Optimization of retroviral vector generation for clinical application

Background For many inherited and acquired diseases of the blood system, gene transfer into hematopoietic cells is a promising strategy to alleviate disease‐related symptoms or even correct genetic alterations. In clinical gene therapy applications, low transduction efficiencies have been a major limitation mainly because of insufficient effective titers of the retroviral supernatants used. Thus, optimization of clinical‐grade vector production under current ‘Good Manufacturing Practice’ (GMP) conditions is a prerequisite for successful gene therapy trials. Methods We established stable retroviral producer clones with single integrations of a retroviral vector encoding for the multidrug‐resistance gene 1 ( MDR1 ). Optimization of vector production in multi‐tray cell factories (MTCFs) 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 be increased up to four‐fold. Plating optimal cell densities of 1×10 4 cells/cm 2 , repetitive harvests of vector supernatant were feasible over four consecutive days. Combining the most advantageous culture and harvest parameters tested, we were able to produce large quantities of serum‐free vector supernatant in 40‐tray MTCFs. Highly efficient gene transfer into primary human CD34 + progenitor cells demonstrated the quality of these vector stocks. Conclusion The large‐scale vector‐production protocol in MTCFs described here is easy to handle, is applicable to a wide range of adherent producer cell lines and, most importantly, complies with current GMP guidelines. Copyright © 2001 John Wiley & Sons, Ltd.