Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34(+) cells

Background Clinically applicable protocols for ex vivo modification of huma n CD34(+) hematopoietic stem/progenitor cells rely on incubation of the tar get cell with supernatant containing recombinant retroviral particles. Alth ough components of the supernatant may have a profound impact on both precl inical and clinical outcome, to date supernatant production has not been pr operly addressed with regard to CD34(+) cells. We wanted to investigate and optimise production conditions for this target using simple, reproducible and clinically applicable procedures and reagents. Methods GP+Am12 under various production conditions and tested for bulk tra nsduction efficiency and endpoint titre on murine and human cell, lines. Ge ne transfer efficiency into CD34(+) cells from mobilised peripheral blood, after a single exposure to retroviral supernatant, was measured by transgen e expression, colony forming assay and long-term culture colony forming ass ay. Retroviral supernatant was obtained under various production conditions from producer and tested for Results Bulk gene transfer or endpoint titre values obtained on cell lines for the different production conditions were not predictive of gene transfe r efficiency into hematopoietic progenitors. Time of virus production appea red to have the greatest impact on gene transfer, peaking at 6 h and decrea sing 2-3-fold at longer time points. Neither the culture vessel used nor th e temperature for virus production had any significant effect on gene trans fer into CD34(+) cells. Supernatant could be produced under defined serum-f ree conditions as efficiently as serum containing conditions for CD34(+) ce ll gene transfer. Conclusions The present data provide important implications for the establi shment of quality controls for small- and large-scale clinical grade supern atant production for gene transfer into human hematopoietic stem/ progenito r cells. Copyright (C) 2001 John Wiley & Sons, Ltd.