# US 2014/0155469 A1 · Adeno-associated-virus rep sequences, vectors and viruses

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- Number: US 2014/0155469 A1
- Document ID: US20140155469A1
- Status: Published application
- Kind: A1
- Application number: 14/112,703
- Filed: 2012-04-19
- Published: 2014-06-05
- Earliest claimed: 2011-04-19
- Assignees: The Research Foundation of State University of New York
- Inventors: Wadie F. Bahou; Patrick Hearing; Varsha Sitaraman
- Web page: https://patentgrep.com/patent/US20140155469A1

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61P 31/12: Antivirals
- C07K 14/005: from viruses
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/86: Viral vectors
- C12Q 1/6897: involving reporter genes operably linked to promoters
- A61K 2039/5256: {expressing foreign proteins}
- C12N 2710/10021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/10033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2710/10041: Use of virus, viral particle or viral elements as a vector
- C12N 2710/10052: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- 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 2840/102: inhibiting translation
- C12N 2840/60: from viruses
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- 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)
- 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)
- 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
- C12Y: ENZYMES

## Abstract

The invention provides adeno-associated virus (AAV) replication (Rep) sequences. In one embodiment, the invention provides nucleotide sequences encoding a chimeric protein, wherein the encoded chimeric protein contains a wild type AAV Rep inhibitory amino acid sequence, and wherein the nucleotide sequences contain a scrambled and/or deoptimized polynucleotide sequence encoding the wild type AAV Rep inhibitory amino acid sequence. The invention provides vectors, cells, and viruses containing the invention's sequences. Also provided are methods for detecting portions of the AAV Rep inhibitory amino acid sequence, which reduce replication and/or infection and/or productive infection by viruses. The invention's compositions and methods are useful for site-specific integration and/or expression of heterologous sequences by recombinant adeno-associated virus (rAAV) vectors and by rAAV virus particles, such as hybrid viruses (e.g., Ad-AAV) comprising such vectors. The invention's compositions and methods find application in, for example, gene therapy and/or vaccines.

## Claims

### Claim at position 1

1-42. (canceled)

### Claim 43

A recombinant nucleotide sequence encoding a chimeric protein,
a) wherein the encoded chimeric protein
i) comprises wild type AAV Rep inhibitory amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28, and
ii) has Rep-mediated nuclease activity, and
b) wherein the recombinant nucleotide sequence comprises a scrambled polynucleotide sequence encoding said wild type AAV Rep inhibitory amino acid sequence.

### Claim 44 (depends on 43)

The recombinant nucleotide sequence of claim 43, wherein
i) said scrambled polynucleotide sequence encoding said wild type SEQ ID NO:22 comprises SEQ ID NO:23,
ii) said scrambled polynucleotide sequence encoding said wild type SEQ ID NO:25 comprises SEQ ID NO:26, and
iii) said scrambled polynucleotide sequence encoding said wild type SEQ ID NO:28 comprises SEQ ID NO:29.

### Claim 45 (depends on 43)

The recombinant nucleotide sequence of claim 43, wherein said scrambled polynucleotide sequence comprises a deoptimized AAV Rep inhibitory nucleotide sequence.

### Claim 46 (depends on 43)

An expression vector comprising the recombinant nucleotide sequence of claim 43.

### Claim 47 (depends on 43)

A recombinant adeno-associated virus (rAAV) comprising the recombinant nucleotide sequence of claim 43.

### Claim 48 (depends on 47)

The recombinant adeno-associated virus (rAAV) of claim 47, wherein the rAAV is infectious.

### Claim 49 (depends on 48)

The recombinant adeno-associated virus (rAAV) of claim 48, wherein the infectious rAAV is replication competent.

### Claim 50 (depends on 49)

The recombinant adeno-associated virus (rAAV) of claim 49, wherein the replication competent rAAV is productive.

### Claim 51 (depends on 47)

The recombinant adeno-associated virus (rAAV) of claim 47, wherein the rAAV is produced by a permissive cell at substantially the same copy number as the copy number of a control AAV that lacks expression of AAV Rep protein.

### Claim 52 (depends on 47)

The recombinant adeno-associated virus (rAAV) of claim 47, wherein the rAAV is characterized by site-specific integration into adeno-associated virus integration site 1 (AAVS1) sequence.

### Claim 53 (depends on 47)

The recombinant adeno-associated virus (rAAV) of claim 47, wherein the rAAV expresses Rep78 protein SEQ ID NO:04 at a reduced level compared to the level expressed by a control hybrid virus that comprises wild type amino acid sequence SEQ ID NO:20 that is encoded by the wild type AAV Rep inhibitory nucleotide sequence listed as SEQ ID NO:17.

### Claim 54 (depends on 47)

The recombinant adeno-associated virus (rAAV) of claim 47, wherein the rAAV is a hybrid virus that comprises at least a portion of a heterologous virus genome sequence.

### Claim 55 (depends on 54)

The recombinant adeno-associated virus (rAAV) of claim 54, wherein the heterologous virus is selected from the group consisting of adenovirus, herpes simplex virus, retrovirus, lentivirus, and baculovirus.

### Claim at position 15

56-60. (canceled)

### Claim 61 (depends on 43)

A method for producing a recombinant adeno-associated virus (rAAV) particle, comprising
a) providing an expression vector comprising the recombinant nucleotide sequence of claim 43,
b) providing an adeno-associated virus (AAV) packaging cell, and
c) transfecting the packaging cell with the expression vector to produce a recombinant adeno-associated virus (rAAV).

### Claim at position 17

62-65. (canceled)

### Claim 66 (depends on 46)

A method for reducing one or more symptoms of disease in a mammalian subject, comprising administering a therapeutically effective amount of the vector of claim 46 to a mammalian subject in need of the therapy.

### Claim at position 19

67-71. (canceled)