Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells
Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells
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DOI：
10.1016/j.omtm.2019.05.013
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发表时间：
2019-09-13
影响因子：
4.7
通讯作者：
Caruso, Manuel
中科院分区：
文献类型：
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作者：
Ghani, Karim;Boivin-Welch, Michael;Caruso, Manuel
γ-逆转录病毒载体是基因治疗中常用的基因递送载体。尽管它的疗效，其强大的增强子有助于在一些造血干细胞（HSC）基因治疗试验的恶性转化。没有病毒增强剂（SIN）的更安全的版本是可用的，但其生产是繁琐的，因为高滴度只能在瞬时转染中获得。我们的目标是开发一个系统，可以很容易地产生高滴度SIN载体从稳定的生产细胞。使用巨细胞病毒增强子-启动子序列产生与减少转录通读的序列（WPRE和强聚腺苷酸化序列）组合的全长基因组RNA，导致瞬时转染中6 × 10（6）感染单位（IU）/mL的SIN GFP载体。将杀稻瘟菌素选择盒掺入逆转录病毒质粒允许在293 Vec包装细胞中产生稳定的克隆，其分别释放2 × 10（7）IU/mL和1.4 × 10（7）IU/mL的SIN GFP和SIN PIGA载体。使用含有长8.9-kb COL 7A 1 cDNA的SIN载体获得1.8 x 10（6）IU/mL的滴度。因此，建立了用于产生稳定的293 Vec衍生的逆转录病毒生产细胞的有效方法，所述逆转录病毒生产细胞释放高滴度SIN载体。
The gamma-retroviral vector is a gene delivery vehicle that is commonly used in gene therapy. Despite its efficacy, its strong enhancers contributed to malignant transformations in some hematopoietic stem cell (HSC) gene therapy trials. A safer version without viral enhancers (SIN) is available, but its production is cumbersome, as high titers can only be obtained in transient transfection. Our aim was to develop a system that could easily generate high-titer SIN vectors from stable producer cells. The use of the cytomegalovirus enhancer-promoter sequence to generate the full-length genomic RNA combined to sequences that decrease transcriptional readthrough (WPRE and strong polyadenylation sequences) led to 6 x 10(6) infectious units (IU)/mL of a SIN GFP vector in transient transfection. The incorporation of a blasticidin selection cassette to the retroviral plasmid allowed the generation of stable clones in the 293Vec packaging cells that release 2 x 10(7) IU/mL and 1.4 x 10(7) IU/mL of a SIN GFP and a SIN PIGA vector, respectively. A titer of 1.8 x 10(6) IU/mL was obtained with a SIN vector containing the long 8.9-kb COL7A1 cDNA. Thus, an efficient process was established for the generation of stable 293Vec-derived retrovirus producer cells that release high-titer SIN vectors.