Adeno-associated virus (AAV) producer cell line and related methods

Patent metadata

Field	Value
Publication number	US-12529074-B2
Application number	US-202318352657-A
Country	US
Kind code	B2
Filing date	Jul 14, 2023
Priority date	Dec 21, 2018
Publication date	Jan 20, 2026
Grant date	Jan 20, 2026

Abstract

The present disclosure relates to a mammalian cell line for producing adeno-associated virus (AAV), suitably including nucleic acids encoding helper genes and AAV genes, under the control of derepressible promoters. The disclosure also relates to isolated nucleic acid molecules that encode such genes, as well as methods of using the mammalian cells for producing AAVs.

First claim

What is claimed is:

1 . An isolated Chinese hamster ovary (CHO) cell or an isolated human embryonic kidney (HEK) cell for producing an adeno-associated virus (AAV), comprising:
a nucleic acid sequence comprising: i) a nucleotide sequence encoding the adenovirus helper E2A and E4Orf6 proteins under control of a first derepressible promoter comprising a functional promoter and two tetracycline operator sequences (TetO 2 ); ii) a nucleotide sequence encoding the AAV gene comprising a) a rep78 protein coding region under control of a second derepressible promoter comprising a p5 promoter and TetO 2 ; b) a cap protein coding region under control of the second derepressible promoter or a p40 native promoter; and c) a rep52 coding region under control of a third derepressible promoter comprised of a p19 promoter and TetO 2 ; iii) a nucleotide sequence encoding adenovirus viral-associated (VA I) non-coding RNA under control of a fourth depressible promoter comprising a functional promoter and TetO 2 ; iv) two adenovirus inverted terminal repeat (ITR) sequences; and v) a repressor element of the first, second, third, and fourth derepressible promoters under the control of a constitutive promoter,
wherein the nucleic acid molecule is integrated into the isolated CHO or isolated HEK cell's genomic DNA and/or is maintained by the isolated CHO or isolated HEK cell extra-chromosomally.

2 . The isolated CHO or isolated HEK cell of claim 1 , wherein the isolated CHO or isolated HEK cell is an isolated CHO or isolated HEK cell culture.

3 . The isolated CHO or isolated HEK cell of claim 2 , wherein the isolated CHO or isolated HEK cell culture is a suspension culture.

4 . The isolated CHO or isolated HEK cell of claim 1 , further comprising an internal ribosome entry site (IRES) element between the e2a and e4orf6 encoding regions.

5 . The isolated CHO or isolated HEK cell of claim 1 , wherein the rep52 coding region is under control of the third derepressible promoter comprising the p19 promoter and TetO 2 contained within an artificial intron.

6 . The isolated CHO or isolated HEK cell of claim 1 , wherein the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.

7 . The isolated CHO or isolated HEK cell of claim 1 , wherein the repressor element is a tetracycline repressor protein.

8 . The isolated CHO or isolated HEK cell of claim 7 , further comprising a nucleic acid encoding a transcriptional repression domain in frame with the nucleic acid encoding the tetracycline repressor protein.

9 . The isolated CHO or isolated HEK cell of claim 1 , further comprising a nucleic acid molecule encoding a gene of interest.

Assignees

• Lonza Walkersville Inc

Inventors

• Gu Bingnan
• Guenther Caitlin M
• Seth Anandita

Classifications

• C12N15/8645

  Adeno-associated virus · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2830/008

  cell type or tissue specific enhancer/promoter combination · CPC title

• C12N2710/10343

  viral genome or elements thereof as genetic vector · CPC title

• C12N2830/006

  tet repressible · CPC title