# US 12,766,200 B2 · Treatment of dystrophinopathies with microdystrophin gene therapy constructs

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- Number: US 12,766,200 B2
- Document ID: US12766200B2
- Status: Granted
- Kind: B2
- Application number: 19/234,245
- Filed: 2025-06-10
- Published: 2025-12-04
- Granted: 2026-10-20
- Earliest claimed: 2019-11-28
- Assignees: REGENXBIO Inc.
- Applicants: Regenxbio Inc.
- Inventors: Chunping Qiao; Devin McDougald; Ye Liu; Olivier Danos
- Web page: https://patentgrep.com/patent/US12766200B2

## CPC

- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- C07K 14/4708: {Duchenne dystrophy}
- C12N 15/86: Viral vectors
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14151: Methods of production or purification of viral material
- C12N 2750/14171: Demonstrated in vivo effect
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are gene constructs that encode a microdystrophin protein for use in gene therapy. The microdystrophin gene constructs and expression cassettes were engineered for improved therapy with respect to efficacy, potency and safety to the subject when expressed by a viral vector in muscle cells and/or CNS cells.

## Claims

### Claim 1

A method of treating a dystrophinopathy in a human subject in need thereof, the method comprising:
administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) particle, wherein the rAAV particle comprises a) a nucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 79, or the reverse complement of the nucleotide sequence, wherein the microdystrophin protein comprises an H3 domain and a C-terminal (CT) domain, wherein the CT domain comprises an α1-syntrophin binding site, and b) a capsid comprising a capsid protein,
wherein the administration results in delivery of a microdystrophin protein to the muscle of the subject.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleotide sequence encoding the microdystrophin protein a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 81, or the reverse complement thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the nucleic acid comprises a transcription regulatory element that promotes expression in muscle, wherein the transcription regulatory element is operably linked to the nucleotide sequence encoding the microdystrophin protein.

### Claim 4 (depends on 3)

The method of claim 3, wherein the transcription regulatory element is a SPc5-12 promoter or a transcriptionally active portion thereof.

### Claim 5 (depends on 3)

The method of claim 3, wherein the nucleic acid comprises a nucleotide sequence comprising from 5′ to 3′:
a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence—the transcription regulatory element sequence—the nucleotide sequence encoding the microdystrophin protein-a polyadenylation sequence-a second AAV ITR sequence.

### Claim 6 (depends on 5)

The method of claim 5, wherein the AAV ITR sequence is an AAV2 ITR sequence.

### Claim 7 (depends on 1)

The method of claim 1, wherein the capsid protein comprises a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 77, b) the amino acid sequence of SEQ ID NO: 77, c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 78, or d) the amino acid sequence of SEQ ID NO: 78.

### Claim 8 (depends on 7)

The method of claim 7, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 77.

### Claim 9 (depends on 1)

The method of claim 1, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, or wherein the subject is a female carrier of DMD or BMD.

### Claim 10

A method of treating a dystrophinopathy in a human subject in need thereof, the method comprising:
administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an rAAV particle, wherein the rAAV particle comprises a) a nucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 1, or the reverse complement of the nucleotide sequence, wherein the microdystrophin protein comprises an H3 domain and a C-terminal (CT) domain, wherein the CT domain comprises an α1-syntrophin binding site, and b) a capsid comprising a capsid protein,
wherein the administration results in delivery of a microdystrophin protein to the muscle of the subject.

### Claim 11 (depends on 10)

The method of claim 10, wherein the nucleotide sequence encoding the microdystrophin protein comprises a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 20, or the reverse complement thereof.

### Claim 12 (depends on 10)

The method of claim 10, wherein the nucleic acid comprises a transcription regulatory element that promotes expression in muscle, wherein the transcription regulatory element is operably linked to the nucleotide sequence encoding the microdystrophin protein.

### Claim 13 (depends on 12)

The method of claim 12, wherein the transcription regulatory element is a SPc5-12 promoter or a transcriptionally active portion thereof.

### Claim 14 (depends on 12)

The method of claim 12, wherein the nucleic acid comprises a nucleotide sequence comprising from 5′ to 3′:
a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence—the transcription regulatory element sequence—the nucleotide sequence encoding the microdystrophin protein—a polyadenylation sequence—a second AAV ITR sequence.

### Claim 15 (depends on 14)

The method of claim 14, wherein the AAV ITR sequence is an AAV2 ITR sequence.

### Claim 16 (depends on 10)

The method of claim 10, wherein the capsid protein comprises a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 77, b) the amino acid sequence of SEQ ID NO: 77, c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 78, or d) the amino acid sequence of SEQ ID NO: 78.

### Claim 17 (depends on 16)

The method of claim 16, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 77.

### Claim 18 (depends on 10)

The method of claim 10, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, or wherein the subject is a female carrier of DMD or BMD.

### Claim 19

A method of delivering a nucleic acid encoding a microdystrophin protein to a cell, the method comprising contacting the cell with an rAAV particle comprising the nucleic acid encoding the microdystrophin protein,
wherein the microdystrophin protein comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 79, wherein the microdystrophin protein comprises an H3 domain and a C-terminal (CT) domain, wherein the CT domain comprises an α1-syntrophin binding site, and wherein the nucleic acid encoding the microdystrophin protein is delivered to the cell.

### Claim 20

A method of delivering a nucleic acid encoding a microdystrophin protein to a cell, the method comprising contacting the cell with an rAAV particle comprising the nucleic acid encoding the microdystrophin protein,
wherein the microdystrophin protein comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 1, wherein the microdystrophin protein comprises an H3 domain and a C-terminal (CT) domain, wherein the CT domain comprises an α1-syntrophin binding site, —and wherein the nucleic acid encoding the microdystrophin protein is delivered to the cell.

### Claim 21 (depends on 5)

The method of claim 5, wherein the transcription regulatory element is SPc5-12, the AAV ITR sequences are AAV2 ITR sequences, and the capsid protein comprises an amino acid sequence of SEQ ID NO: 77.

### Claim 22 (depends on 21)

The method of claim 21, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, or wherein the subject is a female carrier of DMD or BMD.

### Claim 23 (depends on 1)

The method of claim 1, wherein the H3 domain is coupled to an R24 domain via a linker L4 (SEQ ID NO: 12).

### Claim 24 (depends on 14)

The method of claim 14, wherein the transcription regulatory element is SPc5-12, the AAV ITR sequences are AAV2 ITR sequences, and the capsid protein comprises an amino acid sequence of SEQ ID NO: 77.

### Claim 25 (depends on 24)

The method of claim 24, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, or wherein the subject is a female carrier of DMD or BMD.

### Claim 26 (depends on 10)

The method of claim 10, wherein the H3 domain is coupled to an R24 domain via a three amino acid linker.

### Claim 27 (depends on 20)

The method of claim 20, wherein the H3 domain is coupled to an R24 domain via a three amino acid linker.

### Claim 28 (depends on 1)

The method of claim 1, wherein the H3 domain is coupled to an R24 domain via a linker L4 (SEQ ID NO: 12).

### Claim 29 (depends on 10)

The method of claim 10, wherein the H3 domain is coupled to an R24 domain via a linker L4 (SEQ ID NO: 12).

### Claim 30 (depends on 20)

The method of claim 20, wherein the H3 domain is coupled to an R24 domain via a linker L4 (SEQ ID NO: 12).

### Claim 31

A method of treating a dystrophinopathy in a human subject in need thereof, said method comprising:
administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) particle, wherein the rAAV particle comprises
a) a nucleic acid comprising i) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, and ii) a transcription regulatory element comprising the nucleotide sequence of SEQ ID NO: 39; and
b) a capsid comprising a capsid protein,
wherein said administration results in delivery of a microdystrophin protein to the muscle of said subject.

### Claim 32 (depends on 31)

The method of claim 31, wherein the capsid protein comprises a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 77, b) the amino acid sequence of SEQ ID NO: 77, c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 78, or d) the amino acid sequence of SEQ ID NO: 78.

### Claim 33 (depends on 32)

The method of claim 32, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 77.

### Claim 34 (depends on 32)

The method of claim 32, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy, or the subject is a female carrier of DMD or BMD.

### Claim 35 (depends on 34)

The method of claim 34, wherein the dystrophinopathy is DMD.

### Claim 36

A method of treating a dystrophinopathy in a human subject in need thereof, said method comprising:
administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) particle, wherein the rAAV particle comprises a) a nucleic acid comprising from 5′ to 3′: (i) a first AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 73, (ii) a SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39, (iii) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, (iv) a polyadenylation nucleotide sequence of SEQ ID NO: 42, and (v) a second AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 74; and b) a capsid comprising a capsid protein,
wherein said administration results in delivery of a microdystrophin protein to the muscle of said subject.

### Claim 37 (depends on 36)

The method of claim 36, wherein the capsid protein comprises a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 77, b) the amino acid sequence of SEQ ID NO: 77, c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 78, or d) the amino acid sequence of SEQ ID NO: 78.

### Claim 38 (depends on 37)

The method of claim 37, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 77.

### Claim 39 (depends on 37)

The method of claim 37, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy, or the subject is a female carrier of DMD or BMD.

### Claim 40 (depends on 39)

The method of claim 39, wherein the dystrophinopathy is DMD.

### Claim 41

A method of delivering a nucleic acid encoding a microdystrophin protein to a cell, the method comprising contacting the cell with an rAAV particle comprising the nucleic acid encoding the microdystrophin protein and an SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39,
wherein the microdystrophin protein comprises the amino acid sequence of SEQ ID NO: 1, and
wherein the nucleic acid encoding the microdystrophin protein and the SPc5-12 promoter is delivered to the cell.

### Claim 42 (depends on 41)

The method of claim 41, wherein the capsid protein comprises a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 77, b) the amino acid sequence of SEQ ID NO: 77, c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 78, or d) the amino acid sequence of SEQ ID NO: 78.

### Claim 43 (depends on 42)

The method of claim 42, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 77.

### Claim 44

A method of delivering a nucleic acid encoding a microdystrophin protein to a cell, the method comprising contacting the cell with an rAAV particle wherein the rAAV particle comprises a) the nucleic acid comprising from 5′ to 3′: (i) a first AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 73, (ii) a SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39, (iii) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, (iv) a polyadenylation nucleotide sequence of SEQ ID NO: 42, and (v) a second AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 74 and b) a capsid comprising a capsid protein, wherein the nucleic acid encoding the microdystrophin protein is delivered to the cell.

### Claim 45 (depends on 44)

The method of claim 44, wherein the capsid protein comprises a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 77, b) the amino acid sequence of SEQ ID NO: 77, c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 78, or d) the amino acid sequence of SEQ ID NO: 78.

### Claim 46 (depends on 44)

The method of claim 44, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 77.