Generation of adeno-associated virus capsid libraries for insect cells
AssigneeuniQure biopharma B.V.
Inventors- Anggakusuma, Sebastiaan Menno Bosma, Elena Maria Santidrian-Yebra-Pimentel
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Abstract
The present invention relates to means and methods for generating adeno-associated virus capsid libraries using insect host cells. In particular, the invention relates to novel DNA constructs and methods using these constructs for generating adeno-associated virus capsid libraries in insect host cells that minimize the occurrence of cross-packaging and mosaicism in the libraries generated. The invention further relates to method for identifying AAV capsid variants with one or more desired characteristics, such as e.g. the combination of CNS tropism and detargeting of peripheral organs.
Claims21
A linear nucleic acid molecule comprising a nucleic acid construct comprising:
- i) a nucleotide sequence encoding an adeno-associated virus (AAV) capsid variant, wherein the nucleotide sequence encodes an mRNA, translation of which in an insect cell produces the AAV capsid variant, and wherein the nucleotide sequence is operably linked to a promoter for driving expression in an insect cell;
- ii) an enhancer element that is operably linked to the promoter, wherein the enhancer element is dependent on a transcriptional transregulator, and wherein introduction of the transcriptional transregulator into an insect cell comprising the nucleic acid construct, induces transcription from the promoter;
- iii) at least one AAV inverted terminal repeat (ITR) sequence;
- iv) optionally, a reporter gene operably linked to a promoter for driving expression in a mammalian cell;
- and,
- v) optionally, a barcode.
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Dependencies are inferred from the claim text.