# US 2021/0340503 A1 · Recombinant aav production in mammalian cells

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- Number: US 2021/0340503 A1
- Document ID: US20210340503A1
- Status: Published application
- Kind: A1
- Application number: 17/201,524
- Filed: 2021-03-15
- Published: 2021-11-04
- Earliest claimed: 2002-09-23
- Assignees: Applied Genetic Technologies Corporation; The Johns Hopkins University; University of Florida Research Foundation, Inc.
- Applicants: Applied Genetic Technologies Corporation; The Johns Hopkins University; University of Florida Research Foundation, Incorporated
- Inventors: Kyu-Kye Hwang; David R. Knop; James Conway; Barry J. Byrne
- Web page: https://patentgrep.com/patent/US20210340503A1

## 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)
- C12N 15/86: Viral vectors
- C12N 15/8645: {Adeno-associated virus}
- C12N 2710/16643: viral genome or elements thereof as genetic vector
- C12N 2710/16644: Chimeric viral vector comprising heterologous viral elements for production of another viral vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14151: Methods of production or purification of viral material
- C12N 2750/14152: relating to complementing cells and packaging systems for producing virus or viral particles
- 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 includes methods and compositions for the production of high titer recombinant Adeno-Associated Virus (rAAV) in a variety of mammalian cells. The disclosed rAAV are useful in gene therapy applications. Disclosed methods based on co-infection of cells with two or more replication-defective recombinant herpes virus (rHSV) vectors are suitable for high-titer, large-scale production of infectious rAAV.

## Claims

### Claim 1

A method for producing recombinant rAAV in a mammalian cell, the method comprising:
(a) simultaneously infecting the mammalian cell, wherein the mammalian cell is a BHK cell, with:
(i) a first replication-defective rHSV comprising a nucleic acid sequence operably linked to a promoter wherein the nucleic acid comprises an AAV rep gene and an AAV cap gene, wherein the AAV rep and cap genes are integrated into the tk gene of the first rHSV-1; and
(ii) a second replication-defective rHSV comprising a nucleic acid sequence including AAV ITRs and a gene of interest, said gene of interest being operably linked to a promoter;
(b) incubating the infected mammalian cell; and
(c) obtaining rAAV from the cell of step (b), wherein the titer of rAAV produced by the cell is between about 1000 and about 9000 infectious particles (i.p.) per cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the AAV serotype for the cap gene is selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8.

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The method of claim 1, wherein the promoter for the rep gene and the cap gene in the first rHSV is a homologous promoter selected from the group consisting of p5, p19, and p40.

### Claim 5 (depends on 1)

The method of claim 1, wherein the promoter for the rep gene and the cap genes in the first rHSV is a heterologous promoter selected from the group consisting of a CMV promoter, a SV40 early promoter, a Herpes tk promoter, a metallothionine inducible promoter, a mouse mammary tumor promoter, and a chicken β-actin promoter.

### Claim 6 (depends on 1)

The method of claim 1, wherein the second rHSV further comprises a second gene of interest.

### Claim 7 (depends on 1)

The method of claim 1, wherein the gene of interest is flanked by the AAV ITRs.

### Claim 8 (depends on 1)

The method of claim 1, wherein the gene of interest encodes a protein of therapeutic use in humans.

### Claim 9 (depends on 1)

The method of claim 1, wherein the gene of interest encodes a reporter protein that is selected from the group consisting of beta-galactosidase, neomycin phosphoro-transferase, chloramphenicol acetyl transferase, thymidine kinase, luciferase, beta-glucuronidase, xanthine-guanine phosphoribosyl transferase and green fluorescent protein.

### Claim 10 (depends on 1)

The method of claim 1, further comprising infecting the cell with at least one additional virus selected from the group consisting of rHSV, rAAV, and recombinant Adenovirus (rAd).

### Claim 11 (depends on 1)

The method of claim 1, further comprising transfecting the cell with at least one plasmid DNA.

### Claim 12

(canceled)

### Claim 13

A method for producing recombinant Adeno-Associated Virus (rAAV), the method comprising:
(a) simultaneously infecting a mammalian cell, wherein the mammalian cell is selected from the group consisting of a Cos-7 cell and a HT1080 cell, with:
(i) a first replication-defective recombinant herpes simplex virus (rHSV) comprising a nucleic acid sequence operably linked to a promoter wherein the nucleic acid comprises an AAV rep gene and an AAV cap gene; and
(ii) a second replication-defective rHSV comprising a nucleic acid sequence including AAV inverted terminal repeat sequences (ITRs) and a gene of interest, said gene of interest being operably linked to a promoter;
(b) incubating the infected mammalian cell; and
(c) obtaining rAAV from the cell of step (b), wherein the titer of rAAV produced by the cell is between about 1000 and about 9000 infectious particles (i.p.) per cell.

### Claim 14

(canceled)

### Claim 15

(canceled)

### Claim 16 (depends on 13)

The method of claim 13, wherein the AAV serotype for the cap gene is selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8.

### Claim 17

(canceled)

### Claim 18 (depends on 13)

The method of claim 13, wherein the AAV rep and cap genes are integrated into the tk gene of the first rHSV-1.

### Claim 19 (depends on 13)

The method of claim 13, wherein the promoter for the rep gene and the cap genes in the first rHSV is a homologous promoter selected from the group consisting of p5, p19, and p40.

### Claim 20 (depends on 13)

The method of claim 13, wherein the promoter for the rep gene and the cap gene in the first rHSV is a heterologous promoter selected from the group consisting of a CMV promoter, a SV40 early promoter, a Herpes tk promoter, a metallothionine inducible promoter, a mouse mammary tumor promoter, and a chicken β-actin promoter.

### Claim 21 (depends on 13)

The method of claim 13, wherein the second rHSV further comprises a second gene of interest.

### Claim 22 (depends on 13)

The method of claim 13, wherein the gene of interest is flanked by the AAV ITRs.

### Claim 23 (depends on 13)

The method of claim 13, wherein the gene of interest encodes:
(i) a protein of therapeutic use in humans; or
(ii) a reporter protein that is selected from the group consisting of beta-galactosidase, neomycin phosphoro-transferase, chloramphenicol acetyl transferase, thymidine kinase, luciferase, beta-glucuronidase, xanthine-guanine phosphoribosyl transferase and green fluorescent protein.

### Claim 24

(canceled)

### Claim 25 (depends on 13)

The method of claim 13, further comprising infecting the cell with at least one additional virus selected from the group consisting of rHSV, rAAV, and recombinant Adenovirus (rAd).

### Claim 26 (depends on 13)

The method of claim 13, further comprising transfecting the cell with at least one plasmid DNA.

### Claim 27

(canceled)

### Claim 28

(canceled)

### Claim 29

(canceled)