Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (

Ref. Retrovirology 2005, 2:55
). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

Topics:
hematopoietic stem cells, hiv-1, immunologic deficiency syndromes, mice, diagnostic imaging, o(6)-methylguanine-dna methyltransferase, cytokine, infections, luciferases, polymerase chain reaction

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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