysis and visualization is summarized in Table 7 below.

[0230] Table 7. Summary of readouts for statistical analysis and visualization

[0231] [Table 7] Descriptive statistics including group size, mean, SD, and SEM were provided for each parameter.

[0232] Statistical analysis of single time point data Simple comparisons between two groups were performed using the unpaired Welch t-test, or by the Mann-Whitney U test if the assumptions of normality or log-normality were not met.

[0233] Repeated observations of the same subject (longitudinal data) Comparisons between two groups were performed using a two-way mixed-effects model (Mixed Anova) with a Geisser-Greenhouse correction (no assumption of sphericity), followed by Fisher's LSD test between the two groups performed for each time point. Time, group, and the time × group interaction were fixed effects, and individual subjects were random effects. For longitudinal comparisons of more than two groups, a two-way mixed-effects model with Geisser-Greenhouse correction, followed by Dunnett's multiple comparison test, was used. One family was set up per time point. Time, group, and time × group interaction were fixed effects, and individual subjects were random effects. The following sections provide detailed results for some of the parameters listed in Table 6 above.

[0234] result The results of this study provided an indication of the safety and efficacy of the compositions and methods described above. body weight Body weight was significantly lower in HEMO AAV8-Des-mMTM1 mice compared to WT vehicle mice at all time points (Two-way ANOVA: Group: p<0.0001) (Fisher's LSD multiple comparisons: all p<0.001) (Figure 5). Body weight was significantly lower in WT AAV8-Des-mMTM1+AAV8-Des-hMTM1-STOP mice compared to WT vehicle mice at weeks 7–16 (two-way ANOVA: group: p<0.05) (Fisher's LSD multiple comparisons: p<0.05 for all) (Figure 5). Body weight varied significantly among HEMi mice (two-way ANOVA: group effect: NS, group × time interaction effect: p<0.0001). Post-hoc comparisons revealed that body weight of HEMO AAV8-Des-mMTM1 + AAV8 empty capsid mice was significantly reduced at weeks 4 and 5 (Dunnett's multiple comparisons: p<0.05 for both), and that of HEMO AAV8-Des-mMTM1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice was significantly reduced at weeks 5-6 and 8-9 compared to HEMO AAV8-Des-mMTM1 mice (Dunnett's multiple comparisons: all p<0.05).

[0235] mortality rate Data from animals that died or were euthanized before dosing are not shown. No mortality was observed in Groups 1-3, 5, and 7. Two mice each from Groups 4, 6, and Untreated were found to have died before dosing due to factors unrelated to the test substance.

[0236] clinical chemistry All clinical chemistry evaluations were performed only from terminal samples (16 weeks or 5 weeks for naive mice) for all groups, except for ALAT, which was analyzed from both in-life and terminal samples (4–10 weeks). Data from clinical chemistry analyses are shown in Figures 6A–19B. Alkaline phosphatase (AFOS) There was no statistically significant difference in AFOS levels between groups at 16 weeks of age (Fig. 6A). AFOS levels from 5-week-old naive mice are shown in Figure 6B. Alanine aminotransferase (ALAT) ALAT levels were significantly elevated in HEMO AAV8-Des-mMTM1 mice compared with WT vehicle mice at 6 and 16 weeks (two-way ANOVA: group effect: p=0.0005) (Fisher's LSD multiple comparisons: p<0.05 for both) (Figure 7A). ALAT levels were significantly elevated at 10 weeks of age in HEMO AAV8-Des-mMTM1+AAV8-APoE-A1AT-mMTM1 mice compared with HEMO AAV8-Des-mMTM1 mice (one-way ANOVA: group effect: p = 0.0026, Dunnett's post hoc comparison: p < 0.05) (Figure 7A). ALAT levels from 5-week-old untreated mice are shown in Figure 7B. Aspartate aminotransferase (ASAT) ASAT levels were significantly elevated in HEMO AAV8-Des-mMTM1 mice compared with WT vehicle mice at 16 weeks of age (t-test: p<0.01) (FIG. 8A). ASAT levels from 5-week-old naive mice are shown in Figure 8B. albumin There was no statistically significant difference in albumin levels between groups at 16 weeks of age (Fig. 9A). Albumin levels from untreated mice at 5 weeks of age are shown in Figure 9B. γ-glutamyltransferase (GGT) GGT levels were significantly elevated in HEMO AAV8-Des-mMTM1 + AAV8 empty capsid mice and HEMO AAV8-Des-LA1 + AAV8-APoE-A1AT-mMTM1 + AAV8-Des-hMTM1-STOP mice compared with 16-week-old HEMO AAV8-Des-mMTM1 mice (one-way ANOVA: group effect: p = 0.0013, Dunnett's post hoc comparison: p < 0.05 for both mMTM) (Figure 10A). GGT levels from 5-week-old naive mice are shown in Figure 10B. Total protein There was no statistically significant difference in total protein levels between groups at 16 weeks of age (Fig. 11A). Total protein levels from 5-week-old untreated mice are shown in Figure 11B.

[0237] urea There was no statistically significant difference in urea levels between groups at 16 weeks of age (Fig. 12A). Urea levels from untreated mice at 5 weeks of age are shown in Figure 12B. Lactate dehydrogenase (LDH) There was no statistically significant difference in LDH levels between groups at 16 weeks of age (Fig. 13A). LDH levels from naive 5-week-old mice are shown in FIG. 13B. bile acids Bile acid levels were significantly elevated in HEMO AAV8-Des-mMTM1 + AAV8 empty capsid mice compared with 16-week-old HEMO AAV8-Des-mMTM1 mice (one-way ANOVA: group effect: p = 0.0258, Dunnett's post-hoc comparison: p < 0.05) (Figure 14A). However, it should be noted that most bile acid levels were below detection levels in all treatment groups. Bile acid levels from 5-week-old untreated mice are shown in Figure 14B. Total bilirubin There were no statistically significant differences between groups in total bilirubin levels at 16 weeks of age (Figure 15). Total bilirubin data were not available from untreated 5-week-old mice (all values ​​were below the level of detection). Calculated globulin Calculated globulin levels were significantly elevated in HEMO AAV8-Des-mMTM1 mice compared to WT vehicle mice at 16 weeks of age (t-test: p<0.05) (FIG. 16A). Calculated globulin levels from untreated mice at 5 weeks of age are shown in Figure 16B. Calculated albumin / globulin ratio There were no statistically significant differences between groups in calculated albumin / globulin ratio concentrations at 16 weeks of age (FIG. 17A). Albumin / globulin ratio concentrations from 5-week-old untreated mice are shown in Figure 17B. Creatine kinase CK levels were significantly elevated in HEMO AAV8-Des-mMTM1 mice compared to WT vehicle mice at 16 weeks of age (t-test: p<0.05) (FIG. 18A). CK levels from 5-week-old naive mice are shown in FIG. 18B. Creatinine Creatinine levels at 16 weeks of age were not statistically different between groups (Figure 19A). Creatinine levels from untreated mice at 5 weeks of age are shown in Figure 19B.

[0238] hematology Due to the different blood sampling techniques between in-life sampling (saphenous vein, no anesthesia) and terminal sampling (cardiac puncture, pentobarbital anesthesia), statistical analyses were performed separately for in-life samples (weeks 4–10) and terminal samples (week 16). Significant differences in blood parameters, such as total white blood cell count, have been reported to be due to different blood sampling techniques (Hoggatt et al. Exp Hematol. 44(2):132–137.e1. (2016)). Data from hematology analysis are shown in Figures 20A–41B. White blood cell count (WBC) White blood cell counts were significantly elevated in HEMO AAV8-Des-mMTM1 mice compared with WT vehicle mice at 16 weeks of age (t-test, p<0.05) (FIG. 20A). White blood cell counts were significantly elevated in 7-week-old HEMO AAV8-Des-mMTM1 + AAV8 empty capsid mice compared with HEMO AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: NS, group × time interaction effect: p<0.05, Dunnett's post-hoc comparison: p<0.05) (FIG. 20A). White blood cell counts from untreated mice at 4 and 5 weeks of age are shown in Figure 20B. Red blood cell count (RBC) There were no statistically significant differences between groups in red blood cell counts (Figure 21A). Red blood cell counts from untreated mice at 4 and 5 weeks of age are shown in Figure 21B. Hemoglobin (HGB) There were no statistically significant differences between groups in hemoglobin levels (Figure 22A). Hemoglobin levels from untreated mice at 4 and 5 weeks of age are shown in Figure 22B. Hematocrit (HCT) Hematocrit levels were significantly elevated in HEMO AAV8-Des-mMTM1+AAV8-APoE-A1AT-mMTM1 mice compared with 6-week-old HEMO AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: p<0.05, Dunnett's post-hoc comparison: p<0.05) (Figure 23A). Hematocrit levels from untreated mice at 4 and 5 weeks of age are shown in Figure 23B. Mean corpuscular volume (MCV) MCV levels were significantly reduced in HEMIOAAV 8-Des-mMTM 1 mice compared to WT vehicle mice at 4 and 5 weeks of age (t-test, p<0.01 for all) (Figure 24A). MCV levels from untreated mice at 4 and 5 weeks of age are shown in Figure 24B. Mean corpuscular hemoglobin (MCH) There were no statistically significant differences between groups in MCH levels (Figure 25A). MCH levels from untreated mice at 4 and 5 weeks of age are shown in Figure 25B. Mean corpuscular hemoglobin concentration (MCHC) MCHC levels were significantly reduced in 5- and 16-week-old HEMO AAV8-Des-mMTM1+AAV8-APoE-A1AT-mMTM1 mice and in 6-week-old HEMO AAV8-Des-LA1+AAV8-APoE-A1AT-mMTM1+AAV8-Des-hMTM1-STOP mice compared with HEMO AAV8-Des-mMTM1 mice, and were elevated in 7-week-old HEMO AAV8-Des-mMTM1+AAV8-ApoE-A1AT-1+AAV8-Des-hMTM1-STOP mice (all by two-way ANOVA: group effect: NS, group × time interaction effect: p<0.01, Dunnett's post-hoc comparison: p<0.05) (Figure 26A). MCHC levels from untreated mice at 4 and 5 weeks of age are shown in Figure 26B.

[0239] Platelet count (PLT) Platelets or platelet counts were significantly reduced in HEMO AAV8-Des-mMTM1 mice compared with WT vehicle mice at 6 weeks of age (two-way ANOVA: group effect: p<0.05, Dunnett's post-hoc comparison: p<0.01) (Figure 27A). The thrombocyte or platelet counts from untreated mice at 4 and 5 weeks of age are shown in Figure 27B. Neutrophil count (relative) (Neut(%)) There was no statistically significant difference between groups (A) and (B) (Figure 28A). The relative neutrophil counts from untreated in 4- and 5-week-old mice are shown in Figure 28B. Neutrophil count (absolute) (Neut) There were no statistically significant differences between groups in absolute neutrophil counts (Figure 29A). Absolute neutrophil counts from untreated mice at 4 and 5 weeks of age are shown in Figure 29B. Lymphocyte count (relative) (lymph (%)) Relative lymphocyte counts were significantly reduced in 5- and 6-week-old HEMO AAV8-Des-mMTM1+AAV8 empty capsid mice compared with HEMO AAV8-Des-mMTM1 mice (all by two-way ANOVA: group effect: NS, group x time interaction effect: p<0.05, Dunnett's post-hoc comparison: p<0.05) (Figure 30A). The relative lymphocyte counts from untreated mice are shown in Figure 30B. Lymphocyte count (absolute) (lymph) Absolute lymphocyte counts were significantly enhanced in HEMO AAV8-Des-mMTM1 mice compared to WT vehicle mice at 16 weeks of age (t-test, p<0.01) (FIG. 31A). Absolute lymphocyte counts from untreated mice at 4 and 5 weeks of age are shown in Figure 31B. Monocyte count (relative) (Mono(%)) The relative monocyte count was significantly enhanced in WT AAV8-Des-mMTM1+AAV8-Des-hMTM1-STOP mice compared with WT vehicle mice at 16 weeks of age (t-test, p<0.05) (Figure 32A). The relative monocyte counts from untreated mice at 4 and 5 weeks of age are shown in Figure 32B. Monocyte count (absolute) (monocytes) Absolute monocyte numbers were significantly enhanced in HEMO AAV8-Des-mMTM1 mice compared to WT vehicle mice at 16 weeks of age (t-test, p<0.01) (FIG. 33A). Absolute monocyte counts from untreated mice at 4 and 5 weeks of age are shown in Figure 33B. Eosinophil count (relative) (Eos(%)) Eosinophil counts were significantly increased in 4-week-old HEMO AAV8-Des-mMTM1+AAV8 empty capsid mice compared with HEMO AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: p<0.05, Dunnett's post-hoc comparison: p<0.01) (Figure 34A). Eosinophil counts from untreated mice at 4 and 5 weeks of age are shown in Figure 34B. Eosinophil count (absolute) (Eos) Absolute eosinophil counts were significantly increased in 5-week-old HEMO AAV8-Des-mMTM1+AAV8 empty capsid mice compared with HEMO AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: p=0.0001, Dunnett's post-hoc comparison: p<0.05) (Figure 35A). Absolute eosinophil counts from untreated mice at 4 and 5 weeks of age are shown in Figure 35B. Basophil count (relative) (Baso (%)) Relative basophil counts were significantly reduced in 5-week-old HEMO AAV8-Des-mMTM1+AAV8-APoE-A1AT-mMTM1+AAV8-Des-hMTM1-STOP mice compared with HEMO AAV8-Des-mMTM1 mice (two-way ANOVA: 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 from untreated mice at 4 and 5 weeks of age are shown in Figure 36B. Basophil count (absolute) (Baso) There was no statistically significant difference between groups in absolute basophil counts (Figure 37A). Absolute basophil counts from untreated mice at 4 and 5 weeks of age are shown in Figure 37B. - High relative number of unstained cells (LUC (%)). The relative number of large unstained cells was significantly increased in 7-week-old WT AAV8-Des-mMTM1+AAV8-Des-hMTM1-STOP mice 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 numbers of large, unstained cells from untreated mice at 4 and 5 weeks of age are shown in Figure 38B. · Absolute large unstained cell count (LUC) The absolute number of large, unstained cells was significantly reduced in 6-week-old HEMO AAV8-Des-mMTM1+AAV8-APoE-A1AT-mMTM1+AAV8-Des-hMTM1-STOP mice and 10-week-old HEMO AAV8-Des-mMTM1+AAV8-APoE-A1AT-mMTM1 mice compared with HEMO AAV8-Des-mMTM1 mice (two-way ANOVA: group effect: NS, group × time interaction effect: p<0.05, Dunnett's post-hoc comparison: p<0.05) (Figure 39A). The absolute numbers of large unstained cells from untreated mice at 4 and 5 weeks of age are shown in Figure 39B. Reticulocyte count (relative) (reticulocytes (%)) There was no statistically significant difference between groups in relative reticulocyte counts (Figure 40A). The relative reticulocyte counts from untreated mice at 4 and 5 weeks of age are shown in Figure 40B. Reticulocyte count (absolute) (reticulocytes) There was no statistically significant difference between groups (A) and (B). Absolute reticulocyte cell counts (FIG. 41A). The absolute reticulocyte cell counts from untreated mice at 4 and 5 weeks of age are shown in Figure 41B.

[0240] conclusion The results of the above experiments support the safety and efficacy of AAV vectors encoding an MTM1 transgene under the control of liver-tropic regulatory factors, optionally in combination with AAV vectors encoding an MTM1 transgene under the control of muscle-tropic regulatory factors, for the treatment of XLMTM. [Example]

[0241] Treatment of X-linked myotubular myopathy in human patients by co-administration of a pseudotyped AAV2 / 8 vector containing a nucleic acid sequence encoding the myotubularin 1 gene operably linked to a liver-specific promoter and a pseudotyped AAV2 / 8 vector containing a nucleic acid sequence encoding the myotubularin 1 gene operably linked to a desmin promoter Using the compositions and methods of the present disclosure, patients suffering from XLMTM are administered approximately 3 x 10 immunized mice with a pseudotyped AAV2 / 8 vector (FIGS. 1 and 2) comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a liver-specific promoter, and a pseudotyped AAV2 / 8 vector (e.g., resamiridin-virparvovec) comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a desmin promoter. 14 Doses less than 3 × 10 vg / kg (e.g., about 3 × 10 14 vg / kg, 2.9 × 10 14 vg / kg, 2.8 × 10 14 vg / kg, 2.7 × 10 14 vg / kg, 2.6 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.4 × 10 14 vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 1012 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The dose is 0.05 mg / kg or less. When a pseudotyped AAV2 / 8 vector containing a nucleic acid sequence encoding the MTM1 gene operably linked to a liver-specific promoter (Figures 1 and 2) and a pseudotyped AAV2 / 8 vector containing a nucleic acid sequence encoding the MTM1 gene operably linked to a desmin promoter (e.g., resamiligen virparvovec) are simultaneously administered to a patient, the patient exhibits a change from baseline in peak inspiratory pressure. For example, a patient may demonstrate a change from baseline in peak inspiratory pressure by about 24 weeks (e.g., about 20 weeks, about 16 weeks, about 12 weeks, about 8 weeks, or about 4 weeks after co-administration to the patient of a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a liver-specific promoter (Figures 1 and 2) and a pseudotyped AAV2 / 8 vector comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a desmin promoter (e.g., resamiligen virparvovec)). [Example]

[0242] Treatment of X-linked myotubular myopathy in human patients with pseudotyped AAV2 / 8 vectors containing both a nucleic acid sequence encoding the myotubularin 1 gene operably linked to a liver-specific promoter and a nucleic acid sequence encoding the myotubularin 1 gene operably linked to the desmin promoter Using the compositions and methods of the present disclosure, a patient suffering from XLMTM is administered approximately 3×10 HIV-1-associated ... 14 Doses less than 3 × 10 vg / kg (e.g., about 3 × 10 14 vg / kg, 2.9 × 10 14 vg / kg, 2.8 × 10 14 vg / kg, 2.7 × 10 14 vg / kg, 2.6 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.4 × 10 14vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The dose is 0.05 mg / kg or less. When a patient is administered an AAV2 / 8 vector (FIG. 3) comprising both a nucleic acid sequence encoding an MTM1 gene operably linked to a liver-specific promoter and a nucleic acid sequence encoding an MTM1 gene operably linked to a muscle-specific promoter, the patient exhibits a change from baseline in time on mechanical ventilatory support over time. For example, the patient exhibits a change from baseline in time on mechanical ventilatory support over time by about 24 weeks (e.g., about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks) after the patient is administered an AAV2 / 8 vector (FIG. 3) comprising both a nucleic acid sequence encoding an MTM1 gene operably linked to a liver-specific promoter and a nucleic acid sequence encoding an MTM1 gene operably linked to a muscle-specific promoter. [Example]

[0243] Treatment of X-linked myotubular myopathy in human patients with pseudotyped AAV2 / 8 vectors containing nucleic acid sequences encoding the myotubularin 1 gene operably linked to a ubiquitous promoter Using the compositions and methods of the present disclosure, patients suffering from XLMTM are administered approximately 3×10 immunized mice with a pseudotyped AAV2 / 8 vector (FIG. 4) comprising a nucleic acid sequence encoding an MTM1 gene operably linked to a ubiquitous promoter. 14 Doses less than 3 × 10 vg / kg (e.g., about 3 × 10 14 vg / kg, 2.9 × 10 14 vg / kg, 2.8 × 10 14 vg / kg, 2.7 × 10 14 vg / kg, 2.6 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.4 × 10 14 vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The dose is 0.05 mg / kg or less. When a pseudotyped AAV2 / 8 vector (FIG. 4) containing a nucleic acid sequence encoding an MTM1 gene operably linked to a ubiquitous promoter is administered to a patient, the patient achieves functionally independent locus for at least 30 seconds. For example, the patient achieves functionally independent locus by about 24 weeks (e.g., by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks) after administration of a pseudotyped AAV2 / 8 vector (FIG. 4) containing a nucleic acid sequence encoding an MTM1 gene operably linked to a ubiquitous promoter to the patient. [Example]

[0244] Treatment of X-linked myotubular myopathy in human patients with a non-viral vector containing a liver expression construct combined with a pseudotyped AAV2 / 8 vector containing a nucleic acid sequence encoding the myotubularin 1 gene operably linked to the desmin promoter Using the compositions and methods of the present disclosure, patients suffering from XLMTM can be administered a non-viral vector (e.g., lipid nanoparticles) comprising a liver expression construct in combination with a pseudotyped AAV2 / 8 vector (e.g., resamiridin-virparvovec) comprising a nucleic acid sequence encoding the MTM1 gene operably linked to the desmin promoter, wherein the AAV2 / 8 vector delivers approximately 3×10 14 Doses less than 3 × 10 vg / kg (e.g., about 3 × 10 14 vg / kg, 2.9 × 10 14 vg / kg, 2.8 × 10 14 vg / kg, 2.7 × 10 14 vg / kg, 2.6 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.4 × 10 14 vg / kg, 2.3 × 10 14 vg / kg, 2.2 × 10 14 vg / kg, 2.1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 1.9 × 10 14 vg / kg, 1.8 × 10 14 vg / kg, 1.7 × 10 14 vg / kg, 1.6 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.4 × 10 14 vg / kg, 1.3 × 10 14 vg / kg, 1.2 × 10 14 vg / kg, 1.1 × 1014 vg / kg, 1 × 10 14 vg / kg, 1 × 10 14 vg / kg, 1 × 10 13 vg / kg, 1 × 10 12 vg / kg, 1 × 10 11 vg / kg, 1 × 10 10 vg / kg, 1 × 10 9 vg / kg, 1 × 10 8 The dose is 0.05 mg / kg or less. When a non-viral vector (e.g., lipid nanoparticle) containing a liver expression construct is co-administered to a patient with a pseudotyped AAV2 / 8 vector containing a nucleic acid sequence encoding the MTM1 gene operably linked to a desmin promoter (e.g., resamiligen-virparvovec), the patient acquires functional independent locomotion for at least 30 seconds. For example, a patient may acquire a functionally self-sustaining locus for at least 30 seconds by about 24 weeks (e.g., about 20 weeks, about 16 weeks, about 12 weeks, about 8 weeks, or about 4 weeks) after administering to the patient a lipid nanoparticle containing a liver expression construct in combination with a pseudotyped AAV2 / 8 vector containing a nucleic acid sequence encoding the MTM1 gene operably linked to a desmin promoter (e.g., resamirigen virparvovec).

Claims

1. A recombinant adeno-associated viral (AAV) vector comprising a transgene encoding myotubularin 1 (MTM1), wherein the transgene is operably linked to a promoter active in liver tissue.

2. The recombinant AAV vector of claim 1, wherein the promoter is selectively active in liver tissue.

3. The recombinant AAV vector of claim 1 or 2, wherein the promoter comprises a constitutive promoter.

4. The recombinant AAV vector of any one of claims 1 to 3, further comprising a second transgene encoding MTM1.

5. The recombinant AAV vector of claim 4, wherein the second transgene encoding MTM1 is operably linked to a promoter active in muscle tissue.

6. The recombinant AAV vector of any one of claims 1 to 5, which is of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh 10, or AAVrh74 serotype.

7. The recombinant AAV vector according to any one of claims 1 to 6, which is a pseudotyped AAV.

8. 10. A method of treating X-linked myotubular myopathy (XLM™) in a human patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of a recombinant AAV vector of any one of claims 1-7.

9. 9. The method of claim 8, wherein the patient is further administered an anti-cholestatic agent.

10. The method of claim 8 or 9, comprising 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 or selectively active in liver tissue, and (ii) a recombinant AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active or selectively active in muscle tissue.

11. A method for treating XLMTM in a human patient in need of such treatment, comprising administering to the patient therapeutically effective amounts of (i) a non-viral composition comprising a nucleic acid encoding MTM1 operably linked to a promoter that is active or selectively active in liver tissue, and (ii) a recombinant AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active or selectively active in muscle tissue.

12. 12. The method of claim 11, wherein the non-viral composition is a liposome, a vesicle, a synthetic vesicle, an exosome, a synthetic exosome, a dendrimer, or a nanoparticle.

13. A kit comprising (i) a non-viral composition comprising a nucleic acid encoding MTM1, and (ii) a recombinant AAV vector comprising a transgene encoding MTM1 under the control of a promoter that is active or selectively active in muscle tissue, the kit further comprising an insert instructing a user to administer the non-viral composition and the recombinant AAV vector to a patient diagnosed with XLMTM.

Images