# US 12,760,075 B2 · Gene therapy with dysferlin dual vectors

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- Number: US 12,760,075 B2
- Document ID: US12760075B2
- Status: Granted
- Kind: B2
- Application number: 17/924,820
- Filed: 2021-05-12
- Published: 2023-09-07
- Granted: 2026-10-13
- Earliest claimed: 2020-05-13
- Assignees: Research Institute At Nationwide Children's Hospital
- Applicants: Research Institute at Nationwide Children's Hospital
- Inventors: Louise Rodino-Klapac
- Web page: https://patentgrep.com/patent/US12760075B2

## CPC

- C07K 14/4707: {Muscular dystrophy}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- C12N 15/86: Viral vectors
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Recombinant polynucleotides encoding fragments of a human dysferlin protein are described herein. In addition, plasmids, viral vectors, dual vector systems, cells, and compositions comprising such recombinant polynucleotides are further described. Such recombinant polynucleotides, plasmids, viral vectors, dual vector systems, cells, and compositions may be used to treat dysferlinopathies.

## Claims

### Claim 1

A dual adeno-associated viral (AAV) vector system comprising:
(a) a first AAV vector, wherein the first AAV vector comprises a first recombinant polynucleotide comprising a fragment of a human dysferlin (hDYSF) gene, wherein the fragment consists of the nucleotide sequence of SEQ ID NO: 1; and
(b) a second AAV vector, wherein the second AAV vector comprises a second recombinant polynucleotide comprising a fragment of a hDYSF gene, wherein the fragment consists of the nucleotide sequence of SEQ ID NO: 2.

### Claim 2 (depends on 1)

The dual AAV vector system of claim 1, wherein the first and the second recombinant polynucleotides further comprise one or more additional polynucleotide sequences comprising a selection marker, an origin of replication (ORI), or an untranslated region (UTR).

### Claim 3 (depends on 1)

A composition comprising the dual AAV vector system of claim 1 and a pharmaceutically acceptable carrier.

### Claim 4 (depends on 1)

The dual AAV vector system according to claim 1, wherein the first recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6, and wherein the second recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

### Claim 5

An adeno-associated viral (AAV) packaging system comprising:
(a) (i) a first plasmid comprising a first recombinant polynucleotide comprising a fragment of a human dysferlin (hDYSF) gene, wherein the fragment consists of the nucleotide sequence of SEQ ID NO: 1; and
(ii) a second plasmid comprising a second recombinant polynucleotide comprising a fragment of a hDYSF gene, wherein the fragment consists of the nucleotide sequence of SEQ ID NO: 2;
(b) an adenovirus helper plasmid; and
(c) a rep-cap plasmid.

### Claim 6 (depends on 5)

A method for producing an adeno-associated viral (AAV) vector, comprising contacting a cell with the AAV packaging system of claim 5.

### Claim 7 (depends on 6)

The method of claim 6, wherein the cell is a host cell.

### Claim 8 (depends on 5)

A method for producing an adeno-associated viral (AAV) vector, comprising contacting a packaging cell line with the first plasmid or the second plasmid of claim 5, wherein the packaging cell line expresses an adeno-associated viral rep and cap genes.

### Claim 9 (depends on 1)

A cell comprising the dual AAV vector system of claim 1.

### Claim 10

A method of treating a dysferlinopathy, comprising administering to a subject in need thereof:
(a) an effective amount of a first recombinant polynucleotide, wherein the first recombinant polynucleotide comprises a fragment of a human dysferlin (hDYSF) gene, wherein the fragment consists of the nucleotide sequence of SEQ ID NO: 1; and
(b) an effective amount of a second recombinant polynucleotide, wherein the second recombinant polynucleotide comprises a fragment of a hDYSF gene, wherein the fragment consists of the nucleotide sequence of SEQ ID NO: 2.

### Claim 11 (depends on 10)

The method of claim 10, wherein the first recombinant polynucleotide and/or second recombinant polynucleotide is administered intramuscularly or intravenously.

### Claim 12 (depends on 10)

The method of claim 10, wherein the first and second recombinant polynucleotides are administered simultaneously or sequentially.

### Claim 13 (depends on 1)

A method of treating a dysferlinopathy, comprising administering to a subject in need thereof an effective amount of the AAV dual vector system of claim 1.

### Claim 14 (depends on 5)

The AAV packaging system of claim 5, wherein the first plasmid comprising the first recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18, and the second plasmid comprising the second recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 19.

### Claim 15 (depends on 6)

The method of claim 6, further comprising culturing the cell to produce a population of transduced cells, collecting supernatant from the population of transduced cells, and subjecting the supernatant to one or more purification steps to produce a purified AAV vector sample.