Vectors with modified initiation codon for the translation of AAV-REP78 useful for production of AAV
First Claim
1. A nucleic acid molecule comprising:
- (a) a first nucleotide sequence that comprises a single open reading frame (ORF) encoding parvoviral Rep78 and Rep52 proteins, with a translation initiation site of the Rep78 protein being a suboptimal initiation codon selected from the group consisting of ACG, TTG, CTG and GTG; and
(b) an expression control sequence that comprises nonanucleotide sequence SEQ ID NO;
7 upstream of the translation initiation site operably linked to the single ORF.
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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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Citations
39 Claims
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1. A nucleic acid molecule comprising:
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(a) a first nucleotide sequence that comprises a single open reading frame (ORF) encoding parvoviral Rep78 and Rep52 proteins, with a translation initiation site of the Rep78 protein being a suboptimal initiation codon selected from the group consisting of ACG, TTG, CTG and GTG; and (b) an expression control sequence that comprises nonanucleotide sequence SEQ ID NO;
7 upstream of the translation initiation site operably linked to the single ORF. - View Dependent Claims (2, 3, 4)
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- 5. A nucleic acid construct comprising a first nucleotide sequence that comprises a single open reading frame (ORF) encoding parvoviral Rep78 and Rep52 proteins, with a translation initiation site of the Rep78 protein being a suboptimal initiation codon selected from the group consisting of ACG, TTG, CTG and GTG, wherein an expression control sequence for expression in an insect cell is operably linked to the ORF and includes a promoter that is active in insect cells.
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13. An insect cell comprising a first nucleotide sequence that comprises a single ORF encoding parvoviral Rep78 and Rep52 proteins, with a translation initiation site of the Rep78 protein being a suboptimal initiation codon selected from the group consisting of ACG, TTG, CTG and GTG,
which cell does not comprise a parvoviral Rep protein-encoding nucleotide sequence other than said first nucleotide sequence.
Specification