Clinic for Stem Cell Transplantation, Research Department of Cell and Gene Therapy, University Medical Centre Hamburg-Eppendorf , 20246 Hamburg, Germany.
Hum Gene Ther. 2010 Aug;21(8):929-41. doi: 10.1089/hum.2009.042.
Conditional elimination of infused gene-modified alloreactive T cells, using suicide gene activation, has been shown to be an efficient strategy to abrogate severe graft-versus-host disease (GvHD) in the context of adoptive immunotherapy. To overcome shortcomings of the most widely used suicide gene, wild-type (splice-corrected) herpes simplex virus thymidine kinase (scHSVtk), we generated two new variants: the codon-optimized coHSVtk and, by introducing an additional mutation (A168H), the novel TK.007. We transduced human hematopoietic cell lines and primary T cells with retroviral "sort-suicide vectors" encoding combinations of selection markers (tCD34 and OuaSelect) with one of three HSVtk variants. In vitro we observed higher expression levels and sustained long-term expression of TK.007, indicating lower nonspecific toxicity. Also, we noted significantly improved kinetics of ganciclovir (GCV)-mediated killing for TK.007-transduced cells. In an experimental (murine) allogeneic transplantation model, TK.007-transduced T cells mediated severe GvHD, which was readily abrogated by application of GCV (10 mg/kg). Last, we established a modified allotransplantation model that allowed quantitative comparison of the in vivo activities of TK.007 versus scHSVtk. We found that TK.007 mediates both significantly faster and higher absolute killing at low GCV concentrations (10 and 25 mg/kg). In summary, we demonstrate that the novel TK.007 suicide gene combines better killing performance with reduced nonspecific toxicity (as compared with the frequently used splice-corrected wild-type scHSVtk gene), thus representing a promising alternative for suicide gene therapy.
利用自杀基因激活对输注的基因修饰同种异体反应性T细胞进行条件性清除，已被证明是在过继性免疫治疗背景下消除严重移植物抗宿主病（GvHD）的有效策略。为了克服最广泛使用的自杀基因——野生型（剪接校正）单纯疱疹病毒胸苷激酶（scHSVtk）的缺点，我们生成了两个新变体：密码子优化的coHSVtk，以及通过引入额外突变（A168H）得到的新型TK.007。我们用编码选择标记（tCD34和OuaSelect）组合与三种HSVtk变体之一的逆转录病毒“分选自杀载体”转导人造血细胞系和原代T细胞。在体外，我们观察到TK.007的表达水平更高且能长期持续表达，表明其非特异性毒性较低。此外，我们注意到更昔洛韦（GCV）介导的对TK.007转导细胞的杀伤动力学有显著改善。在一个实验性（小鼠）同种异体移植模型中，TK.007转导的T细胞介导了严重的GvHD，通过应用GCV（10 mg/kg）可轻易消除。最后，我们建立了一个改良的同种异体移植模型，用于对TK.007与scHSVtk的体内活性进行定量比较。我们发现，在低GCV浓度（10和25 mg/kg）下，TK.007介导的杀伤速度明显更快，绝对杀伤率更高。总之，我们证明新型TK.007自杀基因（与常用的剪接校正野生型scHSVtk基因相比）具有更好的杀伤性能且非特异性毒性降低，因此是自杀基因治疗的一个有前景的替代方案。
