A NEW SYSTEM FOR STRINGENT, HIGH-TITER VESICULAR STOMATITIS-VIRUS-G PROTEIN-PSEUDOTYPED RETROVIRUS VECTOR INDUCTION BY INTRODUCTION OF CRE RECOMBINASE INTO STABLE PREPACKAGING CELL-LINES

We report here on stable prepackaging cell lines which can be converte d into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction o f Cre recombinase-expressing adenovirus. The generated prepackaging ce ll lines constitutively express the gag-pol genes and contain an induc ible transcriptional unit for the VSV-G gene. From this unit, the intr oduced Cre recombinase excised both a neomycin resistance (Neo(r)) gen e and a poly(A) signal banked by a tandem pair of laxP sequences and i nduced transcription of the VSV-G gene from the same promoter as had b een used for Neo(r) expression. By inserting an mRNA-destabilizing sig nal into the 3' untranslated region of the Neo(r) gene to reduce the a mount of Neo(r) transcript, we were able efficiently to select the clo nes capable of inducing VSV-G at high levels, Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any de tectable infectious virus at all, even after the transduction of a mur ine leukemia virus-based retrovirus vector encoding beta-galactosidase . They reproducibly produced high-titer virus stocks of VSV-G-pseudoty ped retrovirus (1.0 x 10(6) infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G- producing cells are still fully susceptible to transduction by VSV-G p seudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized, We further show that heparin significantly inhibits retransduction of VSV-G pseu dotypes in the culture fluids of packaging cell lines, leading to a tw o-to fourfold increase in the yield of the pseudotypes after induction , This vector-producing system was very stable and should be advantage ous in human gene therapy.