Stable Production of Lentiviral Vectors

Abstract

The present invention provides new stable packaging cell lines and producer cell lines as well as methods to obtain them, and a new method to produce lentiviral vectors using such cell lines. New methods and packaging cell lines of the invention are generated using a baculo-AAV hybrid system for stable expression of structural and regulatory lentiviral proteins, such system comprising a baculoviral backbone containing an integration cassette flanked by AAV ITR, in combination with a plasmid encoding rep protein. This system allows to obtain a stable integration of the structural and regulatory HIV-1 proteins gag/pol and rev. The system allows to obtain a first intermediate including only the structural and regulatory HIV proteins gag/pol and rev, to be used as starting point to obtain stable packaging cell lines as well as producer cell lines.

1 - 21 . (canceled) 
     
     
         22 . A producer cell line containing stably integrated into its genome:
 i. at least one copy of an integration cassette flanked by baculo-adeno associated virus (AAV) inverted terminal repeats (ITRS) including two expression cassettes, wherein the first expression cassette encodes lentiviral gag and pol genes and the second expression cassette encodes a lentiviral rev gene and a selection marker;   ii. at least one copy of an env gene; and   iii. a transfer vector.   
     
     
         23 . A producer cell line according to  claim 22 , wherein the producer cell line further contains at least one copy of a lentiviral tat gene stably integrated into its genome. 
     
     
         24 . A producer cell line according to  claim 22  or  23 , wherein the producer cell line is a human cell line selected from the group consisting of HEK293, HEK293-T, HEK293-SF, TE671, HT1080 and HeLa. 
     
     
         25 . A producer cell line according to  claim 22 , wherein the two expression cassettes are tail-to-tail oriented and each one is driven by a constitutive promoter and a poly A. 
     
     
         26 . A producer cell line according to  claim 25 , wherein the constitutive promoter is selected from the group consisting of CMV, CMV IE, PGK, SV40, eF1α, SFFV, and RSV. 
     
     
         27 . A producer cell line according to  claim 22 , wherein the selection marker is selected from the group consisting of hygromycin, kanamycin, neomycin and zeomycin resistance genes. 
     
     
         28 . A producer cell line according to  claim 22 , wherein the env gene is selected from the group consisting of MLV 4070 env, RD114 env, chimeric envelope protein RD114-TR, chimeric envelope protein RD114-pro, baculovirus GP64 env and GALV env, or derivatives thereof. 
     
     
         29 . A producer cell line according to  claim 22 , wherein the env gene is the gene encoding the chimeric envelope protein RD114-TR. 
     
     
         30 . (canceled) 
     
     
         31 . A producer cell line according to  claim 24 , wherein the human cell line is HEK293-T. 
     
     
         32 . A producer cell line according to  claim 26 , wherein the constitutive promoter is CMV IE. 
     
     
         33 . A producer cell line according to  claim 22 , wherein the selection marker is hygromycin. 
     
     
         34 . A producer cell line according to  claim 22 , wherein the selection marker is cloned downstream of an internal ribosome entry site (IRES). 
     
     
         35 . A producer cell line according to  claim 26 , wherein the constitutive promoters are different. 
     
     
         36 . A producer cell line according to  claim 23 , wherein the tat gene is HIV-1 tat.