US 12,460,226 B2
Compositions and methods for treating duchenne muscular dystrophy
Hansell Stedman, Norristown, PA (US)
Assigned to The Trustees of the University of Pennsylvania, Philadelphia, PA (US)
Appl. No. 17/047,990
Filed by The Trustees of the University of Pennsylvania, Philadelphia, PA (US)
PCT Filed Apr. 16, 2019, PCT No. PCT/US2019/027675
§ 371(c)(1), (2) Date Oct. 15, 2020,
PCT Pub. No. WO2019/204303, PCT Pub. Date Oct. 24, 2019.
Claims priority of provisional application 62/658,464, filed on Apr. 16, 2018.
Prior Publication US 2021/0155956 A1, May 27, 2021
Int. Cl. C12N 15/86 (2006.01); C07K 14/47 (2006.01)
CPC C12N 15/86 (2013.01) [C07K 14/4708 (2013.01); C12N 2750/14121 (2013.01); C12N 2750/14122 (2013.01)] 17 Claims
 
1. A recombinant adeno-associated virus (AAV) having an AAV capsid and a vector genome, wherein the vector genome comprises a nucleic acid sequence encoding a dystrophin superfamily mutant protein comprising a hybrid triple helical repeat under control of regulatory sequences which direct expression thereof, the hybrid triple helical repeat comprising
a first hybrid helix, a second hybrid helix, a third hybrid helix, and a mutant splice junction having joined segments of a first triple helical repeat and a second non-adjacent triple helical repeat of a native dystrophin superfamily protein;
wherein the first triple helical repeat of the native dystrophin superfamily protein has parallel helices A and C and an anti-parallel B helix;
wherein the second non-adjacent triple helical repeat of the native dystrophin superfamily protein has parallel helices A′ and C′ and an anti-parallel B′ helix;
each helix of the hybrid triple helical repeat comprising an N-terminal portion and a C-terminal portion of a helix in the triple helical repeats of the native dystrophin superfamily protein, wherein the hybrid triple helical repeat has parallel first and third helices and an anti-parallel second helix, wherein the N-terminal portion of the first helix and the N-terminal portion of the third helix in the hybrid triple helical repeat are from the first triple helical repeat of the native dystrophin superfamily protein, and the C-terminal portion of the first helix and the C-terminal portion of the third helix are from the second non-adjacent triple helical repeat of the native dystrophin superfamily protein;
the first hybrid helix of the hybrid triple helical repeat comprising an N-terminal portion of helix A fused to a C-terminal portion of helix A′;
the second hybrid helix of the hybrid triple helical repeat comprising an N-terminal portion of helix B′ fused to a C-terminal portion of helix B, wherein helix B and helix B′ are spliced at a position adjacent to the central conserved tryptophan residues at the center of the hydrophobic cores of the first triple helical repeat and the second non-adjacent triple helical repeat joined in the hybrid triple helical repeat, the hydrophobic cores corresponding to the cross-section plane bisecting the tryptophan residues as determined by a Hidden Markov Model and sequence alignment; and
the third hybrid helix of the hybrid triple helical repeat comprising an N-terminal portion of helix C fused to a C-terminal portion of helix C′;
wherein the first hybrid helix and the third hybrid helix are spliced adjacent to the conserved tryptophan residues at the center of the hydrophobic cores of the first triple helical repeat and the second non-adjacent triple helical repeat joined in the hybrid triple helical repeat, the hydrophobic cores corresponding to the cross-section plane bisecting the tryptophan residues as determined by a Hidden Markov Model and sequence alignment.