# US 11,078,464 B2 · High titer recombinant AAV vector production in adherent and suspension cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,078,464 B2
- Document ID: US11078464B2
- Status: Granted
- Kind: B2
- Application number: 14/915,630
- Filed: 2014-08-28
- Published: 2016-08-04
- Granted: 2021-08-03
- Earliest claimed: 2013-08-30
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Huiren Zhao; Grace Ki Jeong Lee; Thomas Wolfe; Cherylene Plewa; Jackie Z. Sheng
- Web page: https://patentgrep.com/patent/US11078464B2

## 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 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 is directed to an in vitro method of producing a recombinant AAV virion in a mammalian host cell that comprises a functional adenoviral E1A gene and rAAV virions made by the method. Host cells are transfected with varying ratios of plasmids comprising the E1A gene and virions.

## Claims

### Claim 1

An in vitro method of producing a recombinant AAV virion in a mammalian host cell, wherein the mammalian host cell is a HEK 293 cell, and wherein the cell is suspended in a transfection media, comprising contacting the cell with polyethylenimine (“PEI”) and:
(i) an accessory construct, wherein said accessory construct is a plasmid (pHelper) consisting essentially of adenoviral E2, E4Orf6, and VAI RNA genes operably linked to an origin of replication element and one or more other regulatory sequences;
(ii) an AAV helper construct, wherein said AAV helper construct is a plasmid (pTrans) consisting essentially of AAV2 rep and AAV1, AAV2, AAV5, AAV8, or AAV9 cap coding regions operably linked to one or more regulatory sequences; and
(iii) an AAV vector construct, wherein said AAV vector construct is a plasmid (pCis) consisting essentially of AAV2 inverted terminal repeats flanking a heterologous gene of interest operably linked to one or more regulatory sequences,
wherein the ratio of pHelper:pTrans:pCis is 1:5:(0.009-0.36) (weight:weight:weight), the PEI is Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000), and the ratio of PEI:DNA is about 3:1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the HEK 293 cell is suspended at a cell density of 2.1-3.0×106 cells/mL.

### Claim 3 (depends on 2)

The method of claim 2, wherein the HEK 293 cell is suspended at a cell density of 2.2-2.7×106 cells/mL.

### Claim 4 (depends on 3)

The method of claim 3, wherein the HEK 293 cell is suspended at a cell density of about 2.5×106 cells/mL.

### Claim 5 (depends on 1)

The method of claim 1, wherein the ratio of pHelper:pTrans:pCis is 1:5:(0.30-0.36) (weight:weight:weight).

### Claim 6 (depends on 5)

The method of claim 5, wherein the ratio of pHelper:pTrans:pCis is 1:5:0.31 (weight:weight:weight).

### Claim 7 (depends on 1)

The method of claim 1, wherein the total amount of plasmid does not exceed 1.5 mg/L of transfection media.

### Claim 8

An in vitro method of producing a recombinant AAV virion in a mammalian host cell, wherein the mammalian host cell is a HEK 293 cell, and wherein the cell adheres to a solid substrate in a transfection media, comprising contacting the cell with calcium phosphate and:
an accessory construct, wherein said accessory construct is a plasmid (pHelper) consisting essentially of adenoviral E2, E4Orf6, and VAI RNA genes operably linked to an origin of replication element and one or more other regulatory sequences;
(ii) an AAV helper construct, wherein said AAV helper construct is a plasmid (pTrans) consisting essentially of AAV2 rep and AAV1, AAV2, AAV5, AAV8, or AAV9 cap coding regions operably linked to one or more regulatory sequences; and
(iii) an AAV vector construct, wherein said AAV vector construct is a plasmid (pCis) consisting essentially of AAV2 inverted terminal repeats flanking a heterologous gene of interest operably linked to one or more regulatory sequences,
wherein the ratio of pHelper:pTrans:pCis is 1:5:(0.009-0.36) (weight:weight:weight).

### Claim 9 (depends on 8)

The method of claim 8, wherein the HEK 293 cell is seeded at a cell density of 2.1-3.0×106 cells/mL.

### Claim 10 (depends on 2)

The method of claim 2, wherein the HEK 293 cell is seeded at a cell density of 2.2-2.7×106 cells/mL.

### Claim 11 (depends on 3)

The method of claim 3, wherein the HEK 293 cell is seeded at a cell density of about 2.5×106 cells/mL.

### Claim 12 (depends on 1)

The method of claim 1, wherein the ratio of pHelper:pTrans:pCis is 1:5:(0.30-0.36) (weight:weight:weight).

### Claim 13 (depends on 5)

The method of claim 5, wherein the ratio of pHelper:pTrans:pCis is 1:5:0.31 (weight:weight:weight).

### Claim 14 (depends on 1)

The method of claim 1, wherein the total amount of plasmid does not exceed 1.5 mg/L of transfection media.