多功能载体同时输送CRISPR/Cas9系统和分子靶向药物用于肝癌的协同精准治疗

The global morbidity and mortality of hepatocellular carcinoma are on the rise. The first-line treatment of advanced hepatocellular carcinoma has the problems of low response rate, drug resistance, and lack of selectivity. It is urgent to develop new treatment strategies to improve therapeutic efficacy as well as response rate and reduce side effects. A recent study indicates that inhibition of the Cdk5 gene increases the sensitivity of hepatocellular carcinoma cells to sorafenib. Our previous studies have shown that knocking out the HPV-E7 or Plk1 through non-viral gene editing can inhibit the growth of cervical cancer cells, and knocking out the PARP1 gene can increase the sensitivity of breast cancer to chemotherapy. Based on these findings, this project aims to develop a multi-functional targeted nanocarrier to co-deliver CRISPR/Cas9 gene editing system and molecularly targeted drug for the synergetic and precise treatment of hepatocellular carcinoma. The project will demonstrate that the nano-formulation can enhance anti-tumor efficacy and improve the safety of the molecularly targeted drug. By optimizing the properties of the multi-functional delivery system, the spatial activity of CRISPR/Cas9 will be controlled to improve the accuracy of gene editing. Moreover, simultaneous knockout of Cdk5 and Plk1 may achieve a synergistic anti-cancer effect and increase the sensitivity of hepatocellular carcinoma cells to the molecularly targeted agent, thereby revealing the superiority of nanocarriers co-delivering gene editing system and molecularly targeted drug for the treatment of hepatocellular carcinoma. This project can provide a novel strategy, guidance and evidence for clinical treatment of hepatocellular carcinoma.

肝癌的全球发病率和死亡率呈上升趋势。晚期肝癌的一线治疗药物存在应答率低、易产生耐药、分布缺乏选择性的问题，亟需寻找新的治疗策略以提高药物的体内输送效率和应答率并降低毒性。近期研究提示，抑制Cdk5基因可增加肝癌细胞对索拉非尼的敏感性。我们前期研究证明，非病毒基因编辑技术敲除HPV E7或Plk1基因可抑制宫颈癌生长，敲除PARP1基因可提高乳腺癌对化疗的敏感性。基于此，本项目拟构建多功能靶向型纳米载体同步输送CRISPR/Cas9系统和分子靶向药用于肝癌的协同精准治疗。项目将证明分子靶向药物的纳米剂型可增强其疗效和安全性，并优化功能协同型纳米载体对Cas9空间活性的调控以提高基因编辑的精准性；将通过Cdk5和Plk1双靶点同时敲除策略以期实现协同抗癌，并增强癌细胞对分子靶向药的敏感性；从而揭示纳米载体同时输送基因编辑系统和分子靶向药用于肝癌治疗的优越性，为肝癌的临床精准治疗提供指导和依据。

肝癌具有高发病率和低生存率的特征。晚期肝癌的一线治疗药物存在应答率低、易产生耐药、分布缺乏选择性的问题，亟需寻找新的治疗策略以提高药物的体内输送效率和应答率并降低毒性。CRISPR/Cas9系统是一种强大的基因组编辑工具，可以在基因组DNA水平实现基因敲除以永久消除靶基因，从而获得更加持久的癌症治疗效果。因此，发展基于CRISPR/Cas9的基因治疗和化疗的联合治疗是一个有前途的协同策略，能够最大限度地提高治疗效果。本项目针对CRISPR/Cas9基因治疗和联合治疗研究存在的瓶颈问题，发展了多种新型功能化纳米药物和纳米载体有效实现肝癌等肝脏疾病的治疗。具体包括：1）膜融合脂质纳米药物递送CRISPR/Cas9基因编辑工具治疗肝细胞癌的研究；2）开发膜融合载体递送肝特异性CRISPR/Cas9基因编辑系统；3）构建逐级靶向纳米载体有效靶向肝细胞实现基因编辑时空调控。总体而言，我们：1）获得了可同步输送CRISPR/Cas9基因编辑系统和分子靶向药物的载体材料；2）优化了多种纳米载药系统，实现了对CRISPR/Cas9活性的空间调控能力，证明了其在精准治疗方面的优势；3）在分子、细胞、模型动物层次，验证了非病毒基因编辑介导的生长因子受体信号传导阻断能够增加肝癌细胞对分子靶向药物（乐伐替尼）的敏感性进而提高疗效，且非病毒基因编辑介导的多靶点阻断能够实现协同的肝癌治疗，阐明了其相互作用机制。在本项目的资助下，已发表论文37篇，申请专利18项，培养/联合培养博士后2名、博士生3名、硕士生4名。项目的实施为肝癌等肝脏疾病的临床精准治疗提供了新思路和新方法，具有科学意义和实际应用价值。

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