促使肿瘤细胞正常化的基因编辑功能化传递系统的研究

This study will be focused on functional delivery systems for tumor targeting and cell nucleus targeting delivery of CRISPR-Cas9 plasmids to realize highly efficient genome editing in targeted tumor cells. To endow the delivery vectors with the targeting capability, aptamers as targeting ligands, will be incorporated to natural polymers through covalent bonds or hybridization of complementary pairs. The multi-functional delivery systems with good biocompatibility will be prepared by self-assembly of aptamer functionalized natural polymers and other functional components such as biomacromolecules and peptides. The efficiency of genome editing on various therapeutic targets include FAK, β-catenin and CDK11 will be evaluated. Through the knockout of corresponding FAK, CTNNB1, CDK11 genes, the CRISPR-Cas9 delivery systems can down-regulate FAK, β-catenin and CDK11 proteins, and thus regulate the expression of other related proteins involved in tumor development through diverse signaling pathways. The genome editing mediated by our delivery systems can alter cellular behavior of tumor cells and suppress the malignant cellular behavior in edited tumor cells to realize normalization of tumor cells. The effects of the genome editing systems on inhibition of cell growth and proliferation, prevention of tumor development, invasion and metastasis, reversal of tumor-induced immune suppression, and inhibition of cancer stemness will be investigated. This study will provide theoretical knowledge on the preparation of the delivery systems of CRISPR-Cas9 plasmids.

项目拟制备CRISPR-Cas9基因编辑体系的功能化载体，通过对CRISPR-Cas9质粒的肿瘤靶向及细胞核靶向传递实现对肿瘤细胞的高效基因编辑。为赋予传递系统靶向功能，将肿瘤细胞及细胞核靶向的核酸适配子通过共价键及杂化互补配对的方式引入到天然高分子中，得到多种适配子功能化天然高分子，通过其与其它功能组分如生物大分子、功能多肽的自组装制备具有良好生物相容性的多功能传递系统。针对与肿瘤细胞行为密切相关的FAK、β-catenin、CDK11这几个肿瘤治疗靶点进行基因编辑，敲除其相应的FAK、CTNNB1、CDK11基因，下调其表达，从而调控其它与肿瘤发展相关的蛋白表达，达到抑制细胞恶性化、使肿瘤细胞“正常化”的目的。研究传递系统对肿瘤细胞的多方面行为的影响，包括抑制细胞生长增殖、防止肿瘤发展侵袭转移、逆转肿瘤免疫抑制、抑制细胞干性转化。本研究将为新型基因编辑传递载体的设计提供理论依据。

项目设计制备了系列高效传递CRISPR-Cas9基因编辑质粒的功能化载体，为赋予传递系统肿瘤靶向等功能，将多种核酸适配子、多肽等功能组分通过共价键及自组装的方式引入基于天然高分子的传递系统中。这些具有理想生物相容性的传递系统可将CRISPR-Cas9质粒靶向传递到肿瘤细胞及其细胞核，实现高效基因编辑。针对与肿瘤发展密切相关的蛋白包括FAK、β-catenin、CXCR4、PPM1D、CDK11进行基因编辑，通过敲除表达这些蛋白的基因，达到了抑制肿瘤细胞生长、防止其侵袭转移、减少干性、逆转肿瘤免疫逃逸及免疫抑制从而激活抗肿瘤免疫的目的，从根本上逆转肿瘤细胞恶性化，促使肿瘤细胞“正常化”。本研究为新型基因编辑传递载体的设计提供了理论依据、并为肿瘤治疗提供了有参考价值的治疗策略。