rAAV PRODUCTION IN INSECT CELLS

An efficient-method of producing a recombinant baculovirus seed stock comprising a gene of interest, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock comprise a gene of interest, said method comprising the steps of:

• (a) introducing a system for production of recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system comprises
• a) a donor plasmid comprising a first antibiotic resistance gene, a counter selective marker and a transfer cassette, and wherein the transfer cassette comprises a left transposon arm, a gene of interest and a right transposon arm;
• b) a helper plasmid comprising a second antibiotic resistance gene;
• c) a bacmid comprising third antibiotic resistance gene and a transposon insertion site located within a reporter cassette;
• (b) growing the transformed bacteria in the presence of three antibiotics to which the first, second and third antibiotic resistance genes confer resistance;
• (c) selecting at least one bacterial colony comprising a recombinant bacmid;
• (d) incubating bacteria from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counter selection against the donor plasmid;
• (e) collecting recombinant bacmids comprising a gene of interest from a bacterial colony after counter selection;
• (f) transforming insect cells with the recombinant bacmids comprising a gene of interest collected in step (e);
• (g) incubating the transformed insect cells and harvesting rBV comprising the gene of interest from the insect cells;
• (h) performing a first passage, wherein a passage comprises the steps of infecting insect cells with rBV comprising the gene of interest and incubating the infected insect cells;
• (i) harvesting rBV comprising the gene of interest from at least two plaques of insect cells and identifying a plaque comprising rBV comprising a gene of interest and wherein the rBV exhibit a low rate of loss of the gene of interest;
• and
• (j) performing a second passage (P1 rBV), wherein the second passage comprises infecting insect cells with rBV from the plaque identified in step (i), incubating the infected insect cells and harvesting a recombinant baculovirus seed stock comprising rBV comprising the gene of interest from the insect cells.

A method of producing a recombinant baculovirus seed stock comprising a stable gene of interest, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock comprise a stable gene of interest, the method comprising the steps of:

• (a) introducing a system for production of recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system comprises:
• i. a donor plasmid comprising a first antibiotic resistance gene, a counter selective marker and a transfer cassette, and wherein the transfer cassette comprises a left transposon arm, a gene of interest and a right transposon arm;
• ii. a helper plasmid comprising a second antibiotic resistance gene;
• iii. a bacmid comprising third antibiotic resistance gene and a transposon insertion site located within a reporter cassette;
• (b) growing the transformed bacteria in the presence of three antibiotics to which the first, second and third antibiotic resistance genes confer resistance;
• (c) selecting at least one bacterial colony comprising a recombinant bacmid;
• (d) incubating bacteria from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counter selection against the donor plasmid;
• (e) collecting recombinant bacmids comprising a gene of interest from a bacterial colony after counter selection;
• (f) transforming insect cells with the recombinant bacmids comprising a gene of interest collected in step (e);
• (g) incubating the transformed insect cells and harvesting rBV comprising the gene of interest from the insect cells;
• (h) performing a first passage, wherein a passage comprises the steps of infecting insect cells with rBV comprising the gene of interest and incubating the infected insect cells;
• (i) harvesting rBV comprising the gene of interest from one or more insect cell plaques and identifying a plaque comprising rBV comprising a stable gene of interest;
• and
• (j) performing a second passage, wherein the second passage comprises infecting insect cells with rBV comprising a stable gene of interest, incubating the infected insect cells and harvesting a recombinant baculovirus seed stock comprising rBV comprising the stable gene of interest from the insect cells.

A method of producing rAAV comprising a gene of interest comprising the steps of:

• (a) providing a first recombinant baculovirus seed stock wherein the first recombinant baculovirus seed stock comprises rBV comprising a stable first gene of interest;
• (b) providing a second recombinant baculovirus seed stock, wherein the second recombinant baculovirus seed stock comprises rBV comprising a stable gene of interest encoding Rep/Cap proteins;
• (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection (MOI) and with the second recombinant baculovirus seed stock at a low multiplicity of infection;
• (d) adding feed to the insect cell media after co-infection;
• (e) incubating the infected insect cells;
• and
• (f) harvesting rAAV comprising the gene of interest from the insect cells.