478. A HIV-1 Based Cross-Packaging System for FIV Vectors

Abstract

Top of pageAbstract FIV lentiviral vectors are surfacing as a common tool for stable transgene delivery in vitro and in vivo. Genetic structural and enzymatic elements inherent to FIV vectors were derived from a parental strain of FIV, which has presumably adapted to be most efficient in the feline host. Despite the successful employment of these vectors for stable transgene delivery in small animal models utilization of FIV vectors in large preclinical animal models such as non-human primates, and eventually clinical studies in humans, remains dubious. Anticipating a need for a safe FIV vector that may overcome species-specific impediments our lab has developed a novel HIV-1 based cross-packaging system that supports packaging, integration and stable expression of FIV vectors. Pertinent to the generation of the cross-packaging system was the incorporation of a small HIV-1 genetic fragment, comprising the RRE, into a FIV vector. Consequently, stable GFP expression was achieved in 293T cells after several cell passages. FIV vectors lacking the HIV-1 RRE were not efficiently cross-packaged with the HIV-1 packaging system as observed by decreased levels of GFP expression and fewer transduced 293T cells. The HIV-1 cross-packaged FIV vectors yielded titers equivalent to those obtained when packaged with a FIV packaging system (>106 IU/mL) on 293T cells. Furthermore, the cross-packaged FIV vectors exhibited transduction efficiencies comparable to standard HIV-1 vectors on primary human epithelial mammary cells. Employing the HIV-1 cross-packaged FIV vectors for testing in preclinical animal models will require reproducible large-scale vector production. To this end we developed a tetracycline-dependent packaging cell line containing the vesicular stomatitis virus glycoprotein (VSV-G) envelope and the HIV-1 packaging cassette to cross-package a stably incorporated conditional self-inactivating (cSIN) FIV vector. The cSIN FIV vector was created by introducing the tetracycline-inducible promoter into the U3 region of the 3' LTR. The stable packaging cell line is currently undergoing characterization. Through ongoing in vitro and in vivo studies we are exploring the potential attributes of cross-packaged FIV vectors.