Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media
Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media
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DOI：
10.1016/j.omtm.2019.11.011
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发表时间：
2020-06-12
影响因子：
4.7
通讯作者：
Throm, Robert E.
中科院分区：
文献类型：
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作者：
Bauler, Matthew;Roberts, Jessica K.;Throm, Robert E.
慢病毒载体越来越多地用于细胞和基因治疗应用，因为它们有效地抑制靶细胞，如造血干细胞和T细胞。由于生产工艺中使用的HEK 293 T细胞的粘附性、血清依赖性，目前的药品生产质量管理规范级慢病毒载体的大规模生产受到限制。为了优化大规模临床级慢病毒载体生产，我们开发了一种改进的生产方案，通过使用市售和化学成分确定的无血清培养基使HEK 293 T细胞悬浮生长。使用优化的转染条件从SJ 293 TS细胞产生滴度与HEK 293 T细胞滴度相当的慢病毒载体，所述优化的转染条件将质粒DNA的所需量减少50%。此外，在1 L下纯化SJ 293 TS衍生的慢病毒载体在起始材料中产生55% +/-14%（n = 138）的转导单位的回收率，比来自粘附HEK 293 T血清依赖性慢病毒载体制备物的历史产量增加超过2倍。SJ 293 TS细胞在4个月的连续培养中稳定地产生慢病毒载体。SJ 293 TS衍生的慢病毒载体有效地转导来自健康供体的原代造血干细胞和T细胞。总体而言，我们的SJ 293 TS细胞系能够在无血清条件下生产高滴度载体，同时减少所需的输入DNA量，从而提供高效的生产选择。
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.