Compositions of dna molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making thereof, and methods of use thereof

A method for treating a disease associated with reduced activity of amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (GDE) in a human patient, the method comprising administering to the patient a DNA molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof, wherein the DNA molecule is contained within a single delivery vector.

A method for treating a disease associated with reduced activity of GDE in a human patient, the method comprising the steps of (i) administering a first dose of a DNA molecule comprising an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof to the patient and (ii) administering a second dose of the DNA molecule to the patient.

A double-stranded DNA molecule comprising in 5′ to 3′ direction of the top strand:

• a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5′ overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
• b. an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof, and
• c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3′ overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.

A double strand DNA molecule comprising in 5′ to 3′ direction of the top strand:

• a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
• b. an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof, and
• c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5′ overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.

A double-stranded DNA molecule comprising in 5′ to 3′ direction of the top strand:

• a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5′ overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
• b. an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof, and
• c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a bottom strand 5′ overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.

A double strand DNA molecule comprising in 5′ to 3′ direction of the top strand:

• a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
• b. an expression cassette comprising a transgene encoding human GDE or a catalytically active fragment thereof, and
• c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3′ overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat.