新型核酸适体-siRNA嵌合体的靶向增敏机制及前列腺癌靶向治疗的研究

Prostate cancer (PCa) incidence continues to rise in China and has become the major disease of adult male reproductive health recently. For localized prostate cancer , dose-escalated radiation therapy has a clear therapeutic benefit; however, escalated doses may also increase injury to non-cancerous tissues. Radiation-sensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery，these agents will also sensitize surrounding normal tissues. RNA interference is a promising new approach for targeting disease associated genes and pathways. The critical challenge for translating RNAi therapy is delivery, particularly for specific cell types. Recently, one delivery approach that is showing some promise is RNA aptamers. Previously, we developed prostate-targeted RNA interference agents which selectively sensitize PSMA-positive cells to IR via aptamer-DNAPK shRNAs, delivered by PSMA aptamers, and selectively reduced DNAPK in PCa cells, xenografts and human prostate tissues.Based on our previous research, here we describe the new generation of aptamer-siRNAs which are full chemical synthesis, easy to generate and avoid random mutation and unfinished transcription compare to our previous method. Moreover, this new aptamer-siRNAs could overcome the cost of certain length synthesis of the nucleotides oligo and could be translated for treatment of PCa. We continue to screen ideal sensitizing siRNA through high-throughput DNA repair pathway siRNA library and design new Aptamer-siRNAs chimeras to selectively silence the target gene in order to sensitize PCa cell. In previous study of this project, we proved the new aptamer-siRNAs could selectively knock down the target genes in vitro and in vivo, and also could sensitize PCa cell to IR. By clarifying the mechanism of new aptamer-siRNAs chimeras, in animal model, we will intravenous inject Aptamer-siRNAs and combine the treatment with IR for PCa target therapy. We believe the new generation PSMA aptamer-siRNAs chimeras could selectively enhance the PCa sensetization for IR combined therapy and has great potential application for future clinical translation.

我国前列腺癌（PCa）发病率持续升高，已成为危害男性生殖健康的重大疾病。升级剂量的放射治疗对局限性 PCa有着明确的疗效，但剂量升级也增加了对癌旁组织的伤害。放射增敏剂可增加电离辐射（IR）的效用，然而未经靶向递送，增敏剂也将使周围的正常组织增敏。RNAi作为新的治疗方法已得到广泛认可, 其所面临的挑战，是如何特异性地将siRNA靶向导入细胞。其中一个方法，是利用RNA核酸适体靶向输送siRNA。申请人在前期工作中，设计PMSA核酸适体-shRNA，结合IR，能有效的靶向抑制PMSA阳性PCa细胞、异植瘤的生长。本项目拟在此基础上，通过DNA 修复途径siRNA文库筛选增敏siRNA。设计新型全化学合成的PSMA Aptamer-siRNA嵌合体，研究并阐明其选择性增敏前列腺癌细胞的作用机制，通过静脉给药，结合IR照射整合治疗，研究其靶向治疗前列腺癌的作用，为临床转化应用提供理论和实验依据

放射治疗是治疗前列腺癌各个阶段的高效治疗手段，可提高从局部疾病的治疗方法到姑息治疗以及远处骨转移患者的生存率。靶向放射增敏剂可以使得这些方法的治疗指数进一步增强。适配体是极其有潜能的用于传递短干扰RNA（siRNA）和短发夹RNA（shRNA）的核酸递送剂。我们前期开发了放射增敏的RNA适体-shRNA嵌合体，在没有转染试剂的情况下选择性地将靶向DNA-PK的shRNA转移到前列腺特异性膜抗原（PSMA）阳性细胞。虽然这些嵌合体是有效的，但是它们的合成需要体外转录，并且其评价仅限于瘤内给药。在本项目中，我们开发了第二代适配体-siRNA嵌合体，可以通过三个独立的化学合成组分的嵌合来组装。新型的适配体-siRNA嵌合体在PSMA阳性的前列腺癌细胞中能靶向敲除DNA-PK，而且不需要额外的转染试剂，并增强了放射介导的细胞死亡的功效。在小鼠模型水平上，静脉内注射嵌合体后，嵌合体能有效地敲低小鼠皮下建立的PSMA阳性肿瘤中的DNA-PK。放射增敏剂的治疗选择性地增强了动物模型PSMA阳性肿瘤的外部放射线疗法的效果。我们的结果表明第二代适配体-siRNA嵌合体可选择性敲除DNA-PK，从而增加了前列腺癌细胞对放射治疗的敏感性，通过静脉给药，结合IR照射的整合治疗，可靶向治疗前列腺癌，为临床转化应用提供理论和实验依据。