A self-inactivating ASLV-based vector system with a clinically applicable split-packaging design for human gene therapy

Accidental insertional activation of proto-oncogenes poses a serious challenge to human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviruses have elucidated distinct target site preferences, highlighting Avian Sarcoma and Leukosis Virus (ASLV) as the retrovirus with the most neutral integration spectrum. In order to reduce vector genotoxicity, changing from currently used vectors based on MLV (gammaretroviral) or HIV-1 (lentiviral) to ASLV (alpharetroviral) would therefore be of potential therapeutic value. However, usage of ASLV vectors for human gene therapy is limited by the lack of appropriate self-inactivating (SIN) vectors and split packaging systems for particle production in mammalian cells. Even though ASLV is not known to replicate in human cells, the presence of coding sequences and intact LTRs in viral particles is not acceptable for clinical applications. We have successfully developed a SIN ASLV-based vector system with a clinically applicable split-packaging design, encoding vector, gag-pol, and env on separate plasmids. Titers in the range of 1 x 10e6 t.u./mL could be obtained by cotransfection in human 293T cells. Codon-optimizing parts of the gag-pol sequence, we were able to improve viral particle titers by at least one order of magnitude. Our newly developed alpharetroviral SIN vectors allowed efficient expression of clinically relevant transgenes such as gp91phox, and were also successfully used to generate murine iPS cells. Furthermore, we efficiently transduced murine lineage-negative cells and human CD34-positive cells, thereby confirming the promising potential of this vector system for gene therapeutic approaches.