Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

中文翻译：

---

使用在无血清培养基中生长的悬浮细胞生产慢病毒载体。

慢病毒载体越来越多地用于细胞和基因治疗应用，因为它们有效地转导了造血干细胞和T细胞等靶细胞。由于在生产过程中使用的HEK293T细胞具有粘附性，血清依赖性，因此目前大规模生产良好生产规范级慢病毒载体的能力受到限制。为了优化大规模临床级慢病毒载体的生产，我们通过使用市场上可买到的和化学成分明确的无血清培养基使HEK293T细胞在悬浮液中生长，从而开发了一种改进的生产方案。使用优化的转染条件，从SJ293TS细胞中产生具有与HEK293T细胞相同效价的慢病毒载体，从而将所需的质粒DNA量减少了50％。此外，以1 L纯化SJ293TS衍生的慢病毒载体可回收起始材料中55％±14％（n = 138）的转导单位，比粘附的HEK293T血清依赖性慢病毒载体的历史产量高出2倍以上准备。在连续培养4个月后，SJ293TS细胞可稳定产生慢病毒载体。SJ293TS衍生的慢病毒载体可有效转导健康捐献者的原代造血干细胞和T细胞。总体而言，我们的SJ293TS细胞系可在无血清条件下进行高滴度载体生产，同时减少所需的输入DNA量，从而提供了高效的生产选择。依附的HEK293T血清依赖性慢病毒载体制剂的历史产量比历史产量增加了2倍以上。在连续培养4个月后，SJ293TS细胞可稳定产生慢病毒载体。SJ293TS衍生的慢病毒载体可有效转导健康捐献者的原代造血干细胞和T细胞。总体而言，我们的SJ293TS细胞系可在无血清条件下进行高滴度载体生产，同时减少所需的输入DNA量，从而提供了高效的生产选择。依附的HEK293T血清依赖性慢病毒载体制剂的历史产量比历史产量增加了2倍以上。在连续培养4个月后，SJ293TS细胞可稳定产生慢病毒载体。SJ293TS衍生的慢病毒载体可有效转导健康捐献者的原代造血干细胞和T细胞。总体而言，我们的SJ293TS细胞系可在无血清条件下进行高滴度载体生产，同时减少所需的输入DNA量，从而提供了高效的生产选择。