# US 10,308,957 B2 · rAAV vectors and methods for transduction of photoreceptors and RPE cells

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- Number: US 10,308,957 B2
- Document ID: US10308957B2
- Status: Granted
- Kind: B2
- Application number: 15/123,515
- Filed: 2015-03-04
- Published: 2016-12-22
- Granted: 2019-06-04
- Earliest claimed: 2014-03-04
- Assignees: University of Florida Research Foundation, Inc.
- Applicants: University of Florida Research Foundation, Inc.
- Inventors: Shannon E. Boye; Sanford L. Boye; Mavis Agbandje-McKenna; Arun Srivastava
- Web page: https://patentgrep.com/patent/US10308957B2

## CPC

- C12N 15/86: Viral vectors
- A61K 9/0048: {Eye, e.g. artificial tears}
- A61K 9/5184: {Virus capsids or envelopes enclosing drugs (liposomes with additional exogenous lipids A61K9/127)}
- C07K 14/005: from viruses
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2750/14122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2810/6027: ssDNA viruses
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)

## Abstract

Disclosed are capsid-modified rAAV particles and expression vectors, as well as compositions and pharmaceutical formulations that comprise them. Also disclosed are methods of preparing and using novel capsid-protein-mutated particle or rAAV vector constructs in a variety of diagnostic and therapeutic applications including, inter alia, as delivery agents for diagnosis, treatment, or amelioration of one or more diseases, disorders, or dysfunctions of the mammalian eye. Also disclosed are methods for subretinal delivery of therapeutic gene constructs to mammalian photoreceptors and retinal pigment epithelial cells, as well as use of the disclosed compositions in the manufacture of medicaments for a variety of in vitro and/or in vivo applications including the treatment of a variety of inherited retinal diseases.

## Claims

### Claim 1

A recombinant adeno-associated viral (rAAV) particle comprising:
a modified capsid protein, wherein the modified capsid protein comprises non-native amino acid substitutions at amino acid residues of a wild-type AAV2 capsid protein as set forth in SEQ ID NO:2, wherein the non-native amino acid substitutions comprise:
(a) Y444F, T491V, Y500F, R585S, R588T, R487G, and Y730F;
(b) Y444F, T491V, Y500F, R585S, and Y730F;
(c) Y444F, T491V, Y500F, R588T, and Y730F; or
(d) Y444F, T491V, Y500F, R585S, R588T, and Y730F,
or at equivalent amino acid positions corresponding thereto in any one of the wild-type AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, or AAV10 capsid proteins, as set forth, respectively, in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10.

### Claim 2 (depends on 1)

The rAAV particle of claim 1, wherein the transduction efficiency of the particle is about 2- to about 50-fold higher in the one or more photoreceptor or RPE cells than that of a particle that comprises a corresponding, unmodified, wild-type capsid protein.

### Claim 3 (depends on 1)

The rAAV particle of claim 1, wherein the particle comprises a polynucleotide comprising a nucleic acid segment that encodes a diagnostic or therapeutic molecule operably linked to a promoter that is capable of expressing the nucleic acid segment in one or more photoreceptors or retinal pigment epithelial cells of a mammalian eye.

### Claim 4 (depends on 3)

The rAAV particle of claim 3, wherein the nucleic acid segment expresses or encodes in one or more photoreceptor cells or RPE cells of a mammalian eye, a polypeptide, a peptide, a ribozyme, a peptide nucleic acid, an siRNA, an RNAi, an antisense oligonucleotide, an antisense polynucleotide, an antibody, an antigen binding fragment, or any combination thereof.

### Claim 5

A nucleic acid vector that encodes a modified capsid protein, wherein the modified capsid protein comprises non-native amino acid substitutions at amino acid residues of a wild-type AAV2 capsid protein as set forth in SEQ ID NO:2, wherein the non-native amino acid substitutions comprise:
(a) Y444F, T491V, Y500F, R585S, R588T, R487G, and Y730F;
(b) Y444F, T491V, Y500F, R585S, and Y730F;
(c) Y444F, T491V, Y500F, R588T, and Y730F; or
(d) Y444F, T491V, Y500F, R585S, R588T, and Y730F,
or at equivalent amino acid positions corresponding thereto in any one of the wild-type AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, or AAV10 capsid proteins, as set forth, respectively, in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10.

### Claim 6 (depends on 5)

The nucleic acid vector of claim 5, wherein the transduction efficiency of a particle comprising the modified capsid protein is about 2- to about 50-fold higher in one or more photoreceptor or RPE cells than that of a particle that comprises a corresponding, unmodified, wild-type capsid protein.

### Claim 7 (depends on 1)

The rAAV particle of claim 1, wherein the non-native amino acid substitutions further comprise an E530K substitution in the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2, or at equivalent amino acid positions corresponding thereto in any one of the wild-type AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, or AAV10 capsid proteins, as set forth, respectively, in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10.

### Claim 8 (depends on 5)

The nucleic acid vector of claim 5, wherein the non-native amino acid substitutions further comprise an E530K substitution in the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2, or at equivalent amino acid positions corresponding thereto in any one of the wild-type AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, or AAV10 capsid proteins, as set forth, respectively, in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10.