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US 12,529,074 B2Granted
Adeno-associated virus (AAV) producer cell line and related methods
AssigneeLonza Walkersville, Inc.
InventorsBingnan Gu, Caitlin M. Guenther, Anandita Seth
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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.
Claims9
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 (TetO2);
- 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 TetO2;
- 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 TetO2;
- 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 TetO2;
- 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.
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