增强摄取及促进内体释放的肿瘤靶向siRNA高效递释系统的设计及其药效学研究

The key to make siRNA therapy into reality is to develop a high efficiency delivery system. The current nanoparticle-mediated delivery system for the clinical application of siRNA remains facing challenges, including formulation stability, safety, low transfection efficiency, etc. In the previous study, a proprietary cRGD-siVEGFR2 (specifically silence vascular endothelial growth factor receptor-2, VEGFR2) was engineered, which can specifically target ανβ3 receptor-expressed tumor tissue in vitro and in vivo, and inhibited angiogenesis and tumor growth. In the present study, we will optimize the XRGD structure based on the mechanism of cellular uptake, endocytosis and endosomal escape, which make XRGD bind with ανβ3 receptor much stronger. With the theory of chemical modifications for oligonucleotide, we will synthesize and screen chemically modified siHVR2-X and siPI3K-X which show much stronger biological activity and much higher gene silence efficacy. Subsequently, XRGD-siRNA molecules were synthesized by combining XRGD with siRNA (siVEGFR2-X or siPI3K-X). In vitro, we will evaluate characteristics of XRGD-siRNA, including cellular uptake, transport, endosomal release and biological function. In vivo, we will establish subcutaneous and in situ glioma mice models to study the bio-distribution, pharmacodynamics and toxicity of XRGD-siRNA. Combining with results in vitro and in vivo, we will value the XRGD-siRNA conjugate as a platform for siRNA to therapy disease in clinic.

小干扰RNA分子（siRNA）临床应用的关键是递释系统。课题前期制备的具有自主知识产权的偶联结构cRGD-siVEGFR2（血管内皮细胞生长因子受体2），能靶向ανβ3受体表达的肿瘤组织，抑制血管生成及肿瘤生长。本课题根据优化cRGD以增强细胞摄取及内体逃逸等理论，设计及筛选得到与ανβ3受体结合活性更强及内体逃逸量最大的XRGD结构；化学修饰及筛选得到沉默效率高及稳定性好的的siVEGFR2-X及siPI3K-X(胞内磷脂酰肌醇激3-酶)分子；化学键偶联XRGD与siRNA，得到XRGD-siRNA分子。体外评价该siRNA递释系统对ανβ3受体表达细胞的靶向性、摄取强度、 细胞内转运、内体释放、及基因沉默等生物学功能及机制；建立小鼠原位脑胶质瘤模型，考察其体内分布、单独及联合两种siRNA给药后的药效学。综合评价XRGD-siRNA偶联物作为siRNA分子肿瘤靶向高效递释系统的可能性。

小干扰RNA分子（siRNA）临床应用的关键是构建高效递释系统。现有纳米载体系统存在制剂不稳定、安全性差、及转染效率低等问题。本课题根据cRGD基团摄取优化及内体逃逸等理论，合成及筛选得到与肿瘤组织亲和活性最强的biRGD（双价cRGD）；根据核酸化学修饰理论，修饰及筛选得到了沉默效率高及生物学活性强的siRNA分子及其修饰规律；联合biRGD与siRNA，合成得到biRGD-siRNA分子。体外评价了该偶联物分子的细胞摄取、转运、内体释放及生物学功能；建立了皮下及原位脑胶质瘤模型，考察分子的体内分布、药效学及毒性。肝脏外组织如肿瘤等siRNA给药系统仍存在难以产业化及因内体逃逸量少致疗效低等问题，是siRNA广泛临床转化的最大瓶颈，也是国内外研究机构和产业界研究的热点和难点问题。通过该结题课题的研究，合成得到的biRGD-siRNA结构，解决了以αvβ3受体为递送靶点实施siRNA治疗肿瘤的给药系统问题、siRNA体内使用的稳定性问题和免疫原性问题。与其他siRNA给药系统体内使用一样，仍然存在biRGD-siRNA经αvβ3受体、以网格蛋白介导机制内吞进入细胞后难以从内体释放致疗效有限的关键核心问题。总结体内外结果，评价了biRGD-siRNA偶联物作为siRNA分子高效递释系统的临床应用的可能性。研究工作共发表科研论文7篇，其中SCI论文4篇（总影响因子26.8），国内核心期刊论文3篇；培养研究生7名，其中博士生2名。