Optimization of retroviral gene transfer protocol to maintain the lymphoidpotential of progenitor cells

We have attempted to improve retrovirus-mediated gene transfer efficacy int o hematopoietic progenitor cells (HPCs) without causing them to lose their lymphoid potential. Highly purified CD34(+) cells on CH-296 fibronectin fra gments have been transduced with three different cytokine combinations. Mur ine CD2 was used as a marker gene. Transgene expression was assayed by FAGS analysis shortly after transduction of CD34(+) cells and after long-term c ulture (LTC) extended by differentiation of various lymphoid lineages: NK c ells, B cells, and dendritic cells. Compared with the historical cytokine m ix, i.e., SCF (stem cell factor) + IL-3 (interleukin 3) + IL-6, the combina tion SCF + FL (Flt-3 ligand) + M-GDF (megakaryocyte growth and differentiat ion factor) + IL-3 significantly improved the total number of viable cells and CD34+ cells after transduction and the Long term-cultured progenitors a fter 6 weeks. In addition, the combination of SCF + FL + M-GDF + IL-3 maint ained more efficiently the lymphoid potential of the progeny of transduced long term-cultured CD34+ cells, as attested by the significantly higher num ber of CD56(+), CD19(+), and CD1a(+) cells recovered when FL and M-GDF were added to SCF + IL-3, Thus, even though additional improvements may still b e needed in transduction of HPCs, these conditions were adopted for a clini cal trial of gene therapy for X-linked severe combined immunodeficiency.