基因修饰细胞疗法具有严重和潜在致命不良事件的固有风险。包括同种反应性细胞的扩增或由于插入诱变引起的恶性转化。减轻基因修饰细胞不受控制的增殖的策略包括共转染自杀基因,例如诱导型胱天蛋白酶9安全开关(ΔiC9)。然而,ΔiC9的激活不能完全消除所有基因修饰的细胞。因此,我们测试了一个独立或一起使用的两个自杀基因系统,以完全消除细胞为目标。第一种方法将ΔiC9与缺乏内源前结构域的诱导型半胱天冬酶8ΔiC8组合。理由是使用具有替代和互补作用机制的第二半胱天冬酶。Jurkat细胞共转导以共表达可由BB同源二聚体激活的ΔiC8,在模型中使用雷帕霉素类似物西罗莫司可激活的ΔiC9来估计诱导型细胞消除的程度。我们发现这两种药物都可以独立激活每种caspase,当两个系统同时激活时,基因修饰细胞的消除增强,细胞再生长明显减少。并行采用了第二种方法,将ΔiC9与RQR8紧凑型自杀基因相结合。RQR8包含CD20模拟表位,以抗CD20单克隆抗体利妥昔单抗为目标,和QBend10,ΔCD34选择标记。同样,当两个系统一起激活时,观察到细胞消除增强,细胞再生长明显减少。使用BB同二聚化剂也注意到剂量滴定效应,而西罗莫司在最低浓度下仍然非常有效。需要进一步的体内研究来验证这些新颖的组合系统,这可能在未来的癌症治疗或再生医学中发挥作用。
Gene-modified cellular therapies carry inherent risks of severe and potentially fatal adverse events, including the expansion of alloreactive cells or malignant transformation due to insertional mutagenesis. Strategies to mitigate uncontrolled proliferation of gene-modified cells include co-transfection of a suicide gene, such as the inducible caspase 9 safety switch (ΔiC9). However, the activation of the ΔiC9 fails to completely eliminate all gene-modified cells. Therefore, we tested a two suicide gene system used independently or together, with the goal of complete cell elimination. The first approach combined the ΔiC9 with an inducible caspase 8, ΔiC8, which lacks the endogenous prodomain. The rationale was to use a second caspase with an alternative and complementary mechanism of action. Jurkat cells co-transduced to co-express the ΔiC8, activatable by a BB homodimerizer, and the ΔiC9 activatable by the rapamycin analog sirolimus were used in a model to estimate the degree of inducible cell elimination. We found that both agents could activate each caspase independently, with enhanced elimination with superior reduction in cell regrowth of gene-modified cells when both systems were activated simultaneously. A second approach was employed in parallel, combining the ΔiC9 with the RQR8 compact suicide gene. RQR8 incorporates a CD20 mimotope, targeted by the anti-CD20 monoclonal antibody rituxan, and the QBend10, a ΔCD34 selectable marker. Likewise, enhanced cell elimination with superior reduction in cell regrowth was observed when both systems were activated together. A dose-titration effect was also noted utilizing the BB homodimerizer, whereas sirolimus remained very potent at minimal concentrations. Further in vivo studies are needed to validate these novel combination systems, which may play a role in future cancer therapies or regenerative medicine.