High-efficiency transduction and long-term gene expression with a murine stem cell retroviral vector encoding the green fluorescent protein in human marrow stromal cells

Bone marrow stromal cells (MSCs) are unique mesenchymal cells that have bee n utilized as vehicles for the delivery of therapeutic proteins in gene the rapy protocols. However, there are several unresolved issues regarding thei r potential therapeutic applications, These include low transduction effici ency, attenuation of transgene expression, and the technical problems assoc iated with drug-based selection markers, To address these issues, we have d eveloped a transduction protocol that yields high-level gene transfer into human MSCs, employing a murine stem cell virus-based bicistronic vector con taining the green fluorescent protein (GFP) gene as a selectable marker, Tr ansduction of MSCs plated at low density for 6 hr per day for 3 days with h igh-titer viral supernatant resulted in a gene transfer efficiency of 80 +/ - 6% (n = 10) as measured by GFP fluorescence. Neither centrifugation nor p hosphate depletion increased transduction efficiency, Assessment of amphotr opic receptor (Pit-2) expression by RT-PCR demonstrated that all MSCs expre ssing the receptor were successfully transduced. Cell cycle distribution pr ofiles measured by propidium iodide staining showed no correlation with the susceptibility of MSCs to transduction by the retroviral vector, Human MSC s sequentially transduced with an adenoviral vector encoding the ecotropic receptor and ecotropic retroviral vector encoding GFP demonstrated that all MSCs are susceptible to retroviral transduction, We further showed that bo th genes of bicistronic vector are expressed for at least 6 months in vitro and that transgene expression did not affect the growth or osteogenic diff erentiation potential of MSCs. Future studies will be directed toward the d evelopment of gene therapy protocols employing this strategy.