# US 9,587,282 B2 · Adeno-associated virus virions with variant capsid and methods of use thereof

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- Number: US 9,587,282 B2
- Document ID: US9587282B2
- Status: Granted
- Kind: B2
- Application number: 14/938,154
- Filed: 2015-11-11
- Published: 2016-06-30
- Granted: 2017-03-07
- Earliest claimed: 2011-04-22
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: David V. Schaffer; Ryan R. Klimczak; James T. Koerber; John G. Flannery; Deniz Dalkara Mourot; Meike Visel; Leah C. T. Byrne
- Web page: https://patentgrep.com/patent/US9587282B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- 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 9/0048: {Eye, e.g. artificial tears}
- A61K 38/1709: {from mammals}
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- A61K 48/0025: {wherein the non-active part clearly interacts with the delivered nucleic acid}
- A61K 48/0041: {the non-active part being polymeric}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 27/00: Drugs for disorders of the senses
- A61P 27/02: Ophthalmic agents
- A61P 27/06: Antiglaucoma agents or miotics
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/005: from viruses
- 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 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- C12N 15/86: Viral vectors
- C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/5058: {Neurological cells}
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/16: Aptamers
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2750/14021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2750/14121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2750/14122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2750/14142: virus or viral particle as vehicle, e.g. encapsulating small organic molecule
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14145: Special targeting system for viral vectors
- C12N 2750/14152: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2810/40: Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, where the AAV virions exhibit greater infectivity of retinal cells, when administered via intravitreal injection, compared to wild-type AAV. The present disclosure further provides methods of delivering a gene product to a retinal cell in an individual, and methods of treating ocular disease.

## Claims

### Claim 1

A recombinant adeno-associated virus (rAAV) virion comprising:
a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence LGETTRP (SEQ ID NO:13), wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein; and
b) a heterologous nucleic acid comprising a nucleotide sequence encoding retinoschisin.

### Claim 2 (depends on 1)

The rAAV virion of claim 1, wherein the retinal cell is a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigmented epithelium cell.

### Claim 3 (depends on 1)

The rAAV virion of claim 1, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.

### Claim 4 (depends on 1)

The rAAV virion of claim 1, wherein the rAAV virion exhibits at least 10-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.

### Claim 5 (depends on 1)

The rAAV virion of claim 1, wherein the rAAV virion exhibits at least 50-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.

### Claim 6 (depends on 1)

A pharmaceutical composition comprising:
a) a recombinant adeno-associated virus virion of claim 1; and
b) a pharmaceutically acceptable excipient.

### Claim 7 (depends on 1)

A method of delivering retinoschisin to a retinal cell in an individual, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to claim 1.

### Claim 8 (depends on 1)

A method of treating retinoschisis, the method comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion according to claim 1.

### Claim 9 (depends on 8)

The method of claim 8, wherein said administering is by intraocular injection.

### Claim 10 (depends on 8)

The method of claim 8, wherein said administering is by intravitreal injection.

### Claim 11

An isolated nucleic acid comprising a nucleotide sequence that encodes a variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion of relative to a corresponding parental AAV capsid protein, and wherein the peptide insertion comprises the amino acid sequence LGETTRP (SEQ ID NO:13), wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.

### Claim 12 (depends on 11)

The isolated nucleic acid of claim 11, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.

### Claim 13 (depends on 11)

An isolated, genetically modified host cell comprising the nucleic acid of claim 11.

### Claim 14

A variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion of relative to a corresponding parental AAV capsid protein, and wherein the peptide insertion comprises the amino acid sequence LGETTRP (SEQ ID NO:13), wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.

### Claim 15 (depends on 14)

The variant AAV capsid protein of claim 14, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.

### Claim 16 (depends on 14)

A nucleic acid comprising a nucleotide sequence encoding the variant AAV capsid protein of claim 14.

### Claim 17 (depends on 1)

The rAAV virion of claim 1, wherein the retinoschisin encoded by the heterologous nucleic acid comprises an amino acid sequence having at least 90% amino acid sequence identity to a contiguous stretch of from about 200 amino acids to 224 amino acids of SEQ ID NO:10.

### Claim 18 (depends on 1)

The rAAV virion of claim 1, wherein the retinoschisin encoded by the heterologous nucleic acid is under transcriptional control of a heterologous promoter.

### Claim 19 (depends on 6)

The pharmaceutical composition of claim 6, wherein the retinoschisin encoded by the heterologous nucleic acid comprises an amino acid sequence having at least 90% amino acid sequence identity to a contiguous stretch of from about 200 amino acids to 224 amino acids of SEQ ID NO:10.

### Claim 20 (depends on 6)

The pharmaceutical composition of claim 6, wherein the retinoschisin encoded by the heterologous nucleic acid is under transcriptional control of a heterologous promoter.

### Claim 21 (depends on 7)

The method of claim 7, wherein the retinoschisin encoded by the heterologous nucleic acid comprises an amino acid sequence having at least 90% amino acid sequence identity to a contiguous stretch of from about 200 amino acids to 224 amino acids of SEQ ID NO:10.

### Claim 22 (depends on 7)

The method of claim 7, wherein the retinoschisin encoded by the heterologous nucleic acid is under transcriptional control of a heterologous promoter.

### Claim 23 (depends on 8)

The method of claim 8, wherein the retinoschisin encoded by the heterologous nucleic acid comprises an amino acid sequence having at least 90% amino acid sequence identity to a contiguous stretch of from about 200 amino acids to 224 amino acids of SEQ ID NO:10.

### Claim 24 (depends on 8)

The method of claim 8, wherein the retinoschisin encoded by the heterologous nucleic acid is under transcriptional control of a heterologous promoter.