Production of adeno-associated virus in insect cells

A method of producing an AAV in an insect cell, comprising:

• (i) providing an insect cell comprising
• a first nucleotide sequence comprising at least one AAV ITR nucleotide sequence,
• a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2 and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell,
• a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell,
• a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and, optionally,
• at least one insect cell-compatible vector,
• wherein at least one of the first, second, third and fourth nucleotide sequences is/are stably integrated in the insect cell and the at least one insect cell-compatible vector, when present, comprises the remainder of the first, second, third and fourth nucleotide sequences which is/are not stably integrated in the insect cell, and
• (ii) maintaining the insect cell under conditions such that AAV is produced.

An insect cell-compatible vector comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence comprising:

• a nine nucleotide sequence of SEQ. ID NO:
• 4 or a nucleotide sequence substantially homologous to SEQ. ID NO:
• 4, located upstream of the initiation codon of the nucleotide sequence encoding AAV VP1 capsid protein, and
• a C at nucleotide position 2 of the nucleotide sequence encoding AAV VP1 capsid protein.

An insect cell comprising:

• a first nucleotide sequence comprising at least one AAV ITR nucleotide sequence,
• a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell,
• a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and
• a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell.

A method of producing an AAV in an insect cell comprising:

• (i) providing at least one insect cell-compatible vector comprising
• a first nucleotide sequence comprising at least one chimeric ITR nucleotide sequence, the ITR nucleotide sequence comprising an AAV backbone and a specific binding and a nicking site of a Rep protein from a parvovirus other than AAV,
• a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell,
• a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and
• a fourth nucleotide sequence comprising a nucleotide sequence encoding a parvoviral Rep protein that can specifically bind and nick the site in the ITR nucleotide sequence within the first nucleotide sequence, operably linked to at least one expression control sequence for expression in an insect cell,
• (ii) introducing said at least one insect cell-compatible vector into an insect cell, and
• (iii) maintaining said insect cell under conditions such that AAV is produced.

A method of producing a parvoviral genome in an insect cell, which method comprises:

• (i) providing at least one insect cell-compatible vector comprising
• a first nucleotide sequence comprising at least one parvoviral ITR,
• a second nucleotide sequence comprising an AAV Rep52 or Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and
• a third nucleotide sequence comprising an AAV Rep78 or Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell,
• (ii) introducing said at least one insect cell-compatible vector into an insect cell, and
• (iii) maintaining said insect cell under conditions such that a parvovirus genome is produced therein.