COMPOSITIONS AND METHODS FOR IMPROVED TREATMENT OF X-LINKED MYOTUBULAR MYOPATHY

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 24, 2022 is named “51037-057WO3_Sequence_Listing_5_24_22_ST25” and is 69,743 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a method for the treatment of cholestatic liver dysfunction associated with current treatments of neuromuscular disorders in patients, such as human patients.

BACKGROUND OF THE INVENTION

X-linked myotubular myopathy (XLMTM) is a fatal monogenic disease of skeletal muscle, resulting from loss-of-function mutations in Myotubularin 1 (MTM1). Approximately one in every 50,000 newborn boys has XLMTM, which typically displays as marked hypotonia and respiratory failure. In extremely rare cases, females can develop a severe form of XLMTM. Survival beyond the postnatal period requires intensive support, including respiratory support (i.e., mechanical ventilation) at birth in 85-90% of patients, ongoing 24-hour ventilator dependence in nearly 50% of patients, and tracheostomy in ˜60% of patients. Until recently, only supportive treatment options, such as ventilator use or a feeding tube, were available. Recently, gene therapy approaches involving the 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 having XLMTM.

SUMMARY OF THE INVENTION

The disclosure provides methods for treating X-linked myotubular myopathy (XLMTM) in a human patient in need thereof. In some embodiments, the patient is administered a therapeutically effective amount of a viral vector containing a transgene encoding myotubularin 1 (MTM1) and an anti-cholestatic agent.

In one aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof, the method including administering to the patient (i) a therapeutically effective amount of a transgene encoding MTM1 and (ii) an anti-cholestatic agent, wherein the anti-cholestatic agent is administered to the patient in one or more doses that commence within about six weeks (e.g., about six weeks before or about six weeks after) of administration of the transgene to the patient.

In a second aspect, the disclosure provides a method of reducing stiffness and/or joint contractures in a human patient diagnosed as having XLMTM, the method comprising administering to the patient (i) a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1 and (ii) an anti-cholestatic agent, wherein the anti-cholestatic agent is administered to the patient in one or more doses that commence within about six weeks (e.g., about six weeks before or about six weeks after) of administration of the viral vector to the patient.

In another aspect, the disclosure provides a method of increasing diaphragm and/or respiratory muscle progression in a human patient diagnosed as having XLMTM, the method comprising administering to the patient (i) a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1 and (ii) an anti-cholestatic agent, wherein the anti-cholestatic agent is administered to the patient in one or more doses that commence within about six weeks (e.g., about six weeks before or about six weeks after) of administration of the viral vector to the patient.

In some embodiments, the anti-cholestatic agent is administered to the patient in one or more doses that commence within about five weeks (e.g., about five weeks before or about five weeks after) of administration of the transgene to the patient, optionally wherein the anti-cholestatic agent is administered to the patient in one or more doses that commence within about four weeks (e.g., about four weeks before or about four weeks after), within about three weeks (e.g., about three weeks before or about three weeks after), within about two weeks (e.g., about two weeks before or about two weeks after), or within about one week (e.g., about one week before or about one week after, about six days before or about six days after, about five days before or about five days after, about four days before or about four days after, about three days before or about three days after, about two days before or about two days after, or about one day before or about one day after) of administration of the transgene to the patient.

In some embodiments, the anti-cholestatic agent is administered to the patient in one or more doses that commence on the same day as administration of the transgene to the patient.

In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof and who has been previously administered an anti-cholestatic agent, the method including administering to the patient a therapeutically effective amount of a transgene encoding MTM1.

In another aspect, the disclosure provides a method of reducing stiffness and/or joint contractures in a human patient diagnosed as having XLMTM and who has been previously administered an anti-cholestatic agent, the method comprising administering to the patient a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1.

In another aspect, the disclosure provides a method of increasing diaphragm and/or respiratory muscle progression in a human patient diagnosed as having XLMTM and who has been previously administered an anti-cholestatic agent, the method comprising administering to the patient a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1.

In some embodiments of either of the foregoing aspects, the method further includes monitoring the patient for development of cholestasis or hyperbilirubinemia.

In some embodiments of either of the foregoing aspects, the patient is monitored for the development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof by evaluating a parameter in a blood sample obtained from the patient, wherein a finding that the parameter is above a reference level identifies the patient as having cholestasis, hyperbilirubinemia, or one or more symptoms thereof.

In some embodiments, the parameter includes the level of a serum bile acid in the blood sample. In some embodiments, the serum bile acid is cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid.

In some embodiments, the parameter includes one or more results of a liver function test.

In some embodiments of any of the foregoing aspects, the parameter includes the level of aspartate aminotransferase or alanine aminotransferase in the blood sample.

In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof, the method including: (a) administering to the patient a transgene encoding MTM1, (b) monitoring the patient for development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and, if the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof, the method including: (a) administering to the patient a viral vector including a transgene encoding MTM1 in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), (b) monitoring the patient for development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and, if the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of reducing stiffness and/or joint contractures in a human patient diagnosed as having XLMTM, the method comprising: (a) administering to the patient a viral vector comprising a transgene encoding MTM1 in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), (b) monitoring the patient for development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and, if the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of increasing diaphragm and/or respiratory muscle progression in a human patient diagnosed as having XLMTM, the method comprising: a) administering to the patient a viral vector comprising a transgene encoding MTM1 in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), (b) monitoring the patient for development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and, if the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof, the method including: (a) administering to the patient a transgene encoding MTM1, (b) determining that the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof, the method including: (a) administering to the patient a viral vector including a transgene encoding MTM1 in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), (b) determining that the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of reducing stiffness and/or joint contractures in a human patient diagnosed as having XLMTM, the method comprising: (a) administering to the patient a viral vector comprising a transgene encoding MTM1 in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), (b) determining that the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of increasing diaphragm and/or respiratory muscle progression in a human patient diagnosed as having XLMTM, the method comprising: (a) administering to the patient a viral vector comprising a transgene encoding MTM1 in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), (b) determining that the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof that is five years old or younger (e.g., 5 years old or younger, 4 years old or younger, 3 years old or younger, 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger), the method including: (a) administering to the patient a therapeutically effective amount of a transgene encoding MTM1, (b) monitoring the patient for development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and, if the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of reducing stiffness and/or joint contractures in a human patient diagnosed as having XLMTM, the method comprising: (a) administering to the patient a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1, (b) monitoring the patient for development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and, if the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of increasing diaphragm and/or respiratory muscle progression in a human patient diagnosed as having XLMTM, the method comprising: (a) administering to the patient a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1, (b) monitoring the patient for development of cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and, if the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of treating XLMTM in a human patient in need thereof that is five years old or younger (e.g., 5 years old or younger, 4 years old or younger, 3 years old or younger, 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger), the method including: (a) administering to the patient a therapeutically effective amount of a transgene encoding MTM1, (b) determining that the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof, and (c) administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of treating or preventing cholestasis or hyperbilirubinemia in a human patient that has XLMTM and who has been previously administered a viral vector including a transgene encoding MTM1 in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), the method including administering to the patient an anti-cholestatic agent.

In another aspect, the disclosure provides a method of treating or preventing cholestasis or hyperbilirubinemia in a human patient that has XLMTM, has been previously administered a transgene encoding MTM1, and that was five years old or younger (e.g., 5 years old or younger, 4 years old or younger, 3 years old or younger, 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger) at the time of administration of the transgene, the method including administering to the patient an anti-cholestatic agent.

In some embodiments of any of the foregoing aspects, the transgene encoding MTM1 was administered to the patient by transduction with a viral vector containing a transgene encoding MTM1.

In some embodiments of any of the foregoing aspects, the patient is five years old or younger (e.g., 5 years old or younger, 4 years old or younger, 3 years old or younger, 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger) at the time of administration of the transgene or viral vector.

In some embodiments of any of the foregoing aspects, the patient is four years old or younger (e.g., 4 years old or younger, 3 years old or younger, 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger) at the time of administration of the transgene or viral vector, optionally wherein the patient is three years old or younger (e.g., 3 years old or younger, 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger), two years old or younger (e.g., 2 years old or younger, 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger), one year old or younger (e.g., 1 year old or younger, 12 months old or younger, 11 months old or younger, 10 months old or younger, 9 months old or younger, 8 months old or younger, 7 months old or younger, 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger), or six months old or younger (e.g., 6 months old or younger, 5 months old or younger, 4 months old or younger, 3 months old or younger, 2 months old or younger, or 1 month old or younger).

In some embodiments of any of the foregoing aspects, the patient was from about 1 month old to about 5 years old (e.g., about 1 month old to about 5 years old, about 2 months old to about 5 years old, about 3 months old to about 5 years old, about 4 months old to about 5 years old, about 5 months old to about 5 years old, about 6 months old to about 5 years old, about 1 year old to about 5 years old, about 2 years old to about 5 years old, about 3 years old to about 5 years old, or about 4 years old to about 5 years old) at the time of administration of the transgene or viral vector.

In some embodiments of any of the foregoing aspects, the viral vector is administered to the patient in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less).

In some embodiments of any of the foregoing aspects, the viral vector is administered to the patient in an amount of less than about 2.5×10¹⁴vg/kg (e.g., in an amount of less than about 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), optionally wherein the viral vector is administered to the patient in an amount of less than about 2×10¹⁴vg/kg (e.g., in an amount of less than about 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), less than about 1.5×10¹⁴vg/kg (e.g., less than about 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less), or less than about 1.4×10¹⁴vg/kg (e.g., less than about 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less).

In some embodiments of any of the foregoing aspects, the viral vector is administered to the patient in an amount of from about 3×10¹³vg/kg to about 2.3×10¹⁴vg/kg, optionally wherein the viral vector is administered to the patient in an amount of from about 8×10¹³vg/kg to about 1.8×10¹⁴vg/kg, from about 1×10¹⁴vg/kg to about 1.6×10¹⁴vg/kg, from about 1.1×10¹⁴vg/kg to about 1.5×10¹⁴vg/kg, or from about 1.2×10¹⁴vg/kg to about 1.4×10¹⁴vg/kg. For example, the viral vector may be administered to the patient in an amount of about 3×10¹³vg/kg, 3.1×10¹³vg/kg, 3.2×10¹³vg/kg, 3.3×10¹³vg/kg, 3.4×10¹³vg/kg, 3.5×10¹³vg/kg, 3.6×10¹³vg/kg, 3.7×10¹³vg/kg, 3.8×10¹³vg/kg, 3.9×10¹³vg/kg, 4×10¹³vg/kg, 4.1×10¹³vg/kg, 4.2×10¹³vg/kg, 4.3×10¹³vg/kg, 4.4×10¹³vg/kg, 4.5×10¹³vg/kg, 4.6×10¹³vg/kg, 4.7×10¹³vg/kg, 4.8×10¹³vg/kg, 4.9×10¹³vg/kg, 5×10¹³vg/kg, 5.1×10¹³vg/kg, 5.2×10¹³vg/kg, 5.3×10¹³vg/kg, 5.4×10¹³vg/kg, 5.5×10¹³vg/kg, 5.6×10¹³vg/kg, 5.7×10¹³vg/kg, 5.8×10¹³vg/kg, 5.9×10¹³vg/kg, 6×10¹³vg/kg, 6.1×10¹³vg/kg, 6.2×10¹³vg/kg, 6.3×10¹³vg/kg, 6.4×10¹³vg/kg, 6.5×10¹³vg/kg, 6.6×10¹³vg/kg, 6.7×10¹³vg/kg, 6.8×10¹³vg/kg, 6.9×10¹³vg/kg, 7×10¹³vg/kg, 7.1×10¹³vg/kg, 7.2×10¹³vg/kg, 7.3×10¹³vg/kg, 7.4×10¹³vg/kg, 7.5×10¹³vg/kg, 7.6×10¹³vg/kg, 7.7×10¹³vg/kg, 7.8×10¹³vg/kg, 7.9×10¹³vg/kg, 8×10¹³vg/kg, 8.1×10¹³vg/kg, 8.2×10¹³vg/kg, 8.3×10¹³vg/kg, 8.4×10¹³vg/kg, 8.5×10¹³vg/kg, 8.6×10¹³vg/kg, 8.7×10¹³vg/kg, 8.8×10¹³vg/kg, 8.9×10¹³vg/kg, 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments of any of the foregoing aspects, the viral vector is administered to the patient in an amount of about 1.3×10¹⁴vg/kg.

In some embodiments of any of the foregoing aspects, the transgene or viral vector is administered to the patient in a single dose including the amount.

In some embodiments of any of the foregoing aspects, the transgene or viral vector is administered to the patient in two or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) doses that, together, comprise the amount.

In some embodiments of any of the foregoing aspects, the two or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) doses are separated from one another by one year or more (e.g., one year or more, one year and six months or more, two years or more, three years or more, four years or more, or five years or more).

In some embodiments of any of the foregoing aspects, the two or more doses are administered to the patient within about 12 months (e.g., about 12 months, about 11 months, about 10 months, about 9 months, about 8 months, about 7 months, about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, or about 1 month) of one another.

In some embodiments of any of the foregoing aspects, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and a synthetic virus.

In some embodiments, the viral vector is an AAV. In some embodiments, the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74 serotype.

In some embodiments, the viral vector is a pseudotyped AAV. In some embodiments, the pseudotyped AAV is AAV2/8 or AAV2/9, optionally wherein the pseudotyped AAV is AAV2/8.

In some embodiments, the transgene encoding MTM1 is operably linked to a muscle specific promoter. In some embodiments, the muscle specific promotor is a desmin promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of ocular paired like homeodomain 3.

In some embodiments, the muscle specific promoter is a desmin promoter.

In some embodiments of any of the foregoing aspects, the viral vector is resamirigene bilparvovec.

In some embodiments of any of the foregoing aspects, the viral vector is administered to the patient by way of intravenous, intramuscular, intradermal, or subcutaneous administration.

In some embodiments of any of the foregoing aspects, the anti-cholestatic agent is selected from the group consisting of a bile acid, a farnesoid X receptor (FXR) ligand, a fibroblast growth factor 19 (FGF-19) mimetic, a Takeda-G-protein-receptor-5 (TGR5) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, a PPAR-alpha agonist, a PPAR-delta agonist, a dual PPAR-alpha and PPAR-delta agonist, an apical sodium-dependent bile acid transporter (ASBT) inhibitor, an immunomodulatory drug, an antifibrotic therapy, and a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor.

In some embodiments, (i) the FXR ligand is obeticholic acid, cilofexor, tropifexor, tretinoin, or EDP-305; (ii) the FGF-19 mimetic is aldafermin; (iii) the TGR5 agonist is INT-777 or INT-767; (iv) the PPAR agonist is bezafibrate, seladelpar, or elafibrinor; (v) the PPAR-alpha agonist is fenofibrate; (vi) the PPAR-delta agonist is seladelpar; (vii) the dual PPAR-alpha and PPAR-delta agonist is elafibranor; (viii) the ASBT inhibitor is odevixibat, maralixibat, or linerixibat; (ix) the immunomodulatory drug is rituximab, abatacept, ustekinumab, infliximab, baricitinib, or FFP-104; (x) the antifibrotic therapy is a vitamin D receptor agonist or simtuzumab; and/or (xi) the NOX inhibitor is setanaxib.

In some embodiments, the bile acid is ursodeoxycholic acid (e.g., ursodiol), nor-ursodeoxycholic acid, or a pharmaceutically acceptable salt thereof. In some embodiments, the bile acid is ursodiol.

In some embodiments of any of the foregoing aspects, the bile acid is administered to the patient in a single dose. In some embodiments, the bile acid is administered to the patient in a plurality of doses.

In some embodiments, the bile acid is administered to the patient in an amount of from about 5 mg/kg/dose to about 20 mg/kg/dose, optionally wherein the bile acid is administered to the patient in an amount of from about 6 mg/kg/dose to about 19 mg/kg/dose, from about 7 mg/kg/dose to about 18 mg/kg/dose, from about 8 mg/kg/dose to about 17 mg/kg/dose, from about 10 mg/kg/dose to about 15 mg/kg/dose, or from about 12 mg/kg/dose to about 13 mg/kg/dose. For example, the bile acid is administered to the patient in an amount of about 5 mg/kg/dose, 6 mg/kg/dose, 7 mg/kg/dose, 8 mg/kg/dose, 9 mg/kg/dose, 10 mg/kg/dose, 11 mg/kg/dose, 12 mg/kg/dose, 13 mg/kg/dose, 14 mg/kg/dose, 15 mg/kg/dose, 16 mg/kg/dose, 17 mg/kg/dose, 18 mg/kg/dose, 19 mg/kg/dose, or 20 mg/kg/dose.

In some embodiments, the bile acid is administered to the patient in an amount of from about 5 mg/kg/dose to about 11 mg/kg/dose, optionally wherein the bile acid is administered to the patient in an amount of from about 6 mg/kg/dose to about 10 mg/kg/dose, or from about 7 mg/kg/dose to about 9 mg/kg/dose. For example, the bile acid is administered to the patient in an amount of about 5 mg/kg/dose, 6 mg/kg/dose, 7 mg/kg/dose, 8 mg/kg/dose, 9 mg/kg/dose, 10 mg/kg/dose, or 11 mg/kg/dose.

In some embodiments, the bile acid is administered to the patient in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) doses per day, week, or month.

In some embodiments, the bile acid is administered to the patient in one or more e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) doses per day, optionally wherein the bile acid is administered to the patient in one dose per day, in two doses per day, three doses per day, four doses per day, or five doses per day.

In some embodiments, the bile acid is administered to the patient in one dose per day.

In some embodiments, the bile acid is administered to the patient in an amount of from about 5 mg/kg/day to about 40 mg/kg/day, optionally wherein (i) the bile acid is administered to the patient in an amount of from about 6 mg/kg/day to about 39 mg/kg/day, from about 8 mg/kg/day to about 37 mg/kg/day, from about 13 mg/kg/day to about 32 mg/kg/day, or from about 20 mg/kg/day to about 25 mg/kg/day, or (ii) the bile acid is administered to the patient in an amount of from about 17 mg/kg/day to about 23 mg/kg/day, from about 18 mg/kg/day to about 22 mg/kg/day, or from about 19 mg/kg/day to about 21 mg/kg/day. For example, the bile acid is administered to the patient in an amount of about 5 mg/kg/day, 6 mg/kg/day, 7 mg/kg/day, 8 mg/kg/day, 9 mg/kg/day, 10 mg/kg/day, 11 mg/kg/day, 12 mg/kg/day, 13 mg/kg/day, 14 mg/kg/day, 15 mg/kg/day, 16 mg/kg/day, 17 mg/kg/day, 18 mg/kg/day, 19 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, or 40 mg/kg/day. In some embodiments, the bile acid is administered to the patient in an amount of 20 mg/kg/day.

In some embodiments, the bile acid is administered to the patient by way of a unit dosage form including 250 mg of the bile acid. In some embodiments of any of the foregoing aspects, the bile acid is administered to the patient by way of a unit dosage form including 500 mg of the bile acid.

In some embodiments of any of the foregoing aspects, the bile is administered to the patient by way of enteral administration.

In some embodiments of any of the foregoing aspects, the patient does not have a history of cholestasis or hyperbilirubinemia. In some embodiments, the patient does not have a history of any underlying liver disease.

In some embodiments of any of the foregoing aspects, the patient was born at greater than or equal to 35 weeks of gestational age and is or was from term age (e.g., adjusted term age) to about 5 years old (e.g., 1 day old to about 5 years old, 2 days old to about 5 years old, 3 days old to about 5 years old, 4 days old to about 5 years old, 5 days old to about 5 years old, 6 days old to about 5 years old, 7 days old to about 5 years old, 8 days old to about 5 years old, 9 days old to about 5 years old, 10 days old to about 5 years old, 11 days old to about 5 years old, 12 days old to about 5 years old, 13 days old to about 5 years old, 14 days old to about 5 years old, 15 days old to about 5 years old, 16 days old to about 5 years old, 17 days old to about 5 years old, 18 days old to about 5 years old, 19 days old to about 5 years old, 20 days old to about 5 years old, 25 days old to about 5 years old, one month old to about 5 years old, two months old to about 5 years old, 3 months old to about 5 years old, 4 months old to about 5 years old, 5 months old to about 5 years old, 6 months old to about 5 years old, 1 year old to about 5 years old, 2 years old to about 5 years old, 3 years old to about 5 years old, and 4 years old to about 5 years old) at the time of administration of the transgene or viral vector.

In some embodiments of any of the foregoing aspects, the patient is male.

In some embodiments of any of the foregoing aspects, the patient requires mechanical ventilatory support, optionally wherein mechanical ventilatory support includes invasive mechanical ventilatory support and noninvasive mechanical ventilatory support.

In some embodiments of any of the foregoing aspects, upon administering the transgene or viral vector to the patient, the patient exhibits a change from baseline in hours of mechanical ventilation support over time, optionally wherein the patient exhibits the change from baseline in hours of mechanical ventilation support over time by about 24 weeks after administration of the transgene or viral vector to the patient, optionally wherein the patient displays the change from baseline in hours of mechanical ventilation support over time by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, upon administering the transgene or viral vector to the patient, the patient achieves functionally independent sitting for at least 30 seconds, optionally wherein the patient achieves the functionally independent sitting by about 24 weeks after administration of the transgene or viral vector to the patient, optionally wherein the patient displays the functionally independent sitting for at least 30 seconds by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, upon administering the transgene or viral vector to the patient, the patient displays a reduction in required mechanical ventilator support to about 16 hours or less per day, optionally wherein the patient displays the reduction in required mechanical ventilator support by about 24 weeks after administration of the viral vector to the patient, optionally wherein the patient displays the reduction in required mechanical ventilator support by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, upon administering the transgene or viral vector to the patient, the patient displays a change from baseline on the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND), optionally wherein the patient displays the change from baseline on the CHOP INTEND by about 24 weeks after administration of the transgene or viral vector to the patient, optionally wherein the patient displays the change from baseline on the CHOP INTEND by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, upon administering the transgene or viral vector to the patient, the patient displays a change from baseline in (MIP), optionally wherein the patient displays the change from baseline in MIP by about 24 weeks after administration of the viral vector to the patient, optionally wherein the patient displays the change from baseline in MIP by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, upon administering the transgene or viral vector to the patient, the patient displays a change from baseline in quantitative analysis of myotubularin expression in a muscle biopsy, optionally wherein the patient displays the change from baseline in quantitative analysis of myotubularin expression in a muscle biopsy by about 24 weeks after administration of the transgene or viral vector to the patient, optionally wherein the patient displays the change from baseline in quantitative analysis of myotubularin expression in a muscle biopsy by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient. In some embodiments, the change from baseline in quantitative analysis of myotubularin expression in a muscle biopsy persists for at least 48 weeks (e.g., 49 weeks, 50 weeks, 51 weeks, 52, weeks, 1 year, or 2 years) after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, upon administering the viral vector to the patient, the patient displays a reduction of stiffness and/or joint contractures, optionally wherein the patient displays the reduction of stiffness and/or joint contractures by about 24 weeks after administration of the viral vector to the patient, optionally wherein the patient displays the reduction of stiffness and/or joint contractures by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, upon administering the viral vector to the patient, the patient displays diaphragm and/or respiratory muscle progression, optionally wherein the patient displays the diaphragm and/or respiratory muscle progression by about 24 weeks after administration of the viral vector to the patient, optionally wherein the patient displays the diaphragm and/or respiratory muscle progression by about 20 weeks, 16 weeks, 12 weeks, 8 weeks, or 4 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, the patient is determined to exhibit cholestasis or one or more symptoms thereof by a finding that the patient exhibits a serum total bile acids level that is greater than 14 μmol/L (e.g., greater than 14 μmol/L, 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, 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, 82 μmol/L, 83 μmol/L, 84 μmol/L, 85 μmol/L, 86 μmol/L, 87 μmol/L, 88 μmol/L, 89 μmol/L, 90 μmol/L, 91 μmol/L, 92 μmol/L, 93 μmol/L, 94 μmol/L, 95 μmol/L, 96 μmol/L, 97 μmol/L, 98 μmol/L, 99 μmol/L, or 100 μmol/L).

In some embodiments of any of the foregoing aspects, the patient is determined to exhibit cholestasis or one or more symptoms thereof by a finding that the patient exhibits one or more parameters in a blood test that is increased or decreased relative to a reference level.

In some embodiments of any of the foregoing aspects, the blood test is a liver function test.

In some embodiments of any of the foregoing aspects, the one or more parameters includes the level of gamma-glutamyl transferase, alkaline phosphatase, aspartate aminotransferase, and/or alanine aminotransferase.

In some embodiments of any of the foregoing aspects, the patient is determined to exhibit hyperbilirubinemia or one or more symptoms thereof by a finding that the patient exhibits a bilirubin level that is greater than 1 mg/dL (e.g., greater than 1 mg/dL, 1.1 mg/dL, 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3. mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL, 3.9 mg/dL, 4 mg/dL, 4.1 mg/dL, 4.2 mg/dL, 4.3 mg/dL, 4.4 mg/dL, 4.5 mg/dL, 4.6 mg/dL, 4.7 mg/dL, 4.8 mg/dL, 4.9 mg/dL, 5 mg/dL, 10 mg/dL, 15 mg/dL, 20 mg/dL, 30 mg/dL, 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL, or 100 mg/dL) in a bilirubin test.

In some embodiments of any of the foregoing aspects, upon administering the viral vector to the patient, the patient displays a bilirubin level that is greater than 1 mg/dL (e.g., greater than 1 mg/dL, 1.1 mg/dL, 1.2 mg/dL, 1.3 mg/dL, 1.4 mg/dL, 1.5 mg/dL, 1.6 mg/dL, 1.7 mg/dL, 1.8 mg/dL, 1.9 mg/dL, 2 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3. mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL, 3.9 mg/dL, 4 mg/dL, 4.1 mg/dL, 4.2 mg/dL, 4.3 mg/dL, 4.4 mg/dL, 4.5 mg/dL, 4.6 mg/dL, 4.7 mg/dL, 4.8 mg/dL, 4.9 mg/dL, 5 mg/dL, 10 mg/dL, 15 mg/dL, 20 mg/dL, 30 mg/dL, 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL, or 100 mg/dL) in a bilirubin test by about 3 weeks after administration of the viral vector to the patient.

In some embodiments of any of the foregoing aspects, the bilirubin level comprises a direct bilirubin level or a total bilirubin level.

In some embodiments of any of the foregoing aspects, the patient is determined to exhibit cholestasis, hyperbilirubinemia, or one or more symptoms thereof by a finding that the patient exhibits a parameter in blood test that is increased relative to a reference level.

In some embodiments of any of the foregoing aspects, the parameter includes the level of a serum bile acid.

In some embodiments of any of the foregoing aspects, the serum bile acid is cholic acid, chenodeoxycholic acid, deoxycholic acid, or ursodeoxycholic acid.

In some embodiments of any of the foregoing aspects, the blood test is a liver function test.

In some embodiments of any of the foregoing aspects, the parameter includes the level of aspartate aminotransferase or alanine aminotransferase.

In one aspect, the disclosure provides a kit including a transgene encoding MTM1 and a package insert, wherein the package insert instructs a user of the kit to administer the transgene to a patient having XLMTM in accordance with the method of any one of the foregoing aspects.

In one aspect, the disclosure provides a kit including a viral vector including a transgene encoding MTM1 and a package insert, wherein the package insert instructs a user of the kit to administer the viral vector to a patient having XLMTM in accordance with the method of any one of the foregoing aspects.

In one aspect, the disclosure provides a kit including an anti-cholestatic agent and a package insert, wherein the package insert instructs a user of the kit to administer the anti-cholestatic agent to a patient to treat or prevent cholestasis or hyperbilirubinemia in accordance with the method of any one of the foregoing aspects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic drawing of an exemplary pseudotyped adeno-associated virus (AAV) 2/8 (AAV2/8) viral vector for the expression of the human myotubularin 1 (hMTM1) gene (e.g., resamirigene bilparvovec). From left to right, the shaded arrows and rectangles represents the nucleic acid sequences encoding a human desmin (hDes) promotor (SEQ ID NO: 3) operatively linked to a Beta-globin Intron, a hMTM1 gene (SEQ ID NO: 4), a Beta-globin poly-adenylation signal (Beta-globin_pA), and flanking AAV2 inverted terminal repeat sequences (ITR). Abbreviations: AAV2_ITR, adeno associated virus 2 inverted terminal repeat; Beta-globin_pA, human Beta-globin polyadenylation signal; hDes, human desmin promotor; hMTM1, human myotubularin complementary DNA.

FIG. 2 is a graph showing changes in total and/or direct bilirubin (fold over the upper limit of normal (ULN)) in human patients treated with 3.0×10¹⁴vg/kg (3e14; light gray) or 1.0×10¹⁴vg/kg (1e14; dark gray) of resamirigene bilparvovec, as described in Example 2, below. Solid rectangles define the 25th to 75th percentiles; crosses define the mean; lower whiskers define the minimum observations above the lower fence (excluding outliers); upper whiskers define the maximum observation below the upper fence (excluding outliers); circles denote outliers, defined as any value above the upper or below the lower fence [greater than 75th percentile+1.5*IQR; or less than 25th percentile−1.5*] where IQR is interquartile range (75th-25th percentiles).

FIG. 3 is a regression plot showing the change from baseline in total bilirubin (mg/dL) in human patients treated with 3.0×10¹⁴vg/kg (light gray solid line) or 1.0×10¹⁴vg/kg (dark gray solid line) of resamirigene bilparvovec, as described in Example 2, below. The regression curve for each group is fitted to all individual subjects dosed at the respective dose (3.0×10¹⁴vg/kg or 1.0×10¹⁴vg/kg of resamirigene bilparvovec; light gray dashed lines and dark gray dashed lines, respectively).

FIGS. 4A-4C are graphs showing respiratory and motor function outcomes after therapy with resamirigene bilparvovec for individual patients. Shown is the time course of ventilator dependence over 24 hours among treated ASPIRO patients on invasive (FIG. 4A), MIP (FIG. 4B), and CHOP INTEND scores (FIG. 4C) for treated ASPIRO patients with a locally estimated scatterplot smoothing regression curve fitted to lower-dose patients, higher-dose patients, and the control group, respectively.

FIGS. 5A-B are heatmaps showing the T-cell and B-cell responses to administration of MTM1 after resamirigene bilparvovec from peripheral blood mononuclear cells (PBMC)/serum samples. FIG. 5A shows an ELISpot assay measuring interferon-γ (IFN-γ) release in response to stimulation of participants PBMCs with MTM1 peptide pool over time in samples from treated participants by mutation type. Shown are IFN-γ cytokine secretion data measured by T cell ELISpot assay. Results are expressed as the number of SFC (spot forming cells) per 10⁶PBMCs. A result was deemed significant if the mean SFC/10⁶cells minus two standard errors is greater than 2× the respective negative control wells, and where p 50.05. Additionally, a 50 SFC/10⁶cut-off was used in determining positive response to AAV8 or MTM1 peptide pool stimulation. Common causes for a result ‘not determined’ include the insufficiency of PBMCs available and failure of samples to respond to stimulation with positive controls. Although a definitive conclusion cannot be made based on the ELISpot data, a number of clinical and histopathological observations do not support a T-cell-mediated immune response as a causative factor in the occurrence of the hepatobiliary severe adverse effects (SAEs). Among the participants that had negative ELISpot results at baseline, participants 12 and 01 later reported cholestatic SAEs. Participant 23 eventually reported SAEs of thrombocytopenia and myocarditis. The baseline sample for participant 09 was positive; over the course of the study he developed severe cholestatic liver dysfunction and fatal sepsis, immune system disorder, and liver disorder. Subjects with only negative (or negative and not determined) results upon dosing included 25, 12 (who eventually developed severe cholestatic liver dysfunction and fatal gastrointestinal bleed), and 06 (who eventually developed severe cholestatic liver dysfunction and fatal sepsis). Patients 11, 33 and 38 did not have ELISpot data. FIG. 5B shows the anti-MTM1 antibody titer overtime in samples from treated participants by mutation type. Shown are anti-MTM1 antibody titer data measured before and after resamirigene bilparvovec dosing of ASPIRO subjects. ‘Not detected’ results are also shown, while presence of antibody titers are shown by heat gradient. MTM1 gene mutations type present in ASPIRO subjects are shown as loss of function (LOF), partial loss of function (PLOF), and inframe exonic deletions (IFED).

FIG. 6 is a diagram of the experimental design for a patient enrollment and dosing trial in the clinical trial ASPIRO.

FIGS. 7A-7D are graphs showing the respiratory and ventilation outcomes after resamirigene bilparvovec. FIGS. 7A and 7B are a graph and quantification, respectively, showing the percent changes from baseline for treated participants as compared with pooled control participants in least-squares mean ventilator hours per 24 hours, while FIGS. 7C and 7D are a graph and quantification, respectively, showing maximal inspiratory pressure (MIP). Participants from the higher-dose cohort (3.0×10¹⁴vg/kg resamirigene bilparvovec) were weaned from ventilation more gradually using a more conservative algorithm with more frequent timepoints compared with the lower-dose cohort (1.0×10¹⁴vg/kg resamirigene bilparvovec). Error bars indicate standard errors. F-tests and associated error bar values are from a mixed effect ANOVA model, indicating a highly significant percent reduction in change from baseline in hours of ventilation per day in treated vs. the control arm over time. Ventilator dependence, data were collected by e-diary (i.e., very frequent reporting) for controls and higher dose participants and collected at discreet site visits for lower-dose participants.

FIGS. 8A and 8B are a graph and quantification, respectively showing motor function after resamirigene bilparvovec. Shown are the changes from baseline in least-squares mean motor function score on the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) scale among treated participants as compared with the pooled control participants. Scores on the CHOP INTEND scale range from 0 to 64, with higher scores indicating better function; a 4-point increment is deemed to be clinically significant. Error bars indicate standard errors.

FIGS. 9A and 9B are a graph and quantification, respectively, showing the achievement of major motor milestones in individual resamirigene bilparvovec-treated and control patients. White boxes start at the age at dosing in the Gene Transfer Clinical Study in X-Linked Myotubular Myopathy (ASPIRO) or age of enrollment (INCEPTUS). The length of the box indicates the patient's time on study. Icons indicate age at motor milestone achievement.

FIGS. 10A-B are a graph and set of photomicrographs, respectively, showing myotubularin (MTM1) protein expression and histopathological changes after resamirigene bilparvovec treatment. FIG. 10A is a graph showing the quantification of an immunoblot of MTM1 protein expression after administration of 1×10¹⁴vg/kg or 3×10¹⁴vg/kg resamirigene bilparvovec, respectively, in muscle biopsy samples from individual patients. FIG. 10B is a set of images showing hematoxylin and eosin (H&E) and nicotinamide adenine dinucleotide (NADH) staining in a muscle biopsy sample taken from a patient administered 1×10¹⁴vg/kg (patient 08) or 3×10¹⁴vg/kg (patient 25) resamirigene bilparvovec, or an age-matched control respectively; at baseline, week 24, and week 48, respectively.

FIG. 11 is a heatmap showing the inflammatory response (e.g., none, very mild, mold, mild to moderate, moderate, moderate to severe, or severe) in patients receiving resamirigene bilparvovec, as assessed by the expression level of CD3 in patients administered a lower (1×10¹⁴vg/kg) or a higher dose (3×10¹⁴vg/kg) of resamirigene bilparvovec.

FIG. 12 is a set of images showing the inflammatory response in patients receiving resamirigene bilparvovec, as assessed with CD3 expression in patients administered 1×10¹⁴vg/kg (patients 17 and 8), at baseline, week 24, and week 48, respectively.

FIG. 13 is a graph showing the Kaplan-Meier analysis of overall survival with the time-to-event analysis based upon the event being death since study enrollment. As of the date cut for the analysis, subjects without event were not included.

FIGS. 14 and 15 are the histopathology of liver biopsies taken from participant 12 day 85 post dose (FIG. 14) and participant 06 during autopsy (FIG. 15). Hemotoxylin and eosin (H&E) stains are shown alongside staining for BSEP, a bile transport protein.

FIG. 14 shows hepatocyte degeneration and giant cell formation, intracellular and extracellular bile collections, bile ductular proliferation, and minimal inflammation.

FIG. 15 shows hepatocyte degeneration, necrosis, and giant cell formation, intracellular and extracellular bile collections, bile ductular proliferation, severe fibrosis, and no significant inflammation.

DEFINITIONS

As used herein, the term “about” refers to a value that is within 10% above or below the value being described. For example, “100 pounds” as used in the context of weight described herein includes quantities that are within 10% above or below 100 lbs. Additionally, when used in the context of a list of numerical quantities, it is to be understood that the term “about,” when preceding a list of numerical quantities, applies to each individual quantity recited in the list.

As used herein, the terms “administering,” “administration,” and the like refer to directly giving a patient a therapeutic agent (e.g., a pharmaceutical composition including a viral vector including a nucleic acid sequence encoding an Myotubularin 1 (MTM1) gene operably linked to a muscle specific promoter) by any effective route. Exemplary routes of administration are described herein and include systemic administration routes, such as intravenous injection, as well as routes of administration directly to the central nervous system of the patient, such as by way of intrathecal injection or intracerebroventricular injection, among others.

As used herein, the term “age-adjusted norms” refers to the process of a normalization of data by age, which is a technique that is used to allow populations of subjects to be compared when the age profiles of the populations are different. As used herein, the term “norm” refers to data that does not undergo a normalization by age, as populations of subjects across age profiles are similar.

As used herein, the terms “alanine aminotransferase” and “ALT” refer to a protein whose amino acid sequence comprises or consists of an 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 an amino acid sequence of a naturally occurring allelic variants of ALT (GPT or GPT2 e.g., splice variants or allelic variants). Human GPT nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_005309.2 (SEQ ID NO: 6), and an exemplary wild-type ALT1 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_005300.1 (SEQ ID NO: 7). Human GPT2 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_001142466.2 (SEQ ID NO: 8), and an exemplary wild-type ALT2 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001135938.1 (SEQ ID NO: 9).

As used herein, the terms “alkaline phosphatase” and “ASP” refer to a protein whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring wild-type ASP protein as well as proteins whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring allelic variants of ASP (e.g., splice variants or allelic variants). Human ASP nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_000478.5 (SEQ ID NO: 10), and an exemplary wild-type ASP amino acid sequence is provided in NCBI RefSeq Acc. No. NP_000469.3 (SEQ ID NO: 11).

As used herein, the term “anti-cholestatic agent” refers to a substance, such as a small molecule that acts to increase bile formation and/or antagonize the effect of hydrophobic bile acids on biological membranes. The term “antagonize,” as used herein with regard to a protein, refers to a molecule that decreases signal transduction resulting from the interaction of the protein with one or more of its binding partners. The antagonist may result in a decrease in the binding of the protein to one or more of its binding partners relative to binding of the two proteins in the absence of the antagonist.

As used herein, the terms “aspartate aminotransferase” and “AST” refer to a protein whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring wild-type AST protein as well as proteins whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring allelic variants of AST (e.g., splice variants or allelic variants). Human AST nucleic acid sequence is provided in NCBI RefSeq Acc. No NM_002079.2 (SEQ ID NO: 12), and an exemplary wild-type ASP amino acid sequence is provided in NCBI RefSeq Acc. No. NP_002070.1 (SEQ ID NO: 13).

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

As used herein, the terms “Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders” and “CHOP INTEND” refer to a validated motor outcome measure developed for the evaluation of weak infants, such as those with a disease of skeletal muscle (e.g., X-linked myotubular myopathy (XLMTM)). CHOP INTEND uses a 0-64-point scale where higher scores indicate better motor function. As used herein, the term “motor function score” refers to a score on the 0-64-point scale of the CHOP INTEND (e.g., a scale of >45 on the CHOP INTEND).

As used herein, the term “cholestasis” refers to a condition where bile cannot flow from the liver to the duodenum. The two clinical distinctions are the “obstructive” type of cholestasis where there is a mechanical blockage in the duct system that can occur from a gallstone or malignancy, and “metabolic” types of cholestasis which are disturbances in bile formation that can occur because of genetic defects or acquired as a side effect of many medications. As used herein, the term “bile” refers to the digestive fluid that is secreted by the liver to aid in the digestion of fats.

As used herein, a “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 some embodiments, a “combination therapy” may include a procedure. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be affected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.

Therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection while a second therapeutic agent of the combination may be administered enterally. In another example, an agent of the therapeutic combination may be administered by intravenous injection and a procedure (e.g., nasobiliary drainage (NBD)) of the therapeutic combination may be performed.

As used herein, the term “dose” refers to the quantity of a therapeutic agent, such as a viral vector described herein, that is administered to a subject at a particular instant for the treatment of a disorder, such as to treat or ameliorate one or more symptoms of a neuromuscular disorder described herein (e.g., XLMTM). A therapeutic agent as 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, therapeutic agent may be administered using one or more unit dosage forms of therapeutic agent, a term that refers to a one or more discrete compositions containing a therapeutic agent that collectively constitute a single dose of the agent.

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 a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, such as clinical results. For example, in the context of treating neuromuscular disorders, such as XLMTM, these terms refer to an amount of the composition sufficient to achieve a treatment response as compared to the response obtained without administration of the composition of interest. An “effective amount,” “therapeutically effective amount,” and the like, of a composition, such as a vector construct of the present disclosure, also include an amount that results in a beneficial or desired result in a subject as compared to a control.

As used herein, the terms “gamma-glutamyl transferase” and “GGT” refers to a protein whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring wild-type GGT protein as well as proteins whose amino acid sequence comprises or consists of an amino acid sequence of a naturally occurring allelic variants of GGT (GGT1, GGT2, and GGT3 e.g., splice variants or allelic variants). Human GGT1 nucleic acid sequence is provided in NCBI RefSeq Acc. No NM_001288833.1 (SEQ ID NO: 14), and an exemplary wild-type GGT1 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_001275762.1 (SEQ ID NO: 15).

As used herein, the term “gestational age” describes how far along a particular pregnancy is and is measured from the first day of a pregnant female subject's last menstrual cycle to the current date. As used herein, the term “labor” (which may also be termed birth) relates to the expulsion of the fetus and placenta from the uterus of a pregnant female subject. For a normal pregnancy, labor may occur at a gestational age of about 40 weeks.

As used herein, the term “hyperbilirubinemia” refers to a condition in which there is a higher-than-normal level of bilirubin in the blood. As used herein, the term “bilirubin” refers to a compound that occurs in the normal catabolic pathway that breaks down heme in vertebrates. This catabolism is a necessary process in the body's clearance of waste products that arise from the destruction of aged or abnormal red blood cells. As used herein, a “bilirubin test” refers to a measurement of the amount of bilirubin in a patient's blood.

As used herein, the term “level” refers to a level of a protein, as compared to a reference. The reference can be any useful reference, as defined herein. By a “decreased level” and an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference (e.g., a decrease or an increase by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or an increase of more than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%, as compared to a reference; a decrease or an increase by less than 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 by more than 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 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). A level of a protein may be expressed in mass/vol (e.g., g/dL, mg/mL, μg/mL, or ng/mL) or percentage relative to total protein in a sample.

As used herein, the terms “liver function test” and “LFT” refers to a hepatic panel (e.g., a group of blood tests that provide information about the state of a patient's liver). A hepatic panel may include measurement of the level of gamma-glutamyl transferase, the level of alkaline phosphatase, the level of aspartate aminotransferase, the level of alanine aminotransferase, the level of albumin, the level of bilirubin, the prothrombin time, the activated partial thromboplastin time, or a combination thereof.

As used herein, the terms “maximal inspiratory pressure” and “MIP” refer to a variable in mechanical ventilation including the total airway pressure delivered, generally used to overcome both respiratory system compliance as well as airway resistance. In as pressure-controlled mode, the MIP includes the sum of the positive-end expiratory pressure and the “delta pressure.” As used herein, the term “delta pressure” refers to a variable in mechanical ventilation including the difference between the MIP and the positive-end expiratory pressure.

As used herein, the term “mechanical ventilatory support” refers to the medical term for artificial ventilation where mechanical means are used to assist or replace spontaneous breathing. As used herein, the term “invasive mechanical ventilatory support” refers to the medical term for artificial ventilation where air is delivered via a tube that is inserted into a patient's windpipe through the mouth or nose and mechanical means are used to assist or replace spontaneous breathing. As used herein, the term “noninvasive mechanical ventillatory support” refers to mechanical ventilatory support in which air is delivered to a patient through a sealed mask that can be placed over the mouth, nose, or the whole face.

As used herein, the term “operably linked” refers to a first molecule joined to a second molecule, wherein the molecules are so arranged 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 adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell.

Additionally, two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present.

As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic compound to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

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 suitable for use with the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which is incorporated herein by reference as it pertains to nucleic acid regulatory elements. Additionally, the term “promoter” may refer to a synthetic promoter, which are regulatory DNA sequences that do not occur naturally in biological systems. Synthetic promoters contain parts of naturally occurring promoters combined with polynucleotide sequences that do not occur in nature and can be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types.

As used herein, a therapeutic agent is considered to be “provided” to a patient if the patient is directly administered therapeutic agent or if the patient is administered a substance that is processed or metabolized in vivo so as to yield therapeutic agent endogenously. For example, a patient, such as a patient having a neuromuscular disorder described herein, may be provided 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., viral vector or cell) that is processed in vivo so as to yield the desired nucleic acid molecule.

As used herein, the terms “patient” and “subject” refer to an organism that receives treatment for a particular disease or condition as described herein (such as a neuromuscular disorder, e.g., XLMTM).

Examples of subjects and patients include mammals, such as humans, receiving treatment for a disease or condition described herein.

By a “reference” is meant any useful reference used to compare protein levels related to cholestatsis, hyperbilirubinemia, or one or more symptoms thereof. The reference can be any sample, standard, standard curve, or level that is used for comparison purposes. The reference can be a normal reference sample or a 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 prior sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject not having cholestatsis, hyperbilirubinemia, or one or more symptoms thereof; a sample from a subject that is diagnosed with cholestatsis, hyperbilirubinemia, or one or more symptoms thereof; a sample from a subject that has been treated for cholestatsis, hyperbilirubinemia, or one or more symptoms thereof; or a sample of a purified protein (e.g., any described herein) at a known normal concentration. By “reference standard or level” is meant a value or number derived from a reference sample. A “normal control value” is a pre-determined value indicative of non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range (“between X and Y”), a high threshold (“no higher than X”), or a low threshold (“no lower than X”). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as “within normal limits” for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject not having cholestatsis, 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 levels of a purified protein, e.g., any described herein, within the normal reference range can also be used as a reference.

As used herein, the term “term age” refers to the age of a patient (e.g., a newborn) born between 37 weeks of gestational age and 42 weeks of gestational age. For example, if the patient was born at 35 weeks of gestational age, the patient is at term age at 14 days old.

As used herein, the term “transgene” refers to a recombinant nucleic acid (e.g., DNA or cDNA) encoding a gene product (e.g., a gene product described herein). The gene product may be an RNA, peptide, or 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(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and/or other functional elements. Embodiments of the disclosure may utilize any known suitable promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and/or other functional elements.

As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a neuromuscular disorder, such as XLMTM, among others. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms (e.g., stiffness and/or joint contractures), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In the context of neuromuscular disorders, such as XLMTM, treatment of a patient may manifest in one or more detectable changes, such as an increase in the concentration of MTM1 protein or nucleic acids (e.g., DNA or RNA, such as mRNA) encoding MTM1, or an increase in MTM1 activity (e.g., by 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. The concentration of MTM1 protein may be determined using protein detection assays known in the art, including ELISA assays described herein.

The concentration of MTM1-encoding nucleic acids may be determined using nucleic acid detection assays (e.g., RNA Seq assays) described herein. Additionally, treatment of a patient suffering from a neuromuscular disorder, such as XLMTM, may manifest in improvements in a patient's muscle function (e.g., skeletal muscle function) as well as improvements in muscle coordination. For example, manifestation of an improvement may include increasing diaphragm and/or respiratory muscle progression.

As used herein, the terms “X-linked myotubular myopathy” and “XLMTM” refer to the genetically inherited neuromuscular disorder that is caused by mutations of the MTM1 gene and is characterized by symptoms including mild to profound muscle weakness, hypotonia (diminished muscle tone), feeding difficulties, and/or severe breathing complications. Human MTM1 has NCBI Gene ID NO 4534. An exemplary wild-type human MTM1 nucleic acid sequence is provided in NCBI RefSeq Acc. No. NM_000252.3 (SEQ ID NO: 1), and an exemplary wild-type myotubularin 1 amino acid sequence is provided in NCBI RefSeq Acc. No. NP_000243.1 (SEQ ID NO: 2).

As used herein, the term “vector” refers to a nucleic acid, e.g., DNA or RNA, that may function as a vehicle for the 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 useful in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/11026, the disclosure of which is incorporated herein by reference. Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of transgenes described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of transgenes contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

Chemical Terms

The chemical terminology used herein is for the purpose of describing various aspects and embodiments of the disclosure and is not intended to be limiting.

In the following chemical definitions, a notation in which an integral number immediately follows an atomic symbol indicates the quantity of atoms of that element that are present in a particular chemical moiety. As will be understood, other atoms, such as hydrogen atoms, or substituent groups described herein, may be present, as necessary, to satisfy the valence of a particular atom. For example, an unsubstituted “C₂alkyl group” has the formula —CH₂CH₃. When used in conjunction with the groups defined herein, a reference to a number of carbon atoms includes the divalent carbon in acetal and ketal groups but does not include the carbonyl carbon in acyl, ester, carbonate, amide, or carbamate groups.

A reference to a number of oxygen, nitrogen, or sulfur atoms in a heteroaryl group only includes those atoms that form a part of a heterocyclic ring.

As used herein, a phrase of the form “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein the alkyl is optionally substituted”). It is not intended to mean that the feature “X” (e.g., alkyl) per se is optional. As described herein, certain compounds may contain one or more “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent, such as any of the substituents or groups described herein. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents that may be used in conjunction with the compounds of the disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions that allow for their production, detection, and, in certain embodiments, recovery, purification, and use for one or more of the purposes disclosed herein.

As used herein, the term “aliphatic” refers to a saturated or unsaturated, straight, branched, or cyclic hydrocarbon. The term “aliphatic” includes, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, and thus incorporates each of these definitions. In some embodiments, “aliphatic” is used to indicate those aliphatic groups having from 1 to 20 carbon atoms.

The aliphatic chain may be, for example, mono-unsaturated, di-unsaturated, tri-unsaturated, or polyunsaturated, or alkynyl. Unsaturated aliphatic groups can be in a cis or trans configuration. In some embodiments, the aliphatic group contains from 1 to about 12 carbon atoms, such as from 1 to about 6 carbon atoms or from 1 to about 4 carbon atoms. In some embodiments, the aliphatic group contains from 1 to about 8 carbon atoms. In some embodiments, the aliphatic group is C₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, or C₁-C₆. The specified ranges used herein indicate an aliphatic group having each member of the range described as an independent species. For example, the term “C₁-C₆aliphatic” as used herein indicates a straight or branched alkyl, alkenyl, or alkynyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms and is intended to mean that each of these is described as an independent species. For example, the term “C₁-C₄aliphatic” as used herein indicates a straight or branched alkyl, alkenyl, or alkynyl group having from 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species. In some embodiments, the aliphatic group is substituted with one or more functional groups that results in the formation of a stable moiety.

As used herein, the term “heteroaliphatic” refers to an aliphatic moiety that contains at least one heteroatom in its chain, such as an amine, carbonyl, carboxy, oxo, thio, phosphate, phosphonate, nitrogen, phosphorus, silicon, or boron atom in place of a carbon atom. In some embodiments, the heteroatom present is nitrogen. In some embodiments, the heteroatom present is oxygen. In some embodiments, the heteroatom present is sulfur. The term “heteroaliphatic” includes, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl moieties. In some embodiments, “heteroaliphatic” is used to indicate a heteroaliphatic group (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having from 1 to 20 carbon atoms. In some embodiments, the heteroaliphatic group is optionally substituted in a manner that results in the formation of a stable moiety. Nonlimiting examples of heteroaliphatic moieties are polyethylene glycol, polyalkylene glycol, amide, polyamide, glycolide, polylactide, polyglycolide, thioether, ether, alkyl-heterocycle-alkyl, —O-alkyl-O-alkyl, and alkyl-O-haloalkyl.

As used herein, the term “acyl” refers to a carbonyl substituent, such as a carbonyl substituent in which the carbonyl carbon is bound to an alkyl group, an alkenyl group, an alkynyl group, an optionally substituted oxygen moiety, an optionally substituted nitrogen moiety, and the like. Exemplary acyl groups include, without limitation, formyl (i.e., a carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl, and butanoyl. Exemplary unsubstituted acyl groups include from 1 to 6, from 1 to 11, or from 1 to 21 carbons.

As used herein, the term “acyloxy” refers to the chemical moiety —OC(O)R in which R is C₁-C₆alkyl, aryl, heteroaryl, C₁-C₆alkyl aryl, or C₁-C₆alkyl heteroaryl.

As used herein, the term “alkyl” refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of 1 to 20 carbon atoms (e.g., 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms). As used herein, the term “alkylene” refers to a divalent alkyl group.

As used herein, the term “alkenyl,” whether recited alone or in combination with other groups, refers to a straight chain or branched hydrocarbon residue having a carbon-carbon double bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms). As used herein, the term “alkenylene” refers to a divalent alkenyl group.

As used herein, the term “alkynyl,” whether recited alone or in combination with other groups, refers to a straight chain or branched hydrocarbon residue having a carbon-carbon triple bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms). As used herein, the term “alkynylene” refers to a divalent alkynyl group.

As used herein, the term “amino” represents —N(R^N1)₂, wherein each R^N1is, independently, H, OH, NO₂, N(R^N2)₂, SO₂OR^N2, SO₂R^N2, SOR^N2, an N-protecting group, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), wherein each of these recited R^N1groups can be optionally substituted; or two R^N1combine to form an alkylene or heteroalkylene, and wherein each R^N2is, independently, H, alkyl, or aryl. The amino groups of the compounds described herein can be an unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e., —N(R^N1)₂).

As used herein, the term “aryl” refers to an aromatic mono- or polycarbocyclic radical of, e.g., 6 to 12, carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, 1,2-dihydronaphthyl, indanyl, and 1H-indenyl.

As used herein, the term “arylalkyl” represents an alkyl group substituted with an aryl group. Exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₁-C₆alkyl C₆-C₁₀aryl, C₁-C₁₀alkyl C₆-C₁₀aryl, or C₁-C₂₀alkyl C₆-C₁₀aryl), such as, benzyl and phenethyl. In some embodiments, the alkyl and the aryl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective groups.

As used herein, the term “bridged cyclyl” refers to a bridged polycyclic group of 5 to 20 atoms, containing from 1 to 3 bridges. Bridged cyclyl includes bridged carbocyclyl (e.g., norbornyl) and bridged heterocyclyl (e.g., 1,4-diazabicyclo[2.2.2]octane).

As used herein, the term “carbocyclyl” refers to a non-aromatic C₃-C₁₂, monocyclic or polycyclic (e.g., bicyclic or tricyclic) structure in which the rings are formed by carbon atoms. Carbocyclyl structures include cycloalkyl groups (e.g., cyclohexyl) and unsaturated carbocyclyl radicals (e.g., cyclohexenyl).

Polycyclic carbocyclyl includes spirocyclic carbocyclyl, bridged carbocyclyl, and fused carbocyclyl. As used herein, the term “carbocyclylene” refers to a divalent carbocyclyl group.

As used herein, the term “cycloalkyl” refers to a saturated, non-aromatic, monovalent mono- or polycarbocyclic radical of 3 to 10, preferably 3 to 6 carbon atoms. This term is further exemplified by radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and adamantyl.

As used herein, the terms “halo” and “halogen” mean a fluorine (fluoro), chlorine (chloro), bromine (bromo), or iodine (iodo) radical.

As used herein, the term “heteroalkyl” refers to an alkyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups. Examples of heteroalkyl groups are an “alkoxy” which, as used herein, refers to alkyl-O— (e.g., methoxy and ethoxy), and an “alkylamino” which, as used herein, refers to —N(alkyl)R^Na, where R^Nais H or alkyl (e.g., methylamino). As used herein, the term “heteroalkylene” refers to a divalent heteroalkyl group.

As used herein, the term “heteroalkenyl” refers to an alkenyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkenyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkenyl groups. Examples of heteroalkenyl groups are an “alkenoxy” which, as used herein, refers to alkenyl-O—. As used herein, the term “heteroalkenylene” refers to a divalent heteroalkenyl group.

As used herein, the term “heteroalkynyl” refers to an alkynyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkynyl group is further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkynyl groups. Examples of heteroalkynyl groups are an “alkynoxy” which, as used herein, refers to alkynyl-O—. As used herein, the term “heteroalkynylene” refers to a divalent heteroalkynyl group.

As used herein, the term “heteroaryl” refers to an aromatic monocyclic or polycyclic structure of 5 to 12 atoms having at least one aromatic ring containing 1, 2, or 3 ring atoms selected from nitrogen, oxygen, and sulfur, with the remaining ring atoms being carbon. In some embodiments, one or two ring carbon atoms of the heteroaryl group are replaced with a carbonyl group. Examples of heteroaryl groups are pyridyl, pyrazoyl, benzooxazolyl, benzoimidazolyl, benzothiazolyl, imidazolyl, oxaxolyl, and thiazolyl. As used herein, the term “heteroarylene” refers to a divalent heteroaryl group.

As used herein, the term “heteroarylalkyl” represents an alkyl group substituted with a heteroaryl group. Exemplary unsubstituted heteroarylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₁-C₆alkyl C₂-C₉heteroaryl, C₁-C₁₀alkyl C₂-C₉heteroaryl, or C₁-C₂₀alkyl C₂-C₉heteroaryl). In some embodiments, the alkyl and the heteroaryl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective groups.

As used herein, the term “heterocyclyl” refers a monocyclic or polycyclic radical (e.g., bicyclic or tricyclic) having 3 to 12 atoms having at least one non-aromatic ring containing 1, 2, 3, or 4 ring atoms selected from N, O, or S, and no aromatic ring containing any N, O, or S atoms. Polycyclic heterocyclyl includes spirocyclic heterocyclyl, bridged heterocyclyl, and fused heterocyclyl. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, thiomorpholinyl, furyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1,3-dioxanyl. As used herein, the term “heterocyclylene” refers to a divalent heterocyclyl group.

As used herein, the term “heterocyclylalkyl” represents an alkyl group substituted with a heterocyclyl group. Exemplary unsubstituted heterocyclylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₁-C₆alkyl C₂-C₉heterocyclyl, C₁-C₁₀alkyl C₂-C₉heterocyclyl, or C₁-C₂₀alkyl C₂-C₉heterocyclyl). In some embodiments, the alkyl and the heterocyclyl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective groups.

As used herein, the term “hydroxyalkyl” refers to an alkyl group substituted with an —OH group.

As used herein, the term “hydroxyl” refers to an —OH group.

As used herein, the term “imine” refers to a ═NR^Ngroup, where R^Nis, e.g., H or alkyl.

As used herein, the term “N-protecting group” refers to those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3rd Edition (John Wiley & Sons, New York, 1999). N-protecting groups include, but are not limited to, acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L, or D, L-amino acids such as alanine, leucine, and phenylalanine; sulfonyl-containing groups such as benzenesulfonyl, and p-toluenesulfonyl; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-20 dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl, arylalkyl groups such as benzyl, triphenylmethyl, and benzyloxymethyl, and silyl groups, such as trimethylsilyl. Preferred N-protecting groups are alloc, formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

As used herein, the term “nitro” refers to an —NO₂group.

As used herein, the term “oxo” refers to an ═O group.

As used herein, the term “sulfonyl” refers to chemical moiety —SO₂—R in which R is hydrogen, aryl, heteroaryl, C₁-C₆alkyl, C₁-C₆alkyl substituted with one or more halogens, such as a —SO₂—CF₃substituent, C₁-C₆alkyl aryl, or C₁-C₆alkyl heteroaryl.

As used herein, the term “sulfonylamino” refers to the chemical moiety —NRSO₂—R′ in which each of R and R′ is independently hydrogen, C₁-C₆alkyl, aryl, heteroaryl, C₁-C₆alkyl aryl, or C₁-C₆alkyl heteroaryl.

As used herein, the term “sulfonyloxy” refers to the chemical moiety —OSO₂—R in which R is hydrogen, C₁-C₆alkyl, C₁-C₆alkyl substituted with one or more halogens, such as a —OSO₂—CF₃substituent, aryl, heteroaryl, C₁-C₆alkyl aryl, or C₁-C₆alkyl heteroaryl.

As used herein, the term “thiol” refers to an —SH group.

The alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl (e.g., cycloalkyl), aryl, heteroaryl, and heterocyclyl groups described herein may be substituted or unsubstituted. When substituted, there will generally be 1 to 4 substituents present, unless otherwise specified. Substituents include, for example: alkyl (e.g., unsubstituted and substituted, where the substituents include any group described herein, e.g., aryl, halo, hydroxy), aryl (e.g., substituted and unsubstituted phenyl), carbocyclyl (e.g., substituted and unsubstituted cycloalkyl), halogen (e.g., fluoro), hydroxyl, heteroalkyl (e.g., substituted and unsubstituted methoxy, ethoxy, or thioalkoxy), heteroaryl, heterocyclyl, amino (e.g., NH₂or mono- or dialkyl amino), azido, cyano, nitro, oxo, sulfonyl, or thiol. Aryl, carbocyclyl (e.g., cycloalkyl), heteroaryl, and heterocyclyl groups may also be substituted with alkyl (unsubstituted and substituted such as arylalkyl (e.g., substituted and unsubstituted benzyl)).

Depictions of Chemical Structures

Compounds of the disclosure may have one or more asymmetric carbon atoms and may exist in the form of optically pure enantiomers, mixtures of enantiomers (e.g., racemates), optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, or mixtures of diastereoisomeric racemates. The optically active forms can be obtained, for example, by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbent or eluant). Accordingly, the compounds disclosed herein may exist in various stereoisomeric forms.

Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. The term “enantiomer” means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of substituents around one or more chiral carbon atoms.

Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as, for example, chiral chromatography and separation methods based thereon. The terms “racemate” and “racemic mixture” refer to a compound containing two enantiomers, wherein such mixtures exhibit no optical activity; i.e., they do not rotate the plane of polarized light. The term “geometric isomer” refers to isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring, or to a bridged bicyclic system. Atoms (other than H) on each side of a carbon-carbon double bond may be in an E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented on the same side of the carbon-carbon double bond) configuration. “R,” “S,” “S*,” “R*,” “E,” “Z,” “cis,” and “trans,” indicate configurations relative to the core molecule.

When the stereochemistry of a compound disclosed herein is named or depicted by structure, the named or depicted stereoisomer is greater than 50% by weight (e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight) relative to its other stereoisomers. For example, when a single enantiomer is named or depicted by structure, the depicted or named enantiomer is greater than 50% by weight (e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight) optically pure. Similarly, when a single diastereomer is named or depicted by structure, the depicted or named diastereomer is greater than 50% by weight (e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight) pure. Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers.

Additionally, when the stereochemistry of a compound disclosed herein is named or depicted by structure, the named or depicted stereoisomer is greater than 50% by mole fraction (e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction) relative to its other stereoisomers. For example, when a single enantiomer is named or depicted by structure, the depicted or named enantiomer is greater than 50% by mole fraction (e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction) relative to the other enantiomer. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is greater than 50% by mole fraction (e.g., at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction) relative to the other diastereomer(s) of the indicated compound. For enantiomeric compounds, percent purity by mole fraction is calculated as the ratio of the molar quantity of the enantiomer of interest relative to the sum of the molar quantities of (i) the enantiomer of interest and (ii) the optical isomer. Similarly, for diastereomeric compounds, percent purity by moles fraction is calculated as the ratio of the molar quantity of the diastereomer of interest relative to the total molar quantities of all diastereomers present for the indicated compound.

When a disclosed compound is named or depicted by structure without indicating the stereochemistry, and the compound has at least one chiral center, it is to be understood that the name or structure encompasses either enantiomer of the compound free from the corresponding optical isomer, a racemic mixture of the compound, or mixtures enriched in one enantiomer relative to its corresponding optical isomer.

When a disclosed compound is named or depicted by structure without indicating the stereochemistry and has two or more chiral centers, it is to be understood that the name or structure encompasses a diastereomer free of other diastereomers, a number of diastereomers free from other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer(s), or mixtures of diastereomers in which one or more diastereomer is enriched relative to the other diastereomers. The present disclosure embraces all of these forms.

Polymorphic Compounds

As will be appreciated by one of skill in the art, many chemical entities can adopt a variety of different solid forms such as, for example, amorphous forms or crystalline forms (e.g., polymorphs, hydrates, solvate). In some embodiments, compounds of the present disclosure may be utilized in any such form, including in any solid form. In some embodiments, compounds described or depicted herein may be provided or utilized in hydrate or solvate form.

DETAILED DESCRIPTION

The present disclosure provides compositions and methods that can be used for treating neuromuscular disorders, particularly X-linked myotubular myopathy (XLMTM). In accordance with the compositions and methods described herein, a patient (e.g., a human patient) having XLMTM may be administered a viral vector, such as an adeno-associated viral (AAV) vector, that contains a transgene encoding Myotubularin 1 (MTM1). The AAV vector may be, for example, a pseudotyped AAV vector, such as an AAV vector containing AAV2 inverted terminal repeats packaged within capsid proteins from AAV8 (AAV2/8). In some embodiments, the transgene is operably linked to a transcription regulatory element, such as a promoter that induces gene expression in a muscle cell. An exemplary promoter that may be used in conjunction with the compositions and methods of the disclosure is a desmin promoter. In some embodiments, the AAV2/8 viral vector comprising a transgene encoding MTM1 is resamirigene bilparvovec.

The present disclosure is based, at least in part, on the discovery of methods of therapeutic and prophylactic treatment that address a significant medical need associated with the existing gene therapy approaches involving the delivery of MTM1 to patients in need thereof (e.g., patients with XLMTM). The present disclosure is also based, in part, on the discovery that the existing gene therapy approaches involving the delivery of MTM1 to patients in need thereof (e.g., patients with XLMTM) are associated with risks, including cholestatic syndromes, such as cholestasis, hyperbilirubinemia, or one or more symptoms thereof. More particularly, the present invention relates to the discovery of a method comprising administration of a viral vector comprising a transgene encoding MTM1 (e.g., resamirigene bilparvovec) and an anti-cholestatic agent (e.g., a bile acid, a farnesoid X receptor (FXR) ligand, a fibroblast growth factor 19 (FGF-19) mimetic, a Takeda-G-protein-receptor-5 (TGR5) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, a PPAR-alpha agonist, a PPAR-delta agonist, a dual PPAR-alpha and PPAR-delta agonist, an apical sodium-dependent bile acid transporter (ASBT) inhibitor, an immunomodulatory drug, an antifibrotic therapy, and a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor) as a prophylactic treatment for cholestatic syndromes associated with the existing gene therapy approaches involving the delivery of MTM1 to patients in need thereof (e.g., patients with XLMTM). In some embodiments, the anti-cholestatic agent is a bile acid. In some embodiments, the bile acid is ursodeoxycholic acid (e.g., ursodiol).

In some embodiments, the disclosure describes a method of treating or preventing cholestasis or hyperbilirubinemia in a human patient that has XLMTM and who has been administered a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1 includes administering to the patient an anti-cholestatic agent.

In some embodiments, the disclosure describes a method of treating XLMTM in a human patient in need thereof and who has been previously administered an anti-cholestatic agent, the method comprising administering to the patient a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1.

In some embodiments, the disclosure describes a method of reducing stiffness and/or joint contractures in a human patient diagnosed as having XLMTM, the method comprising administering to the patient a viral vector comprising a transgene encoding MTM1 and (an anti-cholestatic agent.

In some embodiments, the disclosure describes a method of increasing diaphragm and/or respiratory muscle progression in a human patient diagnosed as having XLMTM, the method comprising administering to the patient a viral vector comprising a transgene encoding MTM1 and (an anti-cholestatic agent.

In some embodiments, the disclosure describes a method of reducing stiffness and/or joint contractures in a human patient diagnosed as having XLMTM and who has been previously administered an anti-cholestatic agent, the method comprising administering to the patient a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1.

In some embodiments, the disclosure describes a method of increasing diaphragm and/or respiratory muscle progression in a human patient diagnosed as having XLMTM and who has been previously administered an anti-cholestatic agent, the method comprising administering to the patient a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1.

The sections that follow provide a description of therapeutic agents and parameters for assessing cholestasis, hyperbilirubinemia, or one or more symptoms thereof that result in the administration of an anti-cholestatic agent described herein. The following sections also describe various transduction agents that may be used in conjunction with the compositions and methods of the disclosure.

Methods of Treatment

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

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

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

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

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

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

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

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

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

In some embodiments, the patient is a child aged 3-5 years old at the time of administration of the viral vector. For example, in some embodiments, the patient is a child that is about 3 years old to about 5 years old (e.g., 3 years old to about 5 years old or 4 years old to about 5 years old). In some embodiments, the patient is 3 years old. In some embodiments, the patient is 4 years old. In some embodiments, the patient is 5 years old.

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

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

In some embodiments, the patient was born at greater than or equal to 35 weeks 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 adjusted term age (e.g., 37 weeks of gestational age or greater) to about 5 years old at the time of administration of the viral vector. For example, if the patient was born at 35 weeks of gestational age, the patient is at term age at 14 days old.

In some embodiments, the patient was born at 35 weeks of gestational age and is between adjusted term age to about 5 years old (e.g., 14 days old to about 5 years old, 15 days old to about 5 years old, 16 days old to about 5 years old, 17 days old to about 5 years old, 18 days old to about 5 years old, 19 days old to about 5 years old, 20 days old to about 5 years old, 25 days old to about 5 years old, one month old to about 5 years old, two months old to about 5 years old, 3 months old to about 5 years old, 4 months old to about 5 years old, 5 months old to about 5 years old, 6 months old to about 5 years old, 1 year old to about 5 years old, 2 years old to about 5 years old, 3 years old to about 5 years old, and 4 years old to about 5 years old) at the time of administration of the viral vector.

In some embodiments, the patient was born at 36 weeks of gestational age and is between adjusted term age to about 5 years old (e.g., 7 days old to about 5 years old, 8 days old to about 5 years old, 9 days old to about 5 years old, 10 days old to about 5 years old, 11 days old to about 5 years old, 12 days old to about 5 years old, 13 days old to about 5 years old, 14 days old to about 5 years old, 15 days old to about 5 years old, 16 days old to about 5 years old, 17 days old to about 5 years old, 18 days old to about 5 years old, 19 days old to about 5 years old, 20 days old to about 5 years old, 25 days old to about 5 years old, one month old to about 5 years old, two months old to about 5 years old, 3 months old to about 5 years old, 4 months old to about 5 years old, 5 months old to about 5 years old, 6 months old to about 5 years old, 1 year old to about 5 years old, 2 years old to about 5 years old, 3 years old to about 5 years old, and 4 years old to about 5 years old) at the time of administration of the viral vector.

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

In some embodiments, the patient is male.

In some embodiments, the patient is female.

X-Linked Myotubular Myopathy

XLMTM is a rare, life-threatening, congenital myopathy caused by a loss-of-function mutation in the MTM1 gene and is characterized in most patients by profound muscle weakness and hypotonia at birth, which results in severe respiratory insufficiency, inability to sit up, stand or walk, and early mortality.

The myopathy associated with XLMTM impairs the development of motor skills such as sitting, standing, and walking. Affected infants may also have difficulties with feeding due to muscle weakness.

Individuals with this condition often do not have the muscle strength to breathe on their own and must be supported with mechanical ventilation. Some affected individuals require mechanical ventilation only periodically, such as during sleep, while others require mechanical ventilation continuously. Patients having XLMTM may also have weakness in the muscles that control eye movement (ophthalmoplegia), weakness in other muscles of the face, and absent reflexes (areflexia).

In XLMTM, muscle weakness often disrupts normal bone development and can lead to fragile bones, an abnormal curvature of the spine (scoliosis), and joint deformities (contractures) of the hips and knees. Patients having XLMTM may have a large head with a narrow and elongated face and a high, arched roof of the mouth (palate). Patients may also have liver disease, recurrent ear and respiratory infections, or seizures.

As a consequence of their severe breathing difficulties, patients having XLMTM usually survive only into early childhood; however, some patients with this condition have lived into adulthood. The compositions and methods of the disclosure provide the important medical benefit of being able to prolong the lifetimes of such patients by restoring functional MTM1 expression. Moreover, the compositions and methods described herein can be used to improve patients' quality of life post-treatment (e.g., reducing stiffness and/or joint contractures or increasing diaphragm and/or respiratory muscle progression), as the disclosure provides a series of guidelines that can be used to determine a patient's eligibility for being weaned off of mechanical ventilation.

Cholestasis and Hyperbilirubinemia

Cholestasis is any condition in which the flow of bile acid from the liver is slowed or blocked, while hyperbilirubinemia is a condition in which there is an accumulation of bilirubin in the blood and serum bile acids appear to remain normal. By contrast, cholestatic syndromes are 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, wherein a finding that the parameter is above a reference level identifies the patient as having cholestasis, hyperbilirubinemia, or one or more symptoms thereof

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

In some embodiments, it is determined that the patient exhibits cholestasis, hyperbilirubinemia, or one or more symptoms thereof and the patient is administered an anti-cholestatic agent.

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

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

In some embodiments, the disclosure provides a method of treating cholestasis in a human patient that has XLMTM and who has been previously administered a viral vector comprising a transgene encoding MTM1 (e.g., resamirigene bilparvovec), the method including administering to the patient an anti-cholestatic agent.

In some embodiments, the disclosure provides a method of treating hyperbilirubinemia in a human patient that has XLMTM and who has been previously administered a viral vector comprising a transgene encoding MTM1 (e.g., resamirigene bilparvovec), the method including administering to the patient an anti-cholestatic agent.

In some embodiments, the disclosure provides a method of preventing cholestasis in a human patient that has XLMTM and who has been previously administered a viral vector comprising a transgene encoding MTM1 (e.g., resamirigene bilparvovec), the method including administering to the patient an anti-cholestatic agent.

In some embodiments, the disclosure provides a method of preventing hyperbilirubinemia in a human patient that has XLMTM and who has been previously administered a viral vector comprising a transgene encoding MTM1 (e.g., resamirigene bilparvovec), the method including administering to the patient an anti-cholestatic agent.

In some embodiments, the patient does not have a history of cholestasis or hyperbilirubinemia.

In some embodiments, the patient does not have a history of any underlying liver disease.

Vectors for Delivery of Exogenous Nucleic Acids to Target Cells
Viral Vectors for Nucleic Acid Delivery

Viral genomes provide a rich source of vectors that can be used for the efficient delivery of 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). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include AAV, retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).

Other viruses useful for delivering polynucleotides encoding antibody light and heavy chains or antibody fragments of the invention include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, 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. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

AAV Vectors for Nucleic Acid Delivery

In some embodiments, nucleic acids of the compositions and methods described herein are incorporated into recombinant AAV (rAAV) vectors and/or virions in order to facilitate their introduction into a cell. rAAV vectors useful in the invention are recombinant nucleic acid constructs that include (1) a transgene to be expressed (e.g., a polynucleotide encoding a MTM1 protein) and (2) viral nucleic acids that facilitate integration and expression of the heterologous genes. The viral nucleic acids may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional inverted terminal repeats (ITRs)) of the DNA into a virion. In typical applications, the transgene encodes MTM1, which is useful for correcting a MTM1 mutation in patients suffering from neuromuscular disorders, such as XLMTM. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV wild type genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype (e.g., derived from serotype 2) suitable for a 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 are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

The nucleic acids and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for example, in U.S. Pat. Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery. rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8 and 9. For targeting muscle cells, rAAV virions that include at least one serotype 1 capsid protein may be particularly useful. rAAV virions that include at least one serotype 6 capsid protein may also be particularly useful, as serotype 6 capsid proteins are structurally similar to serotype 1 capsid proteins, and thus are expected to also result in high expression of MTM1 in muscle cells. rAAV serotype 9 has also been found to be an efficient transducer of muscle cells. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol. 72:2224-2232 (1998); Halbert et al., J.

Virol. 74:1524-1532 (2000); Halbert et al., J. Virol. 75:6615-6624 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

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

AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting 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 can be used in methods of the invention include those capsid hybrids that are generated by molecular breeding of viruses as well as 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).

Resamirigene Bilparvovec

As described herein, a pseudotyped AAV vector including a nucleic acid sequence encoding a MTM1 gene (SEQ ID NO: 4) operably linked to a desmin promotor (SEQ ID NO:3) flanked by AAV2 ITR and packaged within capsid proteins from AAV8 (AAV2/8) as well as the other genetic components listed in Table 1, refers to the compound known by the international proprietary name (INN) of resamirigene bilparvovec.

Resamirigene bilparvovec 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 built by cloning a synthetic DNA sequence complementary to the coding portion (nucleotides 43-1864) of the wild-type human MTM1 transcript (NCBI Ref. Seq NM_000252.3) downstream of the 1.05-kb human desmin enhancer/promoter region. The second intron and polyadenylation sequence of the human p-globin gene (HBB) were inserted upstream and downstream respectively of the MTM1 synthetic cDNA to mediate RNA processing. The expression cassette was flanked by AAV serotype-2 (AAV2) inverted terminal repeats (ITRs). The vector was produced in an AAV8 capsid by two-plasmid transfection in HEK293 cells in suspension culture in bioreactors a full GMP process.

In some embodiments, a method of treating a disorder (e.g., XLMTM) or alleviating one or more symptoms (e.g., stiffness and/or join contractures or need for diaphragm and/or respiratory muscle progression) of a disorder (e.g., XLMTM) in a human patient in need thereof, includes administering to the patient a therapeutically effective amount of resamirigene bilparvovec during a treatment period.

In some embodiments, a method of weaning a human patient off of mechanical ventilation, includes a patient that has previously been administered a therapeutically effective amount of resamirigene bilparvovec. The components of resamirigene bilparvovec are shown in Table 1, below:

TABLE 1

Resamirigene Bilparvovec Nucleic Acid Sequence (SEQ ID NO: 5)

Range

(nucleotides,

relative to
Length

SEQ ID NO: 5)
(nucleotides)
Genetic Component

3080-3198
119
AAV2 ITR

3199-3256
58
Linker sequence

3257-4316
1,060
Human desmin promoter (SEQ ID NO: 3)

4317-4354
38
Linker sequence

4355-4460
106
Human Beta-globin intron

4373-4848
476
Human Beta-globin intron

4458-4902
445
Human Beta-globin intron

4917-6738
1,822
Human MTM1 coding sequence (SEQ ID

NO: 4)

6739-6759
21
Linker sequence

6760-7519
760
Human Beta-globin poly-adenylation

sequence

7520-7551
32
Linker sequence

7552
7,696
AAV2 ITR

As described herein, resamirigene bilparvovec refers to the AAV vector having the nucleic acid sequence of SEQ ID NO: 5, shown below:

TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG
50

GACGTCATTG TCGATCCTGC AGGCGTACGG TAAAAAAAGG CATAGCTAAC
100

AAGGTGTGGA AAAAGAATTA GTGGTTAGAG AGTGAGCTAT TCGTTGAAAC
150

AATTGCGTTC TTGAAACAAT TCTTGCTGGT AAAATGTCAC ATTTTATGTG
200

ACTACAGGTG GAGGATTGGC ACATAACCTA ACCAGTGGGG GAAACAATTG
250

ACCTCTGGAT TTGTCCAAGT GTATAGTAGC ATTTGCCCAA TCGAATGGTC
300

CTGGTAAGGT GTTAATGTTG ACTAGAACCA AAGGTGGAAG TTGCAGGGAA
350

ACTGGTTTAG TACAAGGGTG GACACCAGGC AGTCATCCAG AGGCCCATTA
400

AAGGCCTTGG AATGTTTTTC CGAAGGAGAA TCACTCCCTC TTCTCTCGCT
450

TAAAGTTTTA GGGGATTCAT GAACAGCTGC TGTGGGATAG TTTCATGTCC
500

CTAGCAATTG TAAAGCAACT GAGGGTGGCT TAAACCAGTT TTAGCTTTAG
550

GGTTAGGGTT ACTGGACTAA AATTTGAGAA ATTCATAAAT CTTAAGGAAA
600

TCCATTGTGA GTTTTCATTA TGAGTGCATC CAATGTATAA TTTCCATGAC
650

CCTCCCATGC AAGTGAGCAT GTGAATCAGG AAACGTTACA AGAACCCAAC
700

AAACTCAACC ACTACTAGAC AGGCGATCAC TTCCAGTTAG TATGCAACTT
750

TCTGTGTAAT TTTAGTTACC ATTAAAATCT GGATGACCTT AGTGTAAGGA
800

AAAAATACCT TGAATAGTGT TAAAGATGTA CACTTGGTGT CAGGCATTGT
850

AACATTGATA AATCTGTGTA AGGTGCTTTT TGAAAACTTC AAAGCTGCAT
900

CAAGTCAAGT ACAAGAAAGG CCATGGCTGC TAAAGCTGTT GAAGATGTGG
950

GATGGAACTG GGTCACATTG GTGTTAACAG CGTTGTGCAG AGCCGGCAGG
1000

ATCTTGGTGT GAGCGAACAT TAGTCTATTT AATAAAGCTG TGTGAATGTT
1050

GTAGAGGTGA GGATGCTCAC TTGAAAACTC ACTGAAGAAC ACTTGGCCCC
1100

TTGAACTAAA GTGCTTCTAT CAAGTTCAGT GAGAAATTCC GAATTACAAG
1150

CATAGGTACT AGAAAAGTTT TGAAAAGCAG TATAGAGCAA CATAAGCACA
1200

TTCATAAAAT TAGTGATGTA GAAAGTGAAA TTTCCACGTA TGGTCACTCC
1250

CAGAGAAAAA AAATACGTTT ATTTACCTTT TTTAAAAATA GGGGATTTCA
1300

GGCCGGGTGA GGTGGCTCAC GCCTGTAATC CCAGCACTTT GGGAGGCCCA
1350

GGTGGGCGGA TCACCTGAGG TCAGGAGTTG GAGGGATGGC AAATCCCATC
1400

TCTACAAAAT ATACAAAAAA ATAGCTGGGT GTGTTGGCAG GCGCCTGTAA
1450

TCCCAGCTAC TCGGAAGGCT GAGGCAGGAG AATCCCTGGA ACCAGGGATG
1500

TGGAGGTTGC AGTGAGCCGA GATTGTGTAA CTGCATTCCA GCCTGGGCAA
1550

CAAGAGCAAG ACTCCGTATC AGGAAAAAAA AAAGGGGGGG TTGGATTTCG
1600

CTTGTTGCAT AGGTTGGTCT CAAACTCCTG GCCTCAAGTG ATTCTCCTGC
1650

CTCTGCCTCC CAAAGTGCTG AGATTACAGG TGTGAGGCAC CATGCCAGGT
1700

CTCTTACTGT TTGTAATTAA ATACATACAC ATTTTGTGTG TTTGTGTGCA
1750

CCTTTATAAA GTCAAAGGTG ATAGTAACCC ATTTAAGTTC CTACTCAATT
1800

TTACTTTCCA GGGATAACTA ACTACTTTTT CTTTTTGAGA TGGAGTCTCG
1850

CTGTGTAGCC CAGGCTGGAG TGCAGTGGCA CCATCTCGGC TCACTGCAAG
1900

CTCCTCCTCC CTGGTTCACG CTATTCTCCT GCCTCAGCCT CCCCAACAAC
1950

TAGGACTACA GGCTCACCTC GCCATACCTG GCTAATTTTT TGTATTTTTA
2000

GTAGAGACAG GGTTTCACTG TGTTAGCCAG GATGGTCTCG ATCTCCTGAC
2050

CTTGTGATCC GCCTGCCTCT GCCTCCCAAA GTGCTGGGAT TACAGGCATG
2100

AGCAACCTCA CCCAGCTGGG ATAACTACTT TTTACAGGTT GATATTCTTT
2150

TGGACTTTTC CCCTGTGTAA AAATATACTA TATTTGTTAT GTACATATTA
2200

TGTACATACA GACACAAATT GGACCATTCT CAGTATAATG ATTCTCAGGT
2250

TTTTTTTTTT TTTTTGAGGT GGGGAACTAG ATAATTATGG ACATCTTTCC
2300

ATACTAGCAT ATCAATATCT ACCTCATTCT TTTTAATATT TTTGCTAGTA
2350

TTCCATTGTA TGAATGTCCT ATGATTTACT TAACCTGTCC ATCAATATTT
2400

GTTTCCAGGT TTTTGCTATT ATAATGCTGC TGCAAAGTAC ATCCTCACAC
2450

ATCTTTATTT TGTCTATTCA TATTTCTGTA AGATAGGTTA CTAAAGTTGG
2500

AACTGCCAAA TTAACACTAT CATACTATTT TGTTTTTTAA TTTTAATTTT
2550

TTAAAAAATG TAAAATGTGC AATTTCAAGA GGAGAAACTT GAACACAAGG
2600

AGCAAAATCT ATTTTTATAA CATCCTATTA AAAGCTTGCT TTACATAAAG
2650

ATTTTGAAAG AATAGCATAA ATACAAGATT TCTATTTTAA TTGGATTCTT
2700

AGGGCTAATA AAATAATCAG CCTTAGCACT TATTTATTTA TTTTTTTTGA
2750

GAGGGAGTCT CGCTCTGTTG TCCATGCTGG AGTGCAGTGG CGTGATCTCG
2800

GCTCACTGCA AGCTCCACCT CATGAGTTCA CACCATTCTC CTGCCTCAGT
2850

CTCCCGAGTA GCTGGGACTC CAGGCGCCCT CTACAAAGCC CGTCTAATTT
2900

TTTTTGTATT TTTAGTAGAG ACAGGGTTTC ACTGTGTTAG CCAGGATGGT
2950

CTTGATCTCC TGACCTTGTG ATCTGCCCGC CTCGGCCTCC CAAAGTGCTG
3000

GGATTATAGG CTTGAGCCAC TGCTCCCGGC CAGCACTTAT TTTTATAATT
3050

CTTCATGATT ACTGTGTTAC TGTCCCATGG GCCGCCAGGG CCAGCTAGGT
3100

TGGCCACTCC CTCTCTGCGC GCTCGCTCGC TCACTGAGGC CGGGCGACCA
3150

AAGGTCGCCC GACGCCCGGG CTTTGCCCGG GCGGCCTCAG TGAGCGAGCG
3200

AGCGCGCAGA GAGGGAGTGG CCAACTCCAT CACTAGGGGT TCCTCCTAGC
3250

ACGCGCTACC CCCTGCCCCC CACAGCTCCT CTCCTGTGCC TTGTTTCCCA
3300

GCCATGCGTT CTCCTCTATA AATACCCGCT CTGGTATTTG GGGTTGGCAG
3350

CTGTTGCTGC CAGGGAGATG GTTGGGTTGA CATGCGGCTC CTGACAAAAC
3400

ACAAACCCCT GGTGTGTGTG GGCGTGGGTG GTGTGAGTAG GGGGATGAAT
3450

CAGGGAGGGG GCGGGGGACC CAGGGGGCAG GAGCCACACA AAGTCTGTGC
3500

GGGGGTGGGA GCGCACATAG CAATTGGAAA CTGAAAGCTT ATCAGACCCT
3550

TTCTGGAAAT CAGCCCACTG TTTATAAACT TGAGGCCCCA CCCTCGACAG
3600

TACCGGGGAG GAAGAGGGCC TGCACTAGTC CAGAGGGAAA CTGAGGCTCA
3650

GGGCCAGCTC GCCCATAGAC ATACATGGCA GGCAGGCTTT GGCCAGGATC
3700

CCTCCGCCTG CCAGGCGTCT CCCTGCCCTC CCTTCCTGCC TAGAGACCCC
3750

CACCCTCAAG CCTGGCTGGT CTTTGCCTGA GACCCAAACC TCTTCGACTT
3800

CAAGAGAATA TTTAGGAACA AGGTGGTTTA GGGCCTTTCC TGGGAACAGG
3850

CCTTGACCCT TTAAGAAATG ACCCAAAGTC TCTCCTTGAC CAAAAAGGGG
3900

ACCCTCAAAC TAAAGGGAAG CCTCTCTTCT GCTGTCTCCC CTGACCCCAC
3950

TCCCCCCCAC CCCAGGACGA GGAGATAACC AGGGCTGAAA GAGGCCCGCC
4000

TGGGGGCTGC AGACATGCTT GCTGCCTGCC CTGGCGAAGG ATTGGTAGGC
4050

TTGCCCGTCA CAGGACCCCC GCTGGCTGAC TCAGGGGCGC AGGCCTCTTG
4100

CGGGGGAGCT GGCCTCCCCG CCCCCACGGC CACGGGCCGC CCTTTCCTGG
4150

CAGGACAGCG GGATCTTGCA GCTGTCAGGG GAGGGGAGGC GGGGGCTGAT
4200

GTCAGGAGGG ATACAAATAG TGCCGACGGC TGGGGGCCCT GTCTCCCCTC
4250

GCCGCATCCA CTCTCCGGCC GGCCGCCTGC CCGCCGCCTC CTCCGTGCGC
4300

CCGCCAGCCT CGCCCGGACT CTAGAGGATC CAGATCTAAG CTTCTCTGGT
4350

CACCGATCCT GAGAACTTCA GGGTGAGTCT ATGGGACCCT TGATGTTTTC
4400

TTTCCCCTTC TTTTCTATGG TTAAGTTCAT GTCATAGGAA GGGGAGAAGT
4450

AACAGGGTAC ACATATTGAC CAAATCAGGG TAATTTTGCA TTTGTAATTT
4500

TAAAAAATGC TTTCTTCTTT TAATATACTT TTTTGTTTAT CTTATTTCTA
4550

ATACTTTCCC TAATCTCTTT CTTTCAGGGC AATAATGATA CAATGTATCA
4600

TGCCTCTTTG CACCATTCTA AAGAATAACA GTGATAATTT CTGGGTTAAG
4650

GCAATAGCAA TATTTCTGCA TATAAATATT TCTGCATATA AATTGTAACT
4700

GATGTAAGAG GTTTCATATT GCTAATAGCA GCTACAATCC AGCTACCATT
4750

CTGCTTTTAT TTTATGGTTG GGATAAGGCT GGATTATTCT GAGTCCAAGC
4800

TAGGCCCTTT TGCTAATCAT GTTCATACCT CTTATCTTCC TCCCACAGCT
4850

CCTGGGCAAC GTGCTGGTCT GTGTGCTGGC CCATCACTTT GGCAAAGAAT
4900

TCCGCGGGCG GCCGCAAGTT TCCAGGATGG CTTCTGCATC AACTTCTAAA
4950

TATAATTCAC ACTCCTTGGA GAATGAGTCT ATTAAGAGGA CGTCTCGAGA
5000

TGGAGTCAAT CGAGATCTCA CTGAGGCTGT TCCTCGACTT CCAGGAGAAA
5050

CACTAATCAC TGACAAAGAA GTTATTTACA TATGTCCTTT CAATGGCCCC
5100

ATTAAGGGAA GAGTTTACAT CACAAATTAT CGTCTTTATT TAAGAAGTTT
5150

GGAAACGGAT TCTTCTCTAA TACTTGATGT TCCTCTGGGT GTGATCTCGA
5200

GAATTGAAAA AATGGGAGGC GCGACAAGTA GAGGAGAAAA TTCCTATGGT
5250

CTAGATATTA CTTGTAAAGA CATGAGAAAC CTGAGGTTCG CTTTGAAACA
5300

GGAAGGCCAC AGCAGAAGAG ATATGTTTGA GATCCTCACG AGATACGCGT
5350

TTCCCCTGGC TCACAGTCTG CCATTATTTG CATTTTTAAA TGAAGAAAAG
5400

TTTAACGTGG ATGGATGGAC AGTTTACAAT CCAGTGGAAG AATACAGGAG
5450

GCAGGGCTTG CCCAATCACC ATTGGAGAAT AACTTTTATT AATAAGTGCT
5500

ATGAGCTCTG TGACACTTAC CCTGCTCTTT TGGTGGTTCC GTATCGTGCC
5550

TCAGATGATG ACCTCCGGAG AGTTGCAACT TTTAGGTCCC GAAATCGAAT
5600

TCCAGTGCTG TCATGGATTC ATCCAGAAAA TAAGACGGTC ATTGTGCGTT
5650

GCAGTCAGCC TCTTGTCGGT ATGAGTGGGA AACGAAATAA AGATGATGAG
5700

AAATATCTCG ATGTTATCAG GGAGACTAAT AAACAAATTT CTAAACTCAC
5750

CATTTATGAT GCAAGACCCA GCGTAAATGC AGTGGCCAAC AAGGCAACAG
5800

GAGGAGGATA TGAAAGTGAT GATGCATATC ATAACGCCGA ACTTTTCTTC
5850

TTAGACATTC ATAATATTCA TGTTATGCGG GAATCTTTAA AAAAAGTGAA
5900

GGACATTGTT TATCCTAATG TAGAAGAATC TCATTGGTTG TCCAGTTTGG
5950

AGTCTACTCA TTGGTTAGAA CATATCAAGC TCGTTTTGAC AGGAGCCATT
6000

CAAGTAGCAG ACAAAGTTTC TTCAGGGAAG AGTTCAGTGC TTGTGCATTG
6050

CAGTGACGGA TGGGACAGGA CTGCTCAGCT GACATCCTTG GCCATGCTGA
6100

TGTTGGATAG CTTCTATAGG AGCATTGAAG GGTTCGAAAT ACTGGTACAA
6150

AAAGAATGGA TAAGTTTTGG ACATAAATTT GCATCTCGAA TAGGTCATGG
6200

TGATAAAAAC CACACCGATG CTGACCGTTC TCCTATTTTT CTCCAGTTTA
6250

TTGATTGTGT GTGGCAAATG TCAAAACAGT TCCCTACAGC TTTTGAATTC
6300

AATGAACAAT TTTTGATTAT AATTTTGGAT CATCTGTATA GTTGCCGATT
6350

TGGTACTTTC TTATTCAACT GTGAATCTGC TCGAGAAAGA CAGAAGGTTA
6400

CAGAAAGGAC TGTTTCTTTA TGGTCACTGA TAAACAGTAA TAAAGAAAAA
6450

TTCAAAAACC CCTTCTATAC TAAAGAAATC AATCGAGTTT TATATCCAGT
6500

TGCCAGTATG CGTCACTTGG AACTCTGGGT GAATTACTAC ATTAGATGGA
6550

ACCCCAGGAT CAAGCAACAA CAGCCGAATC CAGTGGAGCA GCGTTACATG
6600

GAGCTCTTAG CCTTACGCGA CGAATACATA AAGCGGCTTG AGGAACTGCA
6650

GCTCGCCAAC TCTGCCAAGC TTTCTGATCC CCCAACTTCA CCTTCCAGTC
6700

CTTCGCAAAT GATGCCCCAT GTGCAAACTC ACTTCTGACC GGTCCGAGGG
6750

CCCAGATCTA ATTCACCCCA CCAGTGCAGG CTGCCTATCA GAAAGTGGTG
6800

GCTGGTGTGG CTAATGCCCT GGCCCACAAG TATCACTAAG CTCGCTTTCT
6850

TGCTGTCCAA TTTCTATTAA AGGTTCCTTT GTTCCCTAAG TCCAACTACT
6900

AAACTGGGGG ATATTATGAA GGGCCTTGAG CATCTGGATT CTGCCTAATA
6950

AAAAACATTT ATTTTCATTG CAATGATGTA TTTAAATTAT TTCTGAATAT
7000

TTTACTAAAA AGGGAATGTG GGAGGTCAGT GCATTTAAAA CATAAAGAAA
7050

TGAAGAGCTA GTTCAAACCT TGGGAAAATA CACTATATCT TAAACTCCAT
7100

GAAAGAAGGT GAGGCTGCAA ACAGCTAATG CACATTGGCA ACAGCCCCTG
7150

ATGCCTATGC CTTATTCATC CCTCAGAAAA GGATTCAAGT AGAGGCTTGA
7200

TTTGGAGGTT AAAGTTTTGC TATGCTGTAT TTTACATTAC TTATTGTTTT
7250

AGCTGTCCTC ATGAATGTCT TTTCACTACC CATTTGCTTA TCCTGCATCT
7300

CTCAGCCTTG ACTCCACTCA GTTCTCTTGC TTAGAGATAC CACCTTTCCC
7350

CTGAAGTGTT CCTTCCATGT TTTACGGCGA GATGGTTTCT CCTCGCCTGG
7400

CCACTCAGCC TTAGTTGTCT CTGTTGTCTT ATAGAGGTCT ACTTGAAGAA
7450

GGAAAAACAG GGGGCATGGT TTGACTGTCC TGTGAGCCCT TCTTCCCTGC
7500

CTCCCCCACT CACAGTGACC GGCCGCTCTA GGAGGAACCC CTAGTGATGG
7550

AGTTGGCCAC TCCCTCTCTG CGCGCTCGCT CGCTCACTGA GGCCGGGCGA
7600

CCAAAGGTCG CCCGACGCCC GGGCTTTGCC CGGGCGGCCT CAGTGAGCGA
7650

GCGAGCGCGC AGAGAGGGAG TGGCCAACCT AGAGGCCGCC AGGGCCATAT
7700

TTCTCAATTT TTAAATTTTT CAAAAAAATT AATCCTTAAT GTGCATATTT
7750

TTGAATTGTT AATATAACTT TTTGAGGTGA TGTCTTCATG TGTTTCAACT
7800

ACTTAAAAAC TTTTAAACAG TATATAATAA AAAATCTTCC AGGCCACTCA
7850

CACCTGTAAT CCCAGCACTT TGGGAGGCTG AGGTGGGCAG ATCACCTGAG
7900

GGCAGGAGTT CGAGACCAGC CTGGCCAATA TATATATATT CATATATTCA
7950

TATATATATA TATATTCATA TATTCATATA TATATATTCA TATATTCATA
8000

TATATATATA TATATATATA TAGCAAAACC TCATCTCTAA TAAAATACAA
8050

AAATTAGCTG AGCGTGGTGA TGGATGCCTG TAGTCCCAGC TACTCGGGAG
8100

GCTGAGGCAG GAGAATCTCT TGAACCTGGG AGGTGGAGGT TGCAGTGAGC
8150

TGAGATGGTG CCACTGCCCT CCAGCCTGAG TGACAGAGCG AGACTCGGTC
8200

TCCAAAAAAA AACAACAAAA AAATCTTCCA TCCTTGTCTC CCATCCACCC
8250

CTTCCCCCCA GCATGTACTT GCAGACTTTA TGCATATACA GTGAGTACTG
8300

TATATACACA AATAATAAAA AAATCATATA TATAATATAT GTAATTCCCC
8350

TTTACATGAA AGGTAGCACA CTGGTCTGTA CAGTCTGTCT GCACTGTGCT
8400

ATTTCACTTT ATATTTTTAT AGTTTGACAG AGTTCTAACA TTTCTTTTTT
8450

TTTTTTTTTA ACAGAGTCTT GTTCCTGATT GTTAAATTTT AAAGCATCCT
8500

AAAGTTTGGT TTCACACTTG AATGAATACC ATGTAAGGAT TCACTTACAT
8550

AGATGTGGTT GCCTGAATCT TAAGAATAAA ATAACATTGT TTGTATTTAT
8600

TTAAATTAGT GTTCCTTTTA TGGTTTGCCT GAAAGCACAA CAAAATCCTC
8650

ACCAAGATAT TACAATTATG ACTCCCATAC AGGTAAACTG TTTAGAGATT
8700

GGCAAGCACC TTTTAATGAA AGGAGTCAGC CAGCTTAGTG TGCAGTATTT
8750

ATTTCTGCCG GAAGAGGGAG CTTCAGGGAC AGACTTTGGT TTAGTCATGA
8800

AGCCTCCAGC ACTCCCAAGC GGTTGTGGTT GACCAAGCAA TTTATGCTTT
8850

TACCTTTCTA CTTCCAGAGG CTTGTTTACT TATCAGTAAG CATTAATTTA
8900

GTGTCCCCTC AGATGCCTTT TACTTTCTTC TTTTCTGCCT AGAATAAGCT
8950

GCTCTTCCAA TTTTGCAGCT ACATGTTTCC ACCCCAGTTG GAATTTCTCC
9000

ATAACATCCA TTGTAGCTAT CCTTCAATCT ACAGCCTCTA TTTCCTGTTA
9050

TAGCTGGTCA GGTCTAATCC CTCAAAATAC TCTGTCCCCT GCTTCCCTTA
9100

TCTGCTGGCC ACCTTTTTCC CCCACATACA CACTGCCATG TCCCACCCTT
9150

CACTCAAGTT GTTCCCTGCC ACCTCAACAA ATTTAAGTCC ATAAAATAGA
9200

GTAAGTGTTC CTGACTGTTA AATTTTAAAG CATCCCAAAG TCTGATTTCA
9250

CACTCGAATG AATACTATGT ACGGATTCAT TTACATAGAT GCGGTTGCAT
9300

GAGTCTTAAC AAAAAAATAA CATTATTTGT ATTTATTCAA AGTACTGTCA
9350

AGATATAATG TCAAGACCTA ATTCAAAGGT TCCACAAAGC CTTCCTTGAC
9400

TGCCCCCAAC GAAGATTATC CATTTTCCCT GAAATCCCAT TGACTTTTCT
9450

ATTTTGTAAG GAGGCTCGTG AGACTCTGTC TAAAAACAAA ACAAAACAAA
9500

AAGAAACAAT CAAACGGCTT GCTTCTGTTC TTTGATCTGC TAGTAAGCAA
9550

AAATTACACA TGGTGACAGG AGCTATGTGA GGCTGTCAGG TTGAATGGGA
9600

GGAGTTTGGG ATCCTGCTTG TGGATGGTTG GAAGAGGCTT TCGGGAAAGA
9650

CAGTATTTAT GTGAGACCTG GAAGATGGGC CTTAGCTTTG CAGAAGGTGG
9700

AGAGGCAGGA AATAGCACGG GGGCCCTGGG GCTGGAAGAC TTGGGCATAT
9750

TTGAGGAACA GAAAGGAGAC CAGCATAACT GAGGTGGGAA AAGCATGTGA
9800

AGAGATGGGG CTGGAGGAGG CCGGGAGTGG TGGCTCACGC CTGTAATCCC
9850

AGCACTTTGG GAGGCCAAGG CAGGCGGATC ATGAGCTCAG GAGATTGAGA
9900

CCATCCTGGC TAACACGGTG AAACCCCCTC TCTACTAAAA ATACAAAAAA
9950

AAAAAAAAAA AAAATTAGCT GGGCGTGGTG GCAGGAGCCT GTAGTCCCAG
10000

CTACCTGGGA GGCTGAGGCA GGAGAATGGC GTGAACCTGG AAGGCTGAGC
10050

TTGCAGTGAG CCGAGATTGC ACCACTGCAC TCCAGCCTGG GAGACAGAGA
10100

GAGACTCCCT CTCAAAAAAA CAAACAAACG AAACAAAACA AAACAAAAAT
10150

TAGCCAGGCG TGGTGGTATG CACCTGTAAT CCCAGCTACT CGGGAGGTTG
10200

AGGCAGGAGA AACGCTTGAA CTCAGGAGGC GGAGGTTGCA GTGAGCCGAG
10250

ACTGCGCCAC TGCACTCCAG CCTGGGTGAC AGAGGGAGAC TCCATCTCAA
10300

AAAAAAAAAT TTTTTTTTTT TTACAAACGG TGTCTCCCTC TGTCGCCCAG
10350

GCTGGAGTGC AGTGGTGTGA TCACAGCTCA CTCCAGCCTC AACCTCCCCA
10400

GCTGAAGCCA TCCTCTTGCC TCAGCCTCCT AAGTAGCTGG GACTACAGGC
10450

GCGCACCTCC AGGCTTGGCT CTTATTCTTT TTATTGTTTT TGAAACTATA
10500

GAACCTATTT TTAAAAAATG TTTTGGTTGT TTTTATTGCT GCTTTTCCTT
10550

TTGGGGTTAG AACACAAGTT TTGATGGGAA ACAGGTTAGA ACACATTCAT
10600

CTCTTCCCAT AGCGATGGTC ATAGAAAAAC GGGGCATATT TATAAACTCT
10650

CAGTTGATCT TAAAATGTGC AAAAGCTGCC GAACTCCTGG GAGTGAGCTC
10700

GAGCCCTGCA GGATCATTGT CACATGTGAG CAAAAGGCCA GCAAAAGGCC
10750

AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC
10800

CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC
10850

CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG
10900

CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT
10950

CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT AGGTATCTCA
11000

GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC
11050

GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA
11100

CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA
11150

TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG
11200

CCTAACTACG GCTACACTAG AAGAACAGTA TTTGGTATCT GCGCTCTGCT
11250

GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC
11300

AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG
11350

CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC
11400

TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT
11450

TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT
11500

AAATCAAGCC CAATCTGAAT AATGTTACAA CCAATTAACC AATTCTGATT
11550

AGAAAAACTC ATCGAGCATC AAATGAAACT GCAATTTATT CATATCAGGA
11600

TTATCAATAC CATATTTTTG AAAAAGCCGT TTCTGTAATG AAGGAGAAAA
11650

CTCACCGAGG CAGTTCCATA GGATGGCAAG ATCCTGGTAT CGGTCTGCGA
11700

TTCCGACTCG TCCAACATCA ATACAACCTA TTAATTTCCC CTCGTCAAAA
11750

ATAAGGTTAT CAAGTGAGAA ATCACCATGA GTGACGACTG AATCCGGTGA
11800

GAATGGCAAA AGTTTATGCA TTTCTTTCCA GACTTGTTCA ACAGGCCAGC
11850

CATTACGCTC GTCATCAAAA TCACTCGCAT CAACCAAACC GTTATTCATT
11900

CGTGATTGCG CCTGAGCGAG ACGAAATACG CGATCGCTGT TAAAAGGACA
11950

ATTACAAACA GGAATCGAAT GCAACCGGCG CAGGAACACT GCCAGCGCAT
12000

CAACAATATT TTCACCTGAA TCAGGATATT CTTCTAATAC CTGGAATGCT
12050

GTTTTTCCGG GGATCGCAGT GGTGAGTAAC CATGCATCAT CAGGAGTACG
12100

GATAAAATGC TTGATGGTCG GAAGAGGCAT AAATTCCGTC AGCCAGTTTA
12150

GTCTGACCAT CTCATCTGTA ACATCATTGG CAACGCTACC TTTGCCATGT
12200

TTCAGAAACA ACTCTGGCGC ATCGGGCTTC CCATACAAGC GATAGATTGT
12250

CGCACCTGAT TGCCCGACAT TATCGCGAGC CCATTTATAC CCATATAAAT
12300

CAGCATCCAT GTTGGAATTT AATCGCGGCC TCGACGTTTC CCGTTGAATA
12350

TGGCTCATAA CACCCCTTGT ATTACTGTTT ATGTAAGCAG ACAGTTTTAT
12400

TGTTCATGAT GATATATTTT TATCTTGTGC AATGTAACAT CAGAGATTTT
12450

GAGACACGGG CCAGAGCTGC A
12500

Methods for the Delivery of Exogenous Nucleic Acids to Target Cells
Transfection Techniques

Techniques that can be used to introduce a transgene, such as a MTM1 transgene described herein, into a target cell are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids (e.g., nucleic acids capable of expression in e.g., neurons, glial cells, or non-neural cells, such as colon and kidney cells). Electroporation of mammalian cells is described in detail, e.g., 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 in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. NUCLEOFECTION™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.

An additional technique useful for the transfection of target cells is the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., J. Vis. Exp. 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, 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. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids are contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig, Top Curr Chem. 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and DEAE-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al., Curr Protoc Mol Biol. 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference.

Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.

Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s). 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.

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 of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications. Their association with the gene vectors (DNA, siRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Ther. 9:102 (2002), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in US2010/0227406, the disclosure of which is incorporated herein by reference.

Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods Cell Biol. 82:309 (2007), the disclosure of which is incorporated herein by reference.

Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein. For example, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., 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

In addition to the above, a variety of tools have been developed that can be used for the incorporation of a gene of interest into a target cell, such as a human cell. One such method that can be used for incorporating polynucleotides encoding target genes into target cells involves the use of transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by 5′ and 3′ excision sites. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites 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 similar excision sites that exist within the nuclear genome of the cell. This allows the gene of interest to be inserted into the cleaved nuclear DNA at the complementary excision sites, and subsequent covalent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the mammalian cell genome completes the incorporation process. In certain cases, the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail in, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail in, e.g., US 2005/0112764), the disclosures of each of which are incorporated herein by reference as they pertain to transposons for use in gene delivery to a cell of interest.

Another tool for the integration of target genes into the genome of a target cell 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. The CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site. In this manner, highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can design a CRISPR/Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order 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 in order to cleave DNA prior to the incorporation of a gene encoding a target gene. The use of CRISPR/Cas to modulate gene expression has been described in, for example, U.S. Pat. No. 8,697,359, the disclosure of which is incorporated herein by reference as it pertains to the use of the CRISPR/Cas system for genome editing. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a gene of interest in a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al., Nat. Rev. Genet. 11:636 (2010); and in Joung et al., Nat. Rev. Mol. Cell Biol. 14:49 (2013), the disclosure of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.

Additional genome editing techniques that can be used to incorporate polynucleotides encoding target genes into the genome of a target cell include the use of ARCUS™ meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA. The use of these enzymes for the incorporation of genes encoding target genes into the genome of a mammalian cell is advantageous in view of the defined structure-activity relationships that have been established for such enzymes. Single chain meganucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a target gene into the nuclear DNA of a target cell. These single-chain nucleases have been described extensively in, for example, U.S. Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.

Pharmaceutical Compositions and Routes of Administration

The gene therapy agents described herein may contain a transgene, such as a transgene encoding MTM1 and may be incorporated into a vehicle for administration into a patient, such as a human patient suffering from a neuromuscular disorder (for example, XLMTM). Pharmaceutical compositions containing vectors, such as viral vectors, that contain the transcription regulatory elements (e.g., a desmin promoter) described herein operably linked to a therapeutic transgene can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

Viral vectors, such as AAV vectors and others described herein, containing the transcription regulatory element operably linked to a therapeutic transgene may be administered to a patient (e.g., a human patient) by a variety of routes of administration. The route of administration may vary, for example, with the onset and severity of disease, and may include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. Intravascular administration includes delivery into the vasculature of a patient. In some embodiments, the administration is into a vessel considered to be a vein (intravenous), and in some administration, the administration is into a vessel considered to be an artery (intraarterial). Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and/or femoral vein. Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and/or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary.

Mixtures of the nucleic acids and viral vectors described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The 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 any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For example, a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur 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 may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

Kits

The compositions described herein can be provided in a kit for use in treating a neuromuscular disorder (e.g., XLMTM). In some embodiments, the kit may include one or more viral vectors as described herein. The kit can include a package insert that instructs a user of the kit, such as a physician of skill 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 some embodiments, the kit may include one or more additional therapeutic agents.

In some embodiments, the kit may include one or more anti-cholestatic agents as described herein. The kit can include a package insert that instructs a user of the kit, such as a physician of skill 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 some embodiments, the kit may include one or more additional therapeutic agents.

Dosing Regimens
Dosing Regimens Involving AAV-MTM1 Vectors

Using the compositions and methods of the disclosure, a patient having a neuromuscular disorder (e.g., XLMTM) may be administered an AAV vector containing a transgene encoding MTM1 (e.g., resamirigene bilparvovec) in an amount of about 1.3×10¹⁴vg/kg. Administration of the vector to the patient in such a quantity can achieve the beneficial effect of augmenting MTM1 expression in the patient, e.g., to within 50% or 200% of wild-type levels, without inducing toxic side effects.

In some embodiments, the AAV vector is administered to the patient in an amount of less than about 3×10¹⁴vg/kg (e.g., in an amount of less than about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less). For example, the AAV vector may be administered to the patient in an amount of about 3×10¹⁴vg/kg, 2.9×10¹⁴vg/kg, 2.8×10¹⁴vg/kg, 2.7×10¹⁴vg/kg, 2.6×10¹⁴vg/kg, 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of less than about 2.5×10¹⁴vg/kg (e.g., in an amount of less than about 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less). For example, the AAV vector may be administered to the patient in an amount of about 2.5×10¹⁴vg/kg, 2.4×10¹⁴vg/kg, 2.3×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of less than about 2×10¹⁴vg/kg (e.g., in an amount of less than about 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less). For example, the AAV vector may be administered to the patient in an amount of about 2×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of less than about 1.5×10¹⁴vg/kg (e.g., less than about 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less). For example, the AAV vector may be administered to the patient in an amount of about 1.5×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of less than about 1×10¹⁴vg/kg (e.g., less than about 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg, or less). For example, the AAV vector may be administered to the patient in an amount of about 1×10¹⁴vg/kg, 1×10¹⁴vg/kg, 1×10¹³vg/kg, 1×10¹²vg/kg, 1×10¹¹vg/kg, 1×10¹⁰vg/kg, 1×10⁹vg/kg, 1×10⁸vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 3×10¹³vg/kg to about 2.3×10¹⁴vg/kg (e.g., in an amount of from about 3×10¹³vg/kg to about 2.3×10¹⁴vg/kg). For example, the AAV vector may be administered to the patient in an amount of about 3×10¹³vg/kg, 3.1×10¹³vg/kg, 3.2×10¹³vg/kg, 3.3×10¹³vg/kg, 3.4×10¹³vg/kg, 3.5×10¹³vg/kg, 3.6×10¹³vg/kg, 3.7×10¹³vg/kg, 3.8×10¹³vg/kg, 3.9×10¹³vg/kg, 4×10¹³vg/kg, 4.1×10¹³vg/kg, 4.2×10¹³vg/kg, 4.3×10¹³vg/kg, 4.4×10¹³vg/kg, 4.5×10¹³vg/kg, 4.6×10¹³vg/kg, 4.7×10¹³vg/kg, 4.8×10¹³vg/kg, 4.9×10¹³vg/kg, 5×10¹³vg/kg, 5.1×10¹³vg/kg, 5.2×10¹³vg/kg, 5.3×10¹³vg/kg, 5.4×10¹³vg/kg, 5.5×10¹³vg/kg, 5.6×10¹³vg/kg, 5.7×10¹³vg/kg, 5.8×10¹³vg/kg, 5.9×10¹³vg/kg, 6×10¹³vg/kg, 6.1×10¹³vg/kg, 6.2×10¹³vg/kg, 6.3×10¹³vg/kg, 6.4×10¹³vg/kg, 6.5×10¹³vg/kg, 6.6×10¹³vg/kg, 6.7×10¹³vg/kg, 6.8×10¹³vg/kg, 6.9×10¹³vg/kg, 7×10¹³vg/kg, 7.1×10¹³vg/kg, 7.2×10¹³vg/kg, 7.3×10¹³vg/kg, 7.4×10¹³vg/kg, 7.5×10¹³vg/kg, 7.6×10¹³vg/kg, 7.7×10¹³vg/kg, 7.8×10¹³vg/kg, 7.9×10¹³vg/kg, 8×10¹³vg/kg, 8.1×10¹³vg/kg, 8.2×10¹³vg/kg, 8.3×10¹³vg/kg, 8.4×10¹³vg/kg, 8.5×10¹³vg/kg, 8.6×10¹³vg/kg, 8.7×10¹³vg/kg, 8.8×10¹³vg/kg, 8.9×10¹³vg/kg, 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 4×10¹³vg/kg to about 2.3×10¹⁴vg/kg, such as an in amount of about 4×10¹³vg/kg, 4.1×10¹³vg/kg, 4.2×10¹³vg/kg, 4.3×10¹³vg/kg, 4.4×10¹³vg/kg, 4.5×10¹³vg/kg, 4.6×10¹³vg/kg, 4.7×10¹³vg/kg, 4.8×10¹³vg/kg, 4.9×10¹³vg/kg, 5×10¹³vg/kg, 5.1×10¹³vg/kg, 5.2×10¹³vg/kg, 5.3×10¹³vg/kg, 5.4×10¹³vg/kg, 5.5×10¹³vg/kg, 5.6×10¹³vg/kg, 5.7×10¹³vg/kg, 5.8×10¹³vg/kg, 5.9×10¹³vg/kg, 6×10¹³vg/kg, 6.1×10¹³vg/kg, 6.2×10¹³vg/kg, 6.3×10¹³vg/kg, 6.4×10¹³vg/kg, 6.5×10¹³vg/kg, 6.6×10¹³vg/kg, 6.7×10¹³vg/kg, 6.8×10¹³vg/kg, 6.9×10¹³vg/kg, 7×10¹³vg/kg, 7.1×10¹³vg/kg, 7.2×10¹³vg/kg, 7.3×10¹³vg/kg, 7.4×10¹³vg/kg, 7.5×10¹³vg/kg, 7.6×10¹³vg/kg, 7.7×10¹³vg/kg, 7.8×10¹³vg/kg, 7.9×10¹³vg/kg, 8×10¹³vg/kg, 8.1×10¹³vg/kg, 8.2×10¹³vg/kg, 8.3×10¹³vg/kg, 8.4×10¹³vg/kg, 8.5×10¹³vg/kg, 8.6×10¹³vg/kg, 8.7×10¹³vg/kg, 8.8×10¹³vg/kg, 8.9×10¹³vg/kg, 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 5×10¹³vg/kg to about 2.3×10¹⁴vg/kg, such as an in amount of about 5×10¹³vg/kg, 5.1×10¹³vg/kg, 5.2×10¹³vg/kg, 5.3×10¹³vg/kg, 5.4×10¹³vg/kg, 5.5×10¹³vg/kg, 5.6×10¹³vg/kg, 5.7×10¹³vg/kg, 5.8×10¹³vg/kg, 5.9×10¹³vg/kg, 6×10¹³vg/kg, 6.1×10¹³vg/kg, 6.2×10¹³vg/kg, 6.3×10¹³vg/kg, 6.4×10¹³vg/kg, 6.5×10¹³vg/kg, 6.6×10¹³vg/kg, 6.7×10¹³vg/kg, 6.8×10¹³vg/kg, 6.9×10¹³vg/kg, 7×10¹³vg/kg, 7.1×10¹³vg/kg, 7.2×10¹³vg/kg, 7.3×10¹³vg/kg, 7.4×10¹³vg/kg, 7.5×10¹³vg/kg, 7.6×10¹³vg/kg, 7.7×10¹³vg/kg, 7.8×10¹³vg/kg, 7.9×10¹³vg/kg, 8×10¹³vg/kg, 8.1×10¹³vg/kg, 8.2×10¹³vg/kg, 8.3×10¹³vg/kg, 8.4×10¹³vg/kg, 8.5×10¹³vg/kg, 8.6×10¹³vg/kg, 8.7×10¹³vg/kg, 8.8×10¹³vg/kg, 8.9×10¹³vg/kg, 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 6×10¹³vg/kg to about 2.3×10¹⁴vg/kg, such as an in amount of about 6×10¹³vg/kg, 6.1×10¹³vg/kg, 6.2×10¹³vg/kg, 6.3×10¹³vg/kg, 6.4×10¹³vg/kg, 6.5×10¹³vg/kg, 6.6×10¹³vg/kg, 6.7×10¹³vg/kg, 6.8×10¹³vg/kg, 6.9×10¹³vg/kg, 7×10¹³vg/kg, 7.1×10¹³vg/kg, 7.2×10¹³vg/kg, 7.3×10¹³vg/kg, 7.4×10¹³vg/kg, 7.5×10¹³vg/kg, 7.6×10¹³vg/kg, 7.7×10¹³vg/kg, 7.8×10¹³vg/kg, 7.9×10¹³vg/kg, 8×10¹³vg/kg, 8.1×10¹³vg/kg, 8.2×10¹³vg/kg, 8.3×10¹³vg/kg, 8.4×10¹³vg/kg, 8.5×10¹³vg/kg, 8.6×10¹³vg/kg, 8.7×10¹³vg/kg, 8.8×10¹³vg/kg, 8.9×10¹³vg/kg, 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, or 2×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 7×10¹³vg/kg to about 2.3×10¹⁴vg/kg, such as an in amount of about 7×10¹³vg/kg, 7.1×10¹³vg/kg, 7.2×10¹³vg/kg, 7.3×10¹³vg/kg, 7.4×10¹³vg/kg, 7.5×10¹³vg/kg, 7.6×10¹³vg/kg, 7.7×10¹³vg/kg, 7.8×10¹³vg/kg, 7.9×10¹³vg/kg, 8×10¹³vg/kg, 8.1×10¹³vg/kg, 8.2×10¹³vg/kg, 8.3×10¹³vg/kg, 8.4×10¹³vg/kg, 8.5×10¹³vg/kg, 8.6×10¹³vg/kg, 8.7×10¹³vg/kg, 8.8×10¹³vg/kg, 8.9×10¹³vg/kg, 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 8×10¹³vg/kg to about 2.3×10¹⁴vg/kg, such as an in amount of about 8×10¹³vg/kg, 8.1×10¹³vg/kg, 8.2×10¹³vg/kg, 8.3×10¹³vg/kg, 8.4×10¹³vg/kg, 8.5×10¹³vg/kg, 8.6×10¹³vg/kg, 8.7×10¹³vg/kg, 8.8×10¹³vg/kg, 8.9×10¹³vg/kg, 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 9×10¹³vg/kg to about 2.3×10¹⁴vg/kg, such as an in amount of 9×10¹³vg/kg, 9.1×10¹³vg/kg, 9.2×10¹³vg/kg, 9.3×10¹³vg/kg, 9.4×10¹³vg/kg, 9.5×10¹³vg/kg, 9.6×10¹³vg/kg, 9.7×10¹³vg/kg, 9.8×10¹³vg/kg, 9.9×10¹³vg/kg, 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of from about 1×10¹⁴vg/kg to about 2.3×10¹⁴vg/kg, such as an in amount 1×10¹⁴vg/kg, 1.1×10¹⁴vg/kg, 1.2×10¹⁴vg/kg, 1.3×10¹⁴vg/kg, 1.4×10¹⁴vg/kg, 1.5×10¹⁴vg/kg, 1.6×10¹⁴vg/kg, 1.7×10¹⁴vg/kg, 1.8×10¹⁴vg/kg, 1.9×10¹⁴vg/kg, 2×10¹⁴vg/kg, 2.1×10¹⁴vg/kg, 2.2×10¹⁴vg/kg, or 2.3×10¹⁴vg/kg.

In some embodiments, the AAV vector is administered to the patient in an amount of about 3×10¹³vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 4×10¹³vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 5×10¹³vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 6×10¹³vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 7×10¹³vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 8×10¹³vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 9×10¹³vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.1×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.2×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.3×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.4×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.5×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.6×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.7×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.8×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 1.9×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.1×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.2×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.3×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.4×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.5×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.6×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.7×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.8×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 2.9×10¹⁴vg/kg. In some embodiments, the AAV vector is administered to the patient in an amount of about 3×10¹⁴vg/kg.

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

In some embodiments, the AAV vector is administered to the patient in two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) doses that, together, comprise the amount (e.g., less than about 3×10¹⁴vg/kg).

In some embodiments, the AAV vector is administered to the patient in two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) doses that each, individually, comprise the amount (e.g., less than about 3×10¹⁴vg/kg).

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

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

Combination Therapies

An AAV vector containing a transgene encoding MTM1 (e.g., resamirigene bilparvovec) described herein can be administered in combination with a one or more additional therapeutic procedures (e.g., nasobiliary drainage (NBD)) and/or agents (e.g., an anti-cholestatic agent) for the treatment of a neuromuscular disorder (e.g., XLMTM).

Therapeutic Procedure

In some embodiments, the one or more additional therapeutic procedures is NBD. NBD is a therapeutic procedure that is performed to help drain bile (e.g., when the bile duct is blocked, a biliary drain may help bile to flow from the liver into the intestine). In some embodiments, NBD is performed with a biliary drain (also known as a biliary stent), which is a thin, hollow, flexible tube with several small holes along the sides. A biliary drain may be inserted in a patient's bile duct to allow it to drain.

Therapeutic Agent

In some embodiments, the one or more additional therapeutic agents is an anti-cholestatic agent (e.g., a bile acid, a farnesoid X receptor (FXR) ligand, a fibroblast growth factor 19 (FGF-19) mimetic, a Takeda-G-protein-receptor-5 (TGR5) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, a PPAR-alpha agonist, a PPAR-delta agonist, a dual PPAR-alpha and PPAR-delta agonist, an apical sodium-dependent bile acid transporter (ASBT) inhibitor, an immunomodulatory drug, an antifibrotic therapy, and a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor) or a combination thereof.

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

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

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

In some embodiments, the anti-cholestatic agent is administered to the patient in one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty, and seventy) doses that commence on the same day (e.g., 24^thhour, on the 23^rdhour, on the 22^ndhour, on the 21^sthour, on the 20^thhour, on the 19^thhour, on the 18^thhour, on the 17^thhour, on the 16^thhour, on the 15^thhour, on the 14^thhour, on the 13^thhour, on the 12^thhour, on the 11^thhour, on the 10^thhour, on the 9^thhour, on the 8^thhour, on the 7^thhour, on the 6^thhour, on the 5^thhour, on the 4^thhour, on the 3^rdhour, on the 2^ndhour, on the 1^sthour, on the 60^thminute, on the 59^thminute, on the 58^thminute, on the 57^thminute, on the 56^thminute, on the 55^thminute, on the 50^thminute, on the 40^thminute, on the 30^thminute, on the 20^thminute, on the 10^thminute, or on the same minute) of administration of the viral vector to the patient.

In some embodiments, the anti-cholestatic agent is a bile acid. In some embodiments, the bile acid is ursodeoxycholic acid or a derivative thereof or nor-ursodeoxycholic acid. In some embodiments, the bile acid is ursodiol.

In some embodiments, the anti-cholestatic agent is an FXR ligand. In some embodiments, the FXR ligand is obeticholic acid, cilofexor, tropifexor, tretinoin, or EDP-305.

In some embodiments, the one or more anti-cholestatic agent is an FGF-19 mimetic. In some embodiment, the FGF-19 mimetic is aldafermin.

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

In some embodiments, the anti-cholestatic agent is a PPAR agonist. In some embodiments, the PPAR agonist is bezafibrate, seladelpar, or elafibrinor.

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

In some embodiments, the anti-cholestatic agent is a PPAR-delta agonist. In some embodiments, the PPAR-delta agonist is seladelpar.

In some embodiments, the anti-cholestatic agent is a dual PPAR-alpha and PPAR-delta agonist.

In some embodiments, the dual PPAR-alpha-delta agonist is elafibranor.

In some embodiments, the one or more anti-cholestatic agent is an ASBT inhibitor. In some embodiments, the ASBT inhibitor is odevixibat, maralixibat, or linerixibat.

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

In some embodiments, the anti-cholestatic agent is an antifibrotic therapy. In some embodiments, the antifibrotic therapy is a vitamin D receptor (VDR) agonist or simtuzumab.

In some embodiments, the anti-cholestatic agent is a NOX inhibitor. In some embodiments, the NOX inhibitor is setanaxib.

In some embodiments, a therapeutically effective amount of a viral vector comprising a transgene encoding MTM1 (e.g., resamirigene bilparvovec) and an anti-cholestatic agent are administered to a patient in need thereof. In some embodiments, a therapeutically effective amount of resamirigene bilparvovec and an anti-cholestatic agent are administered to a patient in need thereof. In some embodiments, a therapeutically effective amount of resamirigene bilparvovec and an anti-cholestatic agent are administered to a patient in need thereof, wherein the anti-cholestatic agent is a bile acid. In some embodiments, a therapeutically effective amount of resamirigene bilparvovec and an anti-cholestatic agent are administered to a patient in need thereof, wherein the anti-cholestatic agent is ursodiol. In some embodiments, a therapeutically effective amount of resamirigene bilparvovec and ursodiol are administered to a patient in need thereof.

Anti-Cholestatic Agent

In some embodiments, a patient is administered an anti-cholestatic agent.

In some embodiments, a patient is administered an anti-cholestatic when a patient is monitored for cholestasis, hyperbilirubinemia, or one or more symptoms thereof and it is determined that the patient exhibits cholestasis or hyperbilirubinemia or one or more symptoms thereof.

In some embodiments, a patient is administered an anti-cholestatic when it is determined that the patient exhibits cholestasis or hyperbilirubinemia or one or more symptoms thereof.

In some embodiments, the anti-cholestatic agent is selected from the list comprising a bile acid, a farnesoid X receptor (FXR) ligand, a fibroblast growth factor 19 (FGF-19) mimetic, a Takeda-G-protein-receptor-5 (TGR5) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, a PPAR-alpha agonist, a PPAR-delta agonist, a dual PPAR-alpha and PPAR-delta agonist, an apical sodium-dependent bile acid transporter (ASBT) inhibitor, an immunomodulatory drug, an antifibrotic therapy, and a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor.