Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34(+) cells emphasize limited transduction at low receptor levels

Successful gene therapy for the treatment of heritable or acquired diseases typically requires high efficiency gene transfer and sustained transgene e xpression. Indirect evidence on the basis of RNA analysis and in vivo compe titive repopulation experiments in animal models suggests a correlation bet ween transduction efficiency and the abundance of retrovirus receptors on t he hematopoietic target cell. However, transduction by oncoretroviral vecto rs is also subject to other factors such as target cell cycle status and th e composition of the virus-containing medium, making it difficult to determ ine the level of receptor expression required for efficient transduction. I n the present study we investigated the impact of receptor expression level on transduction by a vector with a gibbon ape leukemia virus (GALV) envelo pe protein in a tetracycline-inducible tissue culture model that allowed fo r the cell cycle-independent regulated expression of the GALV receptor (Pit 1) in otherwise non-susceptible NIH 3T3 cells. Up-regulation of receptor RN A expression by 4.5-fold resulted in a mean 150-fold increase in transducti on efficiency. We then analyzed cell surface expression of the Pit1 recepto r using a fusion protein consisting of GALV SU portion of the viral envelop e protein linked to the human IgG Fc. These experiments showed that tetracy cline-regulated receptor induction resulted in a dose-dependent increase in binding of fusion protein. At maximum induction fusion protein binding inc reased up to five-fold which paralleled the increase in RNA expression, and correlated with the improved transduction efficiency. Finally, studies of pseudotype-specific fusion protein binding to human CD34-enriched cells rev ealed increased expression of retrovirus receptors after cytokine stimulati on, although overall receptor expression in CD34(+) cells remained lower th an in fibroblast cell lines efficiently transduced by amphotropic and GALV vectors.