# US 9,856,539 B2 · Adeno-associated virus virions with variant capsid and methods of use thereof

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- Number: US 9,856,539 B2
- Document ID: US9856539B2
- Status: Granted
- Kind: B2
- Application number: 14/606,543
- Filed: 2015-01-27
- Published: 2015-08-13
- Granted: 2018-01-02
- 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/US9856539B2

## 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 method of identifying a novel variant recombinant adeno-associated virus (rAAV) virion having greater infectivity of a cell in vivo as compared to the corresponding parental AAV virion, the method comprising:
a) administering a composition comprising a library of variant rAAV virions in an eye of an animal by intravitreal injection, wherein each variant rAAV virion comprises: i) a variant AAV virion capsid protein comprising a peptide of from five to eleven amino acids in length inserted into the GH loop of the corresponding parental AAV capsid protein; and ii) DNA encoding the variant AAV virion capsid protein;
b) isolating a target photoreceptor cell from non-target ocular cells, wherein the target photoreceptor cell has been successfully infected by a variant rAAV virion;
c) polymerase chain reaction (PCR) amplifying DNA encoding an AAV virion capsid protein from the isolated target photoreceptor cells;
d) sequencing the amplified DNA to identify one or more variant AAV virion capsid sequences that have permissive mutations for high infectivity of the target photoreceptor cell as compared to non-target ocular cells;
e) cloning the DNA encoding the variant AAV virion capsid protein; and
f) carrying out error-prone PCR amplification of the cloned DNA, thereby generating a plurality of additional variant rAAV.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the peptide is located between two adjacent amino acids within amino acids corresponding to amino acids 570-611 of the AAV2 capsid protein or the corresponding position in the capsid protein of any of the following AAV serotypes: AAV1, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the variant AAV virion capsid protein comprises 1 to 25 amino acid substitutions.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the variant AAV virion capsid protein is a chimeric capsid protein.

### Claim 5 (depends on 1)

The method of claim 1, further comprising
packaging variant AAV virions from the cloned DNA encoding the variant AAV virion capsid protein.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the increased infectivity is a greater infectivity of said photoreceptor cells following administration of the variant rAAV in vivo as compared to the infectivity of an AAV virion comprising the corresponding parental AAV capsid protein when administered in vivo.