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.