# US 2019/0169237 A1 · Mutant adeno-associated virus virions and methods of use thereof

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- Number: US 2019/0169237 A1
- Document ID: US20190169237A1
- Status: Published application
- Kind: A1
- Application number: 16/274,935
- Filed: 2019-02-13
- Published: 2019-06-06
- Earliest claimed: 2003-06-30
- Assignees: The Regents of the University of California; Integrative Gene Therapeutics
- Applicants: The Regents of the University of California; Integrative Gene Therapeutics
- Inventors: David V. Schaffer; Brian Kaspar; Narendra Maheshri
- Web page: https://patentgrep.com/patent/US20190169237A1

## CPC

- C07K 14/005: from viruses
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/06: Antihyperlipidemics
- A61P 3/08: for glucose homeostasis (pancreatic hormones A61P5/48)
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 5/10: of the posterior pituitary hormones, e.g. oxytocin, ADH
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 13/00: Drugs for disorders of the urinary system
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 31/18: for HIV
- A61P 33/00: Antiparasitic agents
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/04: Immunostimulants
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/86: Viral vectors
- 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
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 provides mutant adeno-associated virus (AAV) that exhibit altered capsid properties, e.g., reduced binding to neutralizing antibodies in serum and/or altered heparin binding and/or altered infectivity of particular cell types. The present invention further provides libraries of mutant AAV comprising one or more mutations in a capsid gene. The present invention further provides methods of generating the mutant AAV and mutant AAV libraries, and compositions comprising the mutant AAV. The present invention further provides recombinant AAV (rAAV) virions that comprise a mutant capsid protein. The present invention further provides nucleic acids comprising nucleotide sequences that encode mutant capsid proteins, and host cells comprising the nucleic acids. The present invention further provides methods of delivering a gene product to an individual, the methods generally involving administering an effective amount of a subject rAAV virion to an individual in need thereof.

## Claims

### Claim 1

An isolated nucleic acid comprising a nucleotide sequence that encodes a mutant adeno-associated virus (AAV) capsid protein, wherein the mutant AAV capsid protein comprises at least one amino acid substitution relative to a wild-type AAV capsid protein.

### Claim 2 (depends on 1)

The isolated nucleic acid of claim 1, wherein the encoded mutant AAV capsid protein exhibits increased heparin binding relative to a wild-type AAV capsid protein.

### Claim 3 (depends on 1)

The isolated nucleic acid of claim 1, wherein the encoded mutant AAV capsid protein exhibits decreased heparin binding relative to a wild-type AAV capsid protein.

### Claim 4 (depends on 1)

The isolated nucleic acid of claim 1, wherein the encoded mutant AAV capsid protein exhibits reduced binding to neutralizing antibody relative to a wild-type AAV capsid protein.

### Claim 5 (depends on 1)

An isolated host cell comprising the nucleic acid of claim 1.

### Claim 6 (depends on 5)

The isolated host cell of claim 5, wherein the host cell is stably transfected with the nucleic acid.

### Claim 7 (depends on 5)

The isolated host cell of claim 5, further comprising a nucleic acid comprising a nucleotide sequence encoding an adeno-associated virus (AAV) rep protein.

### Claim 8 (depends on 7)

The isolated host cell of claim 7, further comprising a recombinant AAV vector.

### Claim 9

An adeno-associated virus (AAV) library, comprising a plurality of nucleic acids comprising a nucleotide sequence that encodes a mutant AAV capsid protein, wherein the mutant AAV capsid protein comprises at least one amino acid substitution relative to a wild-type AAV capsid protein.

### Claim 10

A library of mutant adeno-associated virus (AAV) particles, comprising a plurality of AAV particles comprising an AAV capsid protein comprising at least one amino acid substitution relative to a wild-type AAV capsid protein.

### Claim 11

A recombinant adeno-associated virus (rAAV) virion comprising an AAV capsid protein comprising at least one amino acid substitution relative to a wild-type AAV capsid protein; and a heterologous nucleic acid.

### Claim 12 (depends on 11)

The rAAV virion of claim 11, wherein the rAAV virion exhibits reduced heparin binding relative to wild-type AAV.

### Claim 13 (depends on 11)

The rAAV virion of claim 11, wherein the rAAV virion exhibits increased heparin binding relative to wild-type AAV.

### Claim 14 (depends on 11)

The rAAV virion of claim 11, wherein the rAAV virion exhibits reduced inhibition of infection by neutralizing antibodies relative to the inhibition of infection by neutralizing antibodies of wild-type AAV virion.

### Claim 15 (depends on 11)

The rAAV virion of claim 11, wherein the rAAV virion exhibits increased capacity to cross an endothelial cell layer relative to wild-type AAV virion.

### Claim 16 (depends on 11)

The rAAV virion of claim 11, wherein the rAAV virion exhibits increased infectivity of a non-permissive cell relative to wild-type AAV virion.

### Claim 17

A method of generating a mutant adeno-associated virus (AAV) particle comprising an AAV capsid protein comprising at least one amino acid substitution relative to a wild-type AAV capsid protein, wherein the mutant AAV particle exhibits an altered capsid property, the method comprising:
a) generating a library of AAV mutants comprising a mutation in an AAV cap gene;
b) packaging the AAV library into AAV particles, generating AAV library particles; and
c) selecting the AAV library particles for an altered capsid property.

### Claim 18 (depends on 17)

The method of claim 17, wherein generation of the library of AAV mutants comprises polymerase chain reaction mutagenesis.

### Claim 19 (depends on 18)

The method of claim 18, where the mutagenesis comprises a staggered extension process.

### Claim 20 (depends on 17)

The method of claim 17, wherein generation of the library of AAV mutants comprises oligonucleotide-directed mutagenesis.

### Claim 21 (depends on 17)

The method of claim 17, wherein the selecting step comprises selecting for reduced binding to heparin.

### Claim 22 (depends on 17)

The method of claim 17, wherein the selecting step comprises selecting for increased binding to heparin.

### Claim 23 (depends on 17)

The method of claim 17, wherein the selecting step comprises selecting for reduced binding to neutralizing antibodies.

### Claim 24 (depends on 17)

The method of claim 17, wherein the selecting step comprises selecting for increased infectivity of a non-permissive cell.

### Claim 25 (depends on 17)

The method of claim 17, wherein the selecting step comprises selecting for increased ability to cross an endothelial cell layer.

### Claim 26 (depends on 11)

A method of delivering a gene product to an individual in need thereof, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to claim 11.

### Claim 27 (depends on 26)

The method of claim 26, wherein the gene product is a protein.

### Claim 28 (depends on 26)

The method of claim 26, wherein the gene product is a short interfering RNA.