Development and use of new biological tools based on the use of the Cre/loxP recombination system to produce non-cytotoxic amplicon vectors derived from Herpes Simplex virus type I

Amplicon vectors, derived from herpes simplex virus type I, are a tool of choice for gene transfer. They allow very large DNA to be transported into many cell types without this DNA integrating into the cellular genome. These vectors are defective herpes particles whose genome is a concatemer of plasmid units, whose only viral sequences are the cis signals necessary for its replication and encapsidation. The functions necessary in trans for their production are provided by a helper virus or genome. The result is a viral population consisting of amplicon vectors and helper viruses. The presence of a large number of helper viruses, even defective ones, made these vectors difficult to use in gene therapy. To address this problem, we constructed a helper virus, HSV-1 LaL J, which is defective and whose cis sequences essential for its cleavage-encapsidation, framed by two loxP sites in parallel orientation, are specifically deleted by the Cre recombinase. In parallel, we established a transcomplementing line allowing the production of this virus, as well as a transcomplementing cell line expressing the Cre recombinase, which allows the expression of the HSV-1 LaL J virus, but inhibits its encapsidation. Also, the proteins necessary for the production of amplicon vectors are still synthesized, but the helper virus is no longer produced. We then used the HSV-1 LaL J virus and these new cell lines to produce amplicon vectors. We have thus shown that high titres of amplicon vectors, whose contamination rate by a non-pathogenic helper virus is very low, could be easily obtained. The vectors thus produced are non-cytotoxic for infected cells. Finally, we studied, in vitro, the expression of these vectors.