# US 9,434,928 B2 · Recombinant adeno-associated virus delivery of alpha-sarcoglycan polynucleotides

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- Number: US 9,434,928 B2
- Document ID: US9434928B2
- Status: Granted
- Kind: B2
- Application number: 14/360,243
- Filed: 2012-11-21
- Published: 2014-10-30
- Granted: 2016-09-06
- Earliest claimed: 2011-11-23
- Assignees: Nationwide Children's Hospital, Inc.
- Applicants: Nationwide Children's Hospital, Inc.
- Inventors: Jerry R. Mendell; Louis Chicoine; Louise Rodino-Klapac; Kelly Reed Clark; Thomas J. Preston
- Web page: https://patentgrep.com/patent/US9434928B2

## CPC

- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61K 38/1709: {from mammals}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- A61M 1/3603: {in the same direction}
- A61M 5/1408: {in parallel, e.g. by manifolds or with sequencing valves}
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- C07K 14/4707: {Muscular dystrophy}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- A61M: DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY (introducing media into or onto the bodies of animals A61D7/00; means for inserting tampons A61F13/26; devices for administering food or medicines orally A61J; containers for collecting, storing or administering blood or medical fluids A61J1/05); DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY (surgery A61B; chemical aspects of surgical articles A61L); DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR {(Electrotherapy, e.g. producing anaesthesia by the use of alternating or intermittent currents A61N1/36021)}
- 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

The present invention relates to recombinant adeno-associated virus (rAAV) delivery of an alpha-sarcoglycan gene. The invention provides rAAV products and methods of using the rAAV in the treatment of limb girdle muscular dystrophies such as LGMD2D.

## Claims

### Claim 1

A method of improving muscle function in a patient afflicted with limb girdle muscular dystrophy type 2D (LGMD 2D) comprising the step of perfusing the vasculature of a limb of the patient with an infectious encapsidated recombinant adeno-associated virus (AAV) comprising a AAVrh.74 capsid polynucleotide of SEQ ID NO:2, wherein the genome of the recombinant AAV comprises a gene expression cassette comprising the polynucleotide of SEQ ID NO: 3 encoding an alpha-sarcoglycan under the transcriptional control of a promoter, said cassette flanked by one or more AAV inverted terminal repeats, and wherein the genome lacks AAV rep and cap DNA.

### Claim 2

A method of delivering an alpha-sarcoglycan polynucleotide to an animal in need thereof, comprising the step of perfusing the vasculature of a limb of the animal with an infectious encapsidated recombinant AAV comprising a AAVrh.74 capsid polynucleotide of SEQ ID NO:2, wherein the genome of the recombinant AAV comprises a gene expression cassette comprising the polynucleotide of SEQ ID NO: 3 encoding an alpha-sarcoglycan under the transcriptional control of a promoter, said cassette flanked by one or more AAV inverted terminal repeats, and wherein the genome lacks AAV rep and cap DNA.

### Claim 3

An infectious encapsidated recombinant AAV comprising a AAVrh.74 capsid polynucleotide of SEQ ID NO: 2, wherein the genome of the recombinant AAV comprises a gene expression cassette comprising the polynucleotide of SEQ ID NO: 3 encoding an alpha-sarcoglycan under the transcriptional control of a promoter, said cassette flanked by one or more AAV inverted terminal repeats, and wherein the genome lacks AAV rep and cap DNA.

### Claim 4

A method of delivering a recombinant adeno-associated virus (AAV) comprising:
connecting a recirculation circuit to a vasculature of a limb;
isolating circulation of the limb from the remainder of the body;
adding to the recirculation circuit an infectious encapsidated recombinant AAV comprising a AAVrh.74 capsid polynucleotide of SEQ ID NO:2, wherein the genome of the recombinant AAV comprises a gene expression cassette comprising the polynucleotide of SEQ ID NO: 3 encoding an alpha-sarcoglycan under the transcriptional control of a promoter, said cassette flanked by one or more AAV inverted terminal repeats, and wherein the genome lacks AAV rep and cap DNA;
passing the recombinant AAV through the recirculation circuit and the limb repeatedly; and
disconnecting the recirculation circuit from the limb.

### Claim 5 (depends on 4)

The method of claim 4, further comprising:
removing a volume of blood from the limb after isolating the limb from the remainder of the body;
introducing a non-blood perfusate to the recirculation circuit and the limb prior to adding the recombinant AAV to the recirculation circuit; and
passing the non-blood perfusate through the recirculation circuit and the limb repeatedly as the recombinant AAV is passed through the recirculation circuit and the limb.

### Claim 6 (depends on 5)

The method of claim 5, wherein the non-blood perfusate comprises a buffer solution.

### Claim 7 (depends on 5)

The method of claim 5, further comprising:
removing the non-blood perfusate after repeatedly passing the recombinant AAV and the non-blood perfusate through the recirculation circuit and the limb; and
reintroducing the volume of blood to the limb after removing the non-blood perfustate.