Lemoine François M, Mesel-Lemoine Mariana, Cherai Mustapha, Gallot Géraldine, Vié Henri, Leclercq Virginie, Trébèden-Negre Hélène, Mammès Olivier, Boyer Olivier, Noguiez-Hellin Patricia, Klatzmann David
UPMC/CNRS UMR 7087, CERVI, Hôpital Pitié-Salpêtrière, 83 bd de l'hôpital, 75651 Paris cedex 13, France.
J Gene Med. 2004 Apr;6(4):374-86. doi: 10.1002/jgm.538.
T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells).
To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells.
Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions.
GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.
表达单纯疱疹病毒1型胸苷激酶（HSV1-TK）自杀基因的T细胞可用于控制异基因干细胞移植后的移植物抗宿主病。为了开展基于该策略的临床试验，我们按照药品生产质量管理规范（GMP）构建了一种新型的“分裂基因组”人包装细胞系（1704细胞）。
为了将产生复制能力的逆转录病毒的风险降至最低，对pol进行截短以去除与env重叠的序列。为了提高逆转录病毒感染及转导T细胞的筛选效率，1704细胞生产了高滴度的携带双顺反子Thy1-IRES-TK载体的GALV假型逆转录病毒颗粒，该载体编码CD90糖基磷脂酰肌醇（GPI）锚定膜分子。
使用1704细胞的上清液和优化的转导方案，约50%的原代T细胞被转导，然后可使用针对CD90的临床级免疫磁珠进行纯化（约95%）。超过96%的这些经OKT3/IL-2激活并经CD90选择的T细胞被更昔洛韦杀死。转导T细胞的细胞增殖、细胞因子产生以及转导T细胞克隆的HLA限制性细胞毒性与在相同条件下培养的未转导对应细胞相同。
携带双顺反子Thy1-IRES-TK载体的GALV假型逆转录病毒颗粒，在使用新型人1704包装细胞系且适用于临床试验的条件下，可实现人T细胞的高效转导和快速筛选。
