732. Avian Sarcoma Leukosis Virus Vectors Can Transduce Rhesus Macaque HSCs and Result in a Potentially Safer Integration Profile

Abstract

Retroviral-induced insertional mutagenesis has generated renewed concern due to the occurrence of three cases of T cell lymphoproliferative disease in the otherwise successful French SCID-X1 trial, as well as increasing anecdotal reports documenting oncogenesis after murine retroviral or lentiviral vector based gene transfer. Several genome-wide integration site analyses indicate MLV and HIV based vectors integrate preferentially into regions adjacent to or within human RefSeq genes, thus increasing the possibility of insertional mutagenesis. Intriguingly, the avian sarcoma leukosis virus (ASLV) has been reported to have an unbiased integration pattern in cell lines, but this vector system has not previously been investigated for transduction of repopulating hematopoietic cells. We investigated the efficiency and integration profile of this vector in the clinically-relevant rhesus macaque autologous transplantation model. In this study, ASLV vectors pseduotyped with an amphotropic envelope and expressing marker genes were produced by transfection of vector DNA into the chicken cell line, DF-1. The resulting RCAS (Replication Competent, ALV LTR with A Splice acceptor) vector is able to stably infect human and non-human primate cells but is not replication-competent in these non-avian cells. We obtained vector titers (measured on human HEK-293 cells) in the range of 2 to 9 |[times]| 10E6 cfu/ml for vectors carrying either GFP or a neomycin-resistance marker gene. These ASLV vectors can efficiently and stably transduce rhesus macaque as well as human CD34+ hematopoietic progenitor cells ex vivo at levels of 33% and 40%, respectively. We transplanted two rhesus macaques with ASLV-transduced autologous CD34+ cells. In both animals, a polyclonal hematopoietic reconstitution with sustained gene marking levels in myeloid and lymphoid lineages was observed. Using an optimized linear-amplification mediated PCR (LAM-PCR), we have thus far identified 121 pre-transplant RISs and 199 post-transplant RISs, of which 29.8% and 32.8% were within RefSeq gene coding regions, respectively. These percentages are significantly lower than MLV and HIV based vectors observed previously in our study and similar to those expected for random integration. A comparison of pre-post-transplant RISs revealed no obvious impact of in vivo engraftment on the retroviral integration profile at early time points after transplantation, but followup continues. We report for the first time that a novel vector system of avian origin can be successfully applied in a large animal autologous transplantation model. This vector system is efficient and flexible, with a desirable integration profile compared to standard MLV or lentiviral vectors. Together with our previous findings showing distinctive integration pattern for MLV and HIV based vectors, these results further reinforce the idea that specific integration patterns of different retroviruses may arise because the different integration complexes bind to distinct cellular components.