Patent ID: 8642314

Claim:
A method for the production of a recombinant parvoviral virion, which comprises culturing an insect cell comprising one or more nucleic acid constructs which comprise: (a) a nucleotide sequence comprising a transgene that is flanked by at least one parvoviral inverted terminal repeat (ITR) nucleotide sequence; (b) a first expression cassette comprising a nucleotide sequence encoding at least one of parvoviral Rep proteins Rep78 and Rep68, which nucleotide sequence is operably linked to a first promoter that is capable of driving expression of the at least one of Rep78 and Rep68 proteins in the insect cell; and (c) a second expression cassette comprising a nucleotide sequence encoding a parvoviral capsid protein which is operably linked to a second promoter that is capable of driving expression of the capsid protein in the insect cell; under conditions conducive to the expression of the Rep protein and the capsid protein; wherein (i) the first and second expression cassettes are present on a single nucleic acid construct and are present in equimolar amounts in the insect cell, (ii) the ratio of the Rep78 and/or Rep68 protein expression to the capsid protein expression is regulated by one or more of the following: (A) the first promoter is stronger than the second promoter; (B) the presence of more and/or stronger enhancer elements in the first expression cassette as compared to the second expression cassette; (C) the nucleotide sequence encoding the Rep78 and/or Rep68 protein has a higher codon adaptation index as compared to the nucleotide sequence encoding the capsid protein; (D) temperature optimization of the Rep78 and/or Rep68 protein; and/or (E) a variant Rep78 and/or Rep68 protein with an alteration in the amino acid sequence as compared to the sequence of corresponding wild-type Rep78 and/or Rep68 protein, wherein the sequence alteration results in increased Rep78 and/or Rep68 protein function assessed as increased adeno-associated virus (AAV) production in the insect cell, resulting in expression of the Rep78 and/or Rep68 protein that is greater than expression of the capsid protein.