Recombinant retroviruses with amphotropic and ecotropic host ranges

Claim 1

A stable retroviral packaging cell line which generates helper-free recombinant retrovirus with an amphotropic host range, said stable retroviral packaging cell line comprising packaging cells, wherein the retrovirally derived component of said packaging cells' genome comprises stably incorporated first and second replication-defective retroviral constructs, wherein said first and second replication-defective retroviral constructs: 1) encode and express mutually exclusive regions of the gag, pol, and env genes; 2) were separately and sequentially introduced into the genome of said packaging cells; and wherein the incorporated env gene is derived from an amphotropic virus.

Claim 9

A method of using the packaging cell line according to any one of claims 1, 2, 3, or 4, comprising the step of producing amphotropic retrovirus in said cell line.

Claim 13

A method of making a stable retroviral packaging cell line which generates helper-free recombinant retrovirus with an amphotropic host range, comprising the steps of: a) transfecting mammalian host cells with a first replication-defective retroviral construct, the genome of which comprises: i) sequences derived from the 5' and 3' LTRs of a first retrovirus of interest; (ii) a defective Psi packaging region; (iii) a functional form of at least one region selected from the group consisting of gag and pol; (iv) a defective env region; and (v) a eucaryotic promoter for expressing the functional retroviral genes; b) culturing the transfected cells of step a); c) transfecting the cells cultured in step b) with a second replication-defective retroviral construct, the genome of which comprises: (i) sequences derived from the 5' and 3' LTRs of a second retrovirus of interest; (ii) a defective Psi packaging region; (iii) a functional amphotropic env gene; (iv) defective gag and/or pol regions which corresponds to the gag and/or pol regions which are not defective in the first retroviral construct; and d) culturing the cells transfected in step c) whereby a packaging cell comprising complete but physically separated gag/pol and env coding regions is produced.

Claim 14

A method of making a stable retroviral packaging cell line which generates helper-free recombinant retrovirus with an amphotropic host range, comprising the steps of: a) transfecting mammalian host cells with a first replication-defective retroviral construct, the genome of which comprises: i) sequences derived from the 5' and 3' LTRs of a first retrovirus of interest; (ii) a defective Psi packaging region; (iii) a functional form of at least one region selected from the group consisting of gag and pol; (iv) a defective env region; and (v) a eucaryotic promoter for expressing the functional retroviral genes; b) culturing the transfected cells of step a); c) transfecting the cells cultured in step b) with a second replication-defective retroviral construct, the genome of which comprises: (i) sequences derived from the 5' and 3' LTRs of a second retrovirus of interest; (ii) a defective Psi packaging region; (iii) a functional amphotropic env gene; (iv) defective gag and/or pol regions which corresponds to the gag and/or pol regions which are not defective in the first retroviral construct; and d) culturing the cells transfected in step c) whereby a packaging cell comprising complete but physically separated gag/pol and env coding regions is produced.