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 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.


CONCLUSIONS
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.
Schilz
A J
AJ
EUFETS AG, Idar-Oberstein, Germany.
Kühlcke
K
K
Fauser
A A
AA
Eckert
H G
HG
D018952:Antigens, CD34 / Q000032:analysis; D001709:Biotechnology; D002460:Cell Line; D016895:Culture Media, Serum-Free; D019450:Genes, MDR; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D012190:Retroviridae / Q000235:genetics*
D018952:Antigens, CD34; D016895:Culture Media, Serum-Free