INSERTION OF 2 INDEPENDENT ENHANCERS IN THE LONG TERMINAL REPEAT OF ASELF-INACTIVATING VECTOR RESULTS IN HIGH-TITER RETROVIRAL VECTORS WITH TISSUE-SPECIFIC EXPRESSION

The use of retroviral vectors (RVs) derived from the murine oncoretrov iruses for gene therapy is associated with the risk of malignant trans formation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would incre ase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette contain ing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resultin g retroviral vectors could be transcriptionally trans-activated by tax . In the absence of Tax, however, the viruses showed muscle-specific e xpression. Trans-complementing packaging and indicator cells stably ex pressing Tax were used to isolate high-titer producer cell clones (10( 6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-exp ressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expre ssion of the vectors and the endogenous muscle creatine kinase gene wa s similarly dependent on the maturity of the muscle cultures. One vect or with modified LTRs was also tested in vivo in regenerating muscle a nd showed a delayed pattern of expression in myofibers compared with t he vector containing the wild-type LTRs, These vectors can be easily m odified to contain different tissue-specific enhancer and promoter ele ments and the availability of complementing packaging and indicator ce lls expressing Tax should allow their application in a variety of gene therapy settings.