PO.CH01.04 · 化学
利用PD-L1靶向细胞外囊泡将自杀基因靶向递送至肿瘤细胞用于癌症治疗
Targeted delivery of suicide gene to tumor cells using PD-L1-targeted extracellular vesicles for cancer therapy
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
单纯疱疹病毒胸苷激酶（HSV-TK）基因是一种著名的自杀基因，在用更昔洛韦（GCV）处理时可导致快速分裂细胞死亡。HSV-TK激活GCV并生成GCV三磷酸。细胞DNA聚合酶将激活的GCV三磷酸插入延伸中的DNA，干扰DNA复制，最终导致细胞死亡。然而，自杀基因疗法一直受限于对正常细胞等脱靶细胞的非特异性细胞毒性。在本研究中，我们探索了携带HSV-TK mRNA的PD-L1靶向细胞外囊泡（EV），以将HSV-TK选择性递送至肿瘤细胞。使用慢病毒转导HEK 293FT细胞，转入带GFP标签、含有可被EV转运RNA结合蛋白识别序列的HSV-TK基因。建立了稳定表达HSV-TK基因的HEK 293FT细胞。从稳定细胞中分离EV，并使用点击化学以PD-L1结合肽（CVRARTR）标记，以靶向PD-L1高表达的肿瘤细胞。细胞与EV孵育后HSV-TK mRNA的递送通过在显微镜下观察GFP标签的荧光得到验证。携带HSV-TK mRNA的PD-L1靶向EV在PD-L1高表达的MDA-MB-231肿瘤细胞中发挥了有效的细胞毒性。另一方面，这些EV在PD-L1低表达的肿瘤细胞和正常细胞中表现出低水平的细胞毒性。全身给予EV可抑制小鼠中MDA-MB-231肿瘤异种移植物的生长。这些结果表明，含HSV-TK mRNA的PD-L1靶向EV为自杀基因的靶向递送提供了一条新途径。关键词：HSV-TK；肽；PD-L1；自杀基因疗法
查看英文原文 English abstract
Herpes simplex virus-thymidine kinase (HSV-TK) gene, a well-known suicide gene, causes cell death in rapidly dividing cells when treated with ganciclovir (GCV). The HSV-TK activates GCV and makes GCV-triphosphate. Insertion of activated GCV-triphosphate in elongating DNA by cellular DNA polymerases interferes DNA duplication and eventually leads to cell death. The suicide gene therapy, however, has been limited by non-specific cytotoxicity in off-target cells such as normal cells. In this study, we explored PD-L1-targeted extracellular vesicles (EVs) carrying the HSV-TK mRNA to deliver the HSV-TK selectively into tumor cells. HEK 293FT cells were transduced using lentivirus with the GFP-tagged, HSV-TK gene containing a sequence that is recognized by EV-trafficking RNA-binding protein. Stable HEK 293FT cells expressing the HSV-TK gene were established. EVs were isolated from the stable cells and labeled with a PD-L1-binding peptide (CVRARTR) using click chemistry to target PD-L1-high tumor cells. Delivery of the HSV-TK mRNA after incubation of cells with the EVs was validated by observing the fluorescence of GFP tag under a microscope. The PD-L1-targeted EVs carrying the HSV-TK mRNA exerted an efficient cytotoxicity in PD-L1-high MDA-MB-231 tumor cells. On the other hand, the EVs showed low levels of cytotoxicity in PD-L1-low tumor cells and normal cells. Systemic administration of the EVs inhibited the growth of MDA-MB-231 tumor xenografts in mice. These results demonstrate that the PD-L1-targeted EVs containing the HSV-TK mRNA provide a new avenue for the targeted delivery of a suicide gene. Keywords: HSV-TK; Peptide; PD-L1; Suicide gene therapy
利益披露 Disclosure
G. Park, None..
S. Lee, None..
E. Park, None..
B. Lee, None.