rAAV PRODUCTION IN INSECT CELLS

1 . A system for production of recombinant bacmids with reduced residual DNA, said system comprising:

i. a donor plasmid comprising a first antibiotic resistance gene, a counter selective marker and a transfer cassette, 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.

2 .- 5 . (canceled)

6 . The system of claim 1 , wherein the counter selective marker is an antibiotic sensitive gene.

7 . The system of claim 6 , wherein the antibiotic sensitive gene is selected from the group comprising rpsl, URA 3 and thymidine kinase.

8 . The system of claim 6 , wherein the antibiotic sensitive gene is rpsl.

9 . The system of claim 1 , wherein the donor plasmid does not comprise an active gentamycin resistance gene.

10 . The system of claim 1 , wherein the transfer cassette is optimized to reduce GC rich regions in the transfer cassette.

11 . The system of claim 1 , wherein the donor plasmid comprises neither an active gentamycin resistance gene nor a GC rich region in the transfer cassette.

12 .- 17 . (canceled)

18 . The system of claim 1 , wherein the system is selected from the group comprising a modified Bac-to-Bac system, a modified baculovirus infectious cells (BIC) system and a modified Bac-plus system.

19 - 29 . (canceled)

30 . 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 (P 1 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.

31 . The method of claim 30 , wherein after the first passage at least 80% of the rBV comprise the gene of interest.

32 . (canceled)

33 . The method of claim 30 , wherein said method produces a recombinant baculovirus seed stock after two passages through insect cells, wherein the recombinant baculovirus seed stock is at least 2×10 10 vector genomes/ml (vg/ml).

34 . The method of claim 30 , wherein a bacterial colony comprising a recombinant bacmid is identified by an alteration in reporter activity.

35 .- 38 . (canceled)

40 . The method of claim 30 , wherein the first antibiotic resistance gene, the second antibiotic resistance gene and the third antibiotic resistance gene are selected from the group comprising kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, penicillin resistance genes, streptomycin resistance genes, erythromycin resistance genes and penicillin/streptomycin resistance genes.

41 . The method of claim 30 , wherein donor plasmid comprises an ampicillin resistance gene and rpsl, the helper plasmid comprises a tetracycline resistance gene and the bacmid plasmid comprises a kanamycin resistance gene.

42 . The method of claim 30 , wherein the compound that allows counter selection against the donor plasmid is an antibiotic.

43 .- 45 . (canceled)

46 . The method of claim 30 , wherein the insect cells are selected from the group comprising Sf-RVN cells, Sf 9 cells and Hi 5 cells.

47 . (canceled)

48 . The method of claim 30 , wherein the insect cells are transformed with between about 1 μg and 20 μg bacmids.

49 - 54 . (canceled)

55 . 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.

56 . (canceled)

57 . A recombinant baculovirus seed stock comprising a stable gene of interest produced by the method of claim 55 .

58 .- 95 . (canceled)

96 . 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.

97 .- 100 . (canceled)

101 . The system of claim 1 , wherein the recombinant bacmids with reduced residual DNA are for use in rAAV production.

102 . The system of claim 1 , wherein the helper plasmid is an AAV helper plasmid.