Modified viral vectors and methods of making and using the same
The present invention relates to production of parvoviral vectors to produce adeno-associated virus (AAV) for gene therapy. In particular the invention relates to improvements in parvoviral vectors that increase the packaging capacity, production efficiency, and infectivity of AAV virions that is necessary for large scale manufacturing of AAV for clinical purposes.
1. An insect cell expressing adeno-associated virus (AAV) VP1 protein and AAV VP3 protein and not expressing AAV VP2 protein, wherein the insect cell comprises an exogenous nucleotide sequence of 6.1 kb of at least one gene of interest.
2. The insect cell according to claim 1 , comprising:
(a) a nucleotide sequence encoding AAV VP1 protein and AAV VP3 protein; and/or
(b) a nucleotide sequence encoding AAV VP1 protein and a nucleotide sequence encoding AAV VP3 protein.
3. The insect cell according to claim 2 , wherein the nucleotide sequence as defined in (a) of claim 2 comprises:
(a) an open reading frame encoding AAV VP1 protein; and
(b) an open reading frame encoding AAV VP3 protein.
4. The insect cell according to claim 1 , comprising a nucleotide sequence encoding adeno-associated virus (AAV) VP2 protein with an inactivated VP2 initiation codon.
5. The insect cell according to claim 1 , wherein the insect cell further comprises: (i) a nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell; and optionally, (ii) a nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell.
6. The insect cell according to claim 5 , wherein the insect cell comprises: (a) a first nucleic acid construct comprising (i) nucleotide sequences for AAV VP1 protein and AAV VP3 protein, (ii) the nucleotide sequence comprising a Rep78 or a Rep68 coding sequence and, optionally, (iii) the nucleotide sequence comprising a Rep52 or a Rep40 coding sequence; and, (b) a second nucleic acid construct comprising an exogenous nucleotide sequence of at least 6.1 kb of at least one gene of interest.
7. The insect cell according to claim 6 , wherein the second nucleic acid construct is an insect cell-compatible vector.
8. The insect cell according to claim 7 , wherein the vector is a baculoviral vector.
9. A method for producing an AAV in an insect cell, comprising:
(a) culturing an insect cell according to claim 5 under conditions to produce AAV; and optionally,
(b) recovering the AAV.
10. An AAV virion produced in an insect cell according to claim 1 , the AAV virion comprising in its genome at least one nucleotide sequence encoding a gene product of interest, wherein the at least one nucleotide sequence is not a native AAV nucleotide sequence, and wherein the AAV virion comprises AAV VP1 protein and AAV VP3 protein and does not comprise AAV VP2 protein.
11. The AAV virion according to claim 10 , wherein the at least one nucleotide sequence encoding a gene product of interest is located between two AAV ITR nucleotide sequences.
12. A pharmaceutical composition, comprising the AAV virion according to claim 10 .