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 x 10(4) cells/cm2, 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.
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 x 10(4) cells/cm2, 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.
Authors: Hanno Niess; Jobst C von Einem; Michael N Thomas; Marlies Michl; Martin K Angele; Ralf Huss; Christine Günther; Peter J Nelson; Christiane J Bruns; Volker Heinemann Journal: BMC Cancer Date: 2015-04-08 Impact factor: 4.430