Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
First Claim
1. A nucleic acid construct encoding parvoviral Rep protein Rep78 and Rep52, the nucleic acid construct comprising:
- (a) a biscistronic nucleic acid encoding a parvovirus Rep78 protein and a parvovirus Rep52 protein, the biscistronic nucleic acid comprising a nucleic acid sequence encoding the Rep78 protein that overlaps with a nucleic acid sequence encoding the Rep52 protein, such that the nucleic acid sequence encoding the parvoviral Rep52 protein is not repeated in the biscistronic nucleic acid;
(b) a first expression control sequence that is active in an insect cell operably linked to the sequence encoding Rep78 protein;
(c) a second expression control sequence that is active in an insect cell operably linked to the sequence encoding Rep52 protein.
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Abstract
The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AAV) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.
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20 Claims
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1. A nucleic acid construct encoding parvoviral Rep protein Rep78 and Rep52, the nucleic acid construct comprising:
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(a) a biscistronic nucleic acid encoding a parvovirus Rep78 protein and a parvovirus Rep52 protein, the biscistronic nucleic acid comprising a nucleic acid sequence encoding the Rep78 protein that overlaps with a nucleic acid sequence encoding the Rep52 protein, such that the nucleic acid sequence encoding the parvoviral Rep52 protein is not repeated in the biscistronic nucleic acid; (b) a first expression control sequence that is active in an insect cell operably linked to the sequence encoding Rep78 protein; (c) a second expression control sequence that is active in an insect cell operably linked to the sequence encoding Rep52 protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification