Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

Abstract

Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1␣ (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro, as well as transgene amplification and persistence in vivo. Retrovirus titers of Ͼ10 5 infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10-to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin.

In situ generation of pseudotyped retroviral progeny by adenovirus‐mediated transduction of tumor cells enhances the killing effect of HSV‐tk suicide gene therapy in vitro and in vivo

“…Initial studies of the co-transduction of adeno-retroviral hybrid vectors showed that transient retrovirus-producing cells can be successfully generated and that these could subsequently cause the transduction of neighboring cells [3,7]. Torrent et al [9] also reported a study with a chimeric vector system that resulted in 10-to 50-fold transgene amplification in vivo, although they did not examine the distribution of the transgene or the therapeutic efficacy of their system. In the study reported here, we have altered these hybrid adeno-retroviral vector systems so that the tumor-killing effect of adenovirus-mediated HSV-tk/GCV therapy in situ is enhanced.…”

“…We and others have previously described hybrid vector systems that use adenoviral vectors to deliver retroviral vector and packaging proteins into cells [3][4][5][6][7][8][9]. These systems benefit from the efficient gene transfer characteristics of adenoviral vectors as well as from the stable and long-term gene expression that is typical of retroviral vectors.…”