Micro-dystrophins and related methods of use
Nucleotide sequences including a micro-dystrophin gene are provided. The micro-dystrophin genes may be operatively linked to a regulatory cassette. Methods of treating a subject having, or at risk of developing, muscular dystrophy, sarcopenia, heart disease, or cachexia are also provided. The methods may include administering a pharmaceutical composition including the micro-dystrophin gene and a delivery vehicle to a subject. Further, the methods may include administering the pharmaceutical composition a subject having Duchenne muscular dystrophy or Becker muscular dystrophy.
1. An isolated and purified nucleotide sequence, comprising:
a micro-dystrophin gene encoding a protein comprising:
an amino-terminal actin-binding domain;
a β-dystroglycan binding domain; and
a spectrin-like repeat domain, consisting of five spectrin-like repeats, including spectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16), spectrin-like repeat 17 (SR17), spectrin-like repeat 23 (SR23), and spectrin-like repeat 24 (SR24);
wherein the micro-dystrophin gene is operatively linked to a regulatory cassette.
2. The isolated and purified nucleotide sequence of claim 1 , wherein the protein encoded by the micro-dystrophin gene further comprises at least a portion of a hinge domain.
3. The isolated and purified nucleotide sequence of claim 2 ,
wherein the hinge domain is selected from at least one of a Hinge 1 domain, a Hinge 2 domain, a Hinge 3 domain, and a Hinge 4 domain.
4. The isolated and purified nucleotide sequence of claim 1 , wherein the regulatory cassette is selected from the group consisting of a CK8 promoter and a cardiac troponin T (cTnT) promoter.
5. The isolated and purified nucleotide sequence of claim 1 , wherein the regulatory cassette is a CK8 promoter, and wherein the CK8 promoter has at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:19.
6. The isolated and purified nucleotide sequence of claim 1 , wherein the regulatory cassette is a cTnT promoter, and wherein the cTnT promoter has at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:1.
7. The isolated and purified nucleotide sequence of claim 1 , wherein the micro-dystrophin gene encodes a protein comprising:
at least one of spectrin-like repeat 1 directly coupled to spectrin-like repeat 16, spectrin-like repeat 17 directly coupled to spectrin-like repeat 23, and spectrin-like repeat 23 directly coupled to spectrin-like repeat 24.
8. The isolated and purified nucleotide sequence of claim 3 , which comprises a Hinge 1 domain (H1) and a Hinge 4 domain (H4).
9. A pharmaceutical composition, comprising:
the isolated and purified nucleotide sequence of claim 1 ; and
a delivery vehicle.
10. The pharmaceutical composition of claim 9 , wherein the delivery vehicle comprises an adeno-associated virus (AAV) vector or a recombinant adeno-associated virus (rAAV) vector.
11. The delivery vehicle of claim 10 , wherein the adeno-associated virus (AAV) vector consists of serotype AAV6, AAV8, or AAV9.
12. The delivery vehicle of claim 10 , wherein the recombinant adeno-associated virus (rAAV) vector consists of rAAV6, rAAV8, rAAV9, or rAAV2/6.
13. The delivery vehicle of claim 11 , wherein the adeno-associated virus (AAV) vector is serotype AAV9.
14. The delivery vehicle of claim 12 , wherein the recombinant adeno-associated virus (rAAV) vector is rAAV9.