基于合成致死新策略的siRNA传递系统构建及其肝肿瘤穿透效应的机制研究

Gene target selection and drug delivery system design have a great influence on the antitumor therapeutic efficacy of siRNA drug. In the past decades, a lot of siRNAs were developed to inhibit oncogene, with beneficial antitumor efficacy. However, off-target effect of siRNA induced serious toxicity to normal tissues and organs, limiting the clinical application. In addition, traditional siRNA nanocarriers can only delivery drug to the periphery of tumor tissue, unable to reach the hypoperfusion area of tumor and penetrate all cancer cells. Our preliminary study showed that synthetic lethality had promising to avoid the toxicity of siRNA, however, the therapeutic effect on hepatocellular carcinoma (HCC) and safety need to be studied further. In this study, HCC was used as disease model. First, specific CDK1 silencing siRNA was designed and selected based on synthetic lethality between oncogene MYC and cyclin-dependent kinase 1 (CDK1). Then, tumor penetrating lipid nanocarrier (DTP-LNP) encapsulated CDK1-siRNA was developed according to the natural tumor bioelectrical field. DTP-LNP was expected to achieve HCC deep penetration and ultra-high siRNA transfection, killing MYC overexpressed HCC tumor cell specifically. This study will provide a new RNA interference strategy with maximized HCC therapeutic efficiency and minimized toxicity.

基因靶点选择和药物递送系统设计是影响siRNA药物抗肿瘤疗效的关键因素。目前，针对致癌基因设计的siRNA，其脱靶效应会对正常组织产生严重的毒副作用；而传统的纳米载体仅能将药物递送至肿瘤边缘，无法到达肿瘤内部低灌注区发挥药效；限制了siRNA药物的疗效和临床应用。我们的前期研究提示，合成致死策略有望解决siRNA的用药毒性，但其用于肝癌的疗效和安全性仍有待研究。为此，本研究拟基于周期蛋白依赖性激酶（CDK1）与致癌基因MYC构成合成致死关系的机理，合成和筛选专属性沉默CDK1的siRNA（CDK1-siRNA）。随后，利用肿瘤存在生物电场的生理特性，制备具有肿瘤组织穿透作用的CDK1-siRNA脂质纳米载体(DTP-LNP)，实现siRNA对肝癌组织的高效渗透和转染，通过与MYC基因协同作用，达到特异性杀灭MYC突变型肝癌的目的。以期为肝癌临床治疗提供一种高效低毒化的siRNA递送新策略。

基因靶点选择和药物递送系统设计是影响siRNA药物抗肿瘤疗效的关键因素。本项目针对siRNA肿瘤治疗药物存在的脱靶毒性以及递送载体设计难点，采用与MYC具有合成致死作用的CDK1-siRNA，设计并构建了一种具有相转变功能的脂质液晶纳米载体DCLC，用于高效克服siRNA递送过程中的细胞屏障，提高siRNA疗效并消除脱靶副作用。研究结果表明，DCLC脂质液晶纳米载体可包载siRNA避免其被血清降解，并且促进肿瘤细胞的摄取。DCLC在胞内内涵体酸性环境下通过相转变作用与内涵体发生高效的膜融合，释放出游离的siRNA至细胞质中与mRNA结合，实现了克服细胞屏障和siRNA的高效递送的效果。此外，以CDK1-siRNA作为治疗药物，利用其与肝癌MYC基因之间的合成致死作用，分别在细胞水平和动物水平实现了高效的CDK1基因沉默，显著性抑制HepG2肝癌细胞的增殖及其移植瘤的生长，且不对正常肝细胞产生影响，消除了脱靶的副作用。为提高临床肝癌的抗肿瘤疗效开辟一种RNA干扰治疗新策略，达到了预期的研究目标。