Optimization of retroviral vector generation for clinical application

Background For many inherited and acquired diseases of the blood system, ge ne transfer into hematopoietic cells is a promising strategy to alleviate d isease-related symptoms or even correct genetic alterations. In clinical ge ne therapy applications, low transduction efficiencies have been a major,, limitation mainly because of insufficient effective titers of the retrovira l supernatants used. Thus, optimization of clinical-grade vector production under current 'Good Manufacturing Practice' (GMP) conditions is a prerequi site for successful gene therapy trials. Methods We established stable retroviral producer clones with single integr ations of a retroviral vector encoding for the multidrug-resistance gene 1 (MDR1). Optimization of vector production in multi-tray cell factories (MTC Fs) was studied with particular regard to harvest medium, cell density and harvest time point. Results We demonstrated that high-titer vector stocks could be produced in serum-free medium. By reducing the volume of harvest medium, titers could b e increased up to four-fold. Plating optimal cell densities of I X 10(4) ce lls/cm(2), repetitive harvests of vector supernatant were feasible over fou r consecutive days. Combining the most advantageous culture and harvest par ameters tested, we were able to produce large quantities of serum-free vect or supernatant in 40-tray MTCFs. Highly efficient gene transfer into primar y human CD34(+) progenitor cells demonstrated the quality of these vector s tocks. Conclusion The large-scale vector-production protocol in MTCFs described he re is easy to handle, is applicable to a wide range of adherent producer ce ll lines and, most importantly, complies with current GMP guidelines. Copyr ight (C) 2001 John Wiley & Sons, Ltd.