Schilz A J, Kühlcke K, Fauser A A, Eckert H G
EUFETS AG, Idar-Oberstein, Germany.
J Gene Med. 2001 Sep-Oct;3(5):427-36. doi: 10.1002/jgm.204.
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.
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.
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.
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.
对于许多血液系统的遗传性和获得性疾病，将基因导入造血细胞是缓解疾病相关症状甚至纠正基因改变的一种有前景的策略。在临床基因治疗应用中，低转导效率一直是一个主要限制因素，主要原因是所用逆转录病毒上清液的有效滴度不足。因此，在当前“良好生产规范”（GMP）条件下优化临床级载体生产是基因治疗试验成功的先决条件。
我们建立了稳定的逆转录病毒生产克隆，其单个整合了编码多药耐药基因1（MDR1）的逆转录病毒载体。针对收获培养基、细胞密度和收获时间点，研究了多盘细胞工厂（MTCF）中载体生产的优化。
我们证明可以在无血清培养基中生产高滴度载体储备液。通过减少收获培养基的体积，滴度可提高至四倍。接种最佳细胞密度为1×10⁴个细胞/cm²，连续四天重复收获载体上清液是可行的。结合测试的最有利的培养和收获参数，我们能够在40盘MTCF中大量生产无血清载体上清液。高效基因转移到原代人CD34⁺祖细胞中证明了这些载体储备液的质量。
本文所述的MTCF中大规模载体生产方案易于操作，适用于多种贴壁生产细胞系，最重要的是，符合当前GMP指南。
