# US 11,898,170 B2 · Cell culture methods involving HDAC inhibitors or rep proteins

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- Number: US 11,898,170 B2
- Document ID: US11898170B2
- Status: Granted
- Kind: B2
- Application number: 16/496,110
- Filed: 2018-03-22
- Published: 2020-01-30
- Granted: 2024-02-13
- Earliest claimed: 2017-03-22
- Assignees: Ultragenyx Pharmaceutical Inc.
- Applicants: Ultragenyx Pharmaceutical Inc.
- Inventors: Aubrey R. Tiernan; Christopher Tipper
- Web page: https://patentgrep.com/patent/US11898170B2

## 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 2330/51: Specially adapted vectors
- C12N 2510/02: Cells for production
- C12N 2511/00: Cells for large scale production
- 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 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 invention relates to methods of culturing cells, generating cell lines, and delivering polynucleotides to cells involving the use of HDAC inhibitors and/or adeno-associated virus (AAV) rep proteins.

## Claims

### Claim 1

A method of developing a stable cell line comprising a recombinant AAV (rAAV) vector sequence stably integrated into chromosome 19 for producing rAAV, the method comprising:
a) providing a rAAV vector comprising the rAAV vector sequence and a rep mRNA selected from the group consisting of rep68 mRNA, rep78 mRNA, and a combination thereof to a HeLa cell culture;
b) culturing the HeLa cell culture under conditions that allow for integration of the rAAV vector into the HeLa cell genome;
c) identifying a HeLa cell that contains the rAAV vector sequence integrated into chromosome 19 of the HeLa cell genome; and
d) culturing the mammalian cell under conditions that allow for generation of a cell line that produces rAAV,
wherein the rAAV produced by the cell line is at a titer of at least 8.5×1010 GC/ml when the HeLa cell culture of d) is cultured for at least four days.

### Claim 2 (depends on 1)

The method of claim 1, wherein the delivering of the rAAV vector and the rep mRNA is performed using electroporation, chemical-based transfection, microinjection, or cell squeezing.

### Claim 3 (depends on 2)

The method of claim 2, wherein the chemical-based transfection comprises exposure to lipids, calcium phosphate, cationic polymers, DEAE-dextran, or activated dendrimers.

### Claim 4 (depends on 1)

The method of claim 1, wherein the delivering of the rAAV vector and the rep mRNA is performed using magnetic beads or nanoparticles.

### Claim 5 (depends on 1)

The method of claim 1, wherein the rAAV vector sequence is integrated into the HeLa cell genome at a 2-kb region on the long arm of chromosome 19 (19q13.3-qter).

### Claim 6 (depends on 1)

The method of claim 1, wherein the amount of rep mRNA provided to the HeLa cell is between 0.75 μg to 1 μg per 106 cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the rAAV produced by the HeLa cell line is at a titer of about 8.85×1010 GC/ml to about 1.4 to 1011 GC/ml.

### Claim 8 (depends on 1)

A stable HeLa cell line generated using the method of claim 1.

### Claim 9 (depends on 1)

A plurality of rAAV particles produced by the stable HeLa cell line developed using the method of claim 1.

### Claim 10 (depends on 1)

The method of claim 1, wherein the HeLa cell culture is a HeLa S3 cell culture.