Highly productive packaging lines

Claim 2

A packaging cell line for the production of defective infectious retroviruses, wherein said packaging cell line is a human cell comprising, in its genome, at least two retroviral vectors: (a) wherein the first retroviral vector comprises, under the control of a LTR (Long Terminal Repeat) type promoter, the sequence of retroviral gag/pol genes, a mutated or normal IRES (Internal Ribosome Entry Sites) sequence, the sequence of a first selection gene, and a polyadenylation sequence; (b) wherein the second retroviral vector comprises, under the control of a promoter, the sequence of a gene coding for an envelope protein, a mutated or normal IRES sequence, the sequence of a second selection gene and a polyadenylation sequence; and (c) said first and second vectors being free of a retroviral packaging sequence.

Claim 3

The packaging cell-line according to claim 1 , wherein the first selection gene is selected from a blasticidine S resistance gene (BSR gene) and a zeomycine resistance gene (ZeoR gene).

Claim 4

A method for producing high titres of recombinant viruses in the packaging cell-lines as defined in claim 1 , comprising: (a) infecting or transfecting said cell line by a recombinant vector comprising: (a1) a gene of therapeutic interest X, under the control of a promoter; (a2) a nucleotide sequence Y, the expression of which complements a deficient function in the packaging cell; and (a3) a packaging sequence Ψ, (b) selecting said cell line in a culture medium causing cell death when the sequence Y is not expressed.

Claim 5

A packaging cell line for the production of defective infectious retroviruses, wherein said packaging cell line is a human cell comprising, in its genome, at least a first and a second viral constructs, said first viral construct comprising, operably linked, a promoter, the sequence of retroviral gag/pol genes, a mutated or normal IRES (Internal Ribosome Entry Sites) sequence, the sequence of a first selection gene and a polyadenylation sequence, said second viral construct comprising, operably linked, a promoter, the sequence of a gene coding for an envelope protein, a mutated or normal IRES sequence, the sequence of a second selection gene and a polyadenylation sequence, said first and second constructs being free of a retroviral packaging sequence.

Claim 6

A packaging cell line for the production of defective infectious retroviruses, wherein said packaging cell line is a human cell comprising, in its genome, at least a first and a second viral constructs, said first viral construct comprising, operably linked, a promoter, the sequence of retroviral gag/pol genes, a mutated or normal IRES (Internal Ribosome Entry Sites) sequence, the sequence of a first selection gene and a polyadenylation sequence, said second viral construct comprising, operably linked, a promoter, the sequence of a gene coding for an envelope protein, a mutated or normal IRES sequence, the sequence of a second selection gene and a polyadenylation sequence, said first and second constructs being free of a retroviral packaging sequence.

Claim 7

The cell line of claim 4 , wherein the envelope protein encoded by said second viral construct is a 4070A envelope protein.

Claim 8

The cell of claim 4 , wherein the IRES sequence in said first and second viral constructs is a mutated IRES sequence.

Claim 9

A packaging cell line for the production of defective infectious viruses, wherein said packaging cell line is a human cell comprising, in its genome, at least a first a second viral constructs, said first viral construct comprising, operably linked, a retroviral LTR (Long Terminal Repeat) type promoter sequence, the sequence of retroviral gag/pol genes, a mutated or normal IRES (Internal Ribosome Entry Sites) sequence, the sequence of a first selection gene selected from the group consisting of a blasticidine S resistance gene (BSR gene) and a zeomycine resistance gene (ZeoR gene), and a polyadenylation sequence, said second viral construct comprising, operably linked, a cytomegalovirus (CMV) promoter, the sequence of a gene coding for an envelope protein, a mutated or normal IRES sequence, the sequence of a phleomycine resistance gene and a polyadenylation sequence, said first and second constructs being free of a retroviral packaging sequence.

Claim 10

A packaging cell line for the production of defective infectious viruses, wherein said packaging cell line is a human cell comprising, in its genome, at least a first a second viral constructs, said first viral construct comprising, operably linked, a retroviral LTR (Long Terminal Repeat) type promoter sequence, the sequence of retroviral gag/pol genes, a mutated or normal IRES (Internal Ribosome Entry Sites) sequence, the sequence of a first selection gene selected from the group consisting of a blasticidine S resistance gene (BSR gene) and a zeomycine resistance gene (ZeoR gene), and a polyadenylation sequence, said second viral construct comprising, operably linked, a cytomegalovirus (CMV) promoter, the sequence of a gene coding for an envelope protein, a mutated or normal IRES sequence, the sequence of a phleomycine resistance gene and a polyadenylation sequence, said first and second constructs being free of a retroviral packaging sequence.

Claim 11

The cell line of claim 7 , wherein the envelope protein encoded by said second viral construct is a 4070A envelope protein.

Claim 12

The cell line of claim 7 , wherein the IRES sequence in said first and second viral constructs is a mutated IRES sequence.

Claim 13

The cell line of claim 7 , wherein the LTR type promoter sequence in said first viral constructs is the LTR of a FB29 Friend virus.

Claim 14

A method of producing recombinant retroviruses, comprising infecting or transfecting a packaging cell line of claim 4 with a recombinant retroviral vector and collecting the recombinant retroviruses produced.