基于细胞体内自组装过程的核酸蛋白质复合纳米颗粒的设计合成

基于RNA的各种生物技术，已被证实是在基因水平操控表型的强有力的工具。然而，为了彻底实现RNA疗法的潜力，一个重大挑战是在体内有效地将RNA递送给靶标细胞。尽管基于RNA的递送策略已有很多成功的案例，但这些方法往往需要大量的合成工作。一些基于纳米载药平台的方法也存在潜在的生物不相容性和细胞毒性。在本项目中，申请人设计了一种细胞体内组装的合成策略，合成核酸蛋白质复合纳米颗粒来递送CRISPR/Cas9-sgRNA复合物进行针对性的基因编辑和基因治疗。申请人拟计划使用合成生物学的方法将两亲性蛋白质、Cas9蛋白质和与Cas9结合的靶向sgRNA在同一细胞载体中原位共表达并自组装成RNA-蛋白质复合物纳米颗粒。该体系将提供一种制备具有良好生物相容性的RNA纳米药物的新方法，为低成本、大规模、可控制备RNA纳米药物打下坚实的基础。

RNA-based biotechnologies, such as small interfering RNA (siRNA) and CRISPR/Cas9 platform, have proven to be attractive tools for phenotype manipulation at genetic level, and have the potential to develop therapeutics targeting numerous genetic diseases, including cancer. However, a barrier of realizing the potential of therapeutic RNA is to effectively deliver RNA to target cells. Although various delivery strategies have been developed, such as RNA-based conjugation chemistry and nanoparticle-based delivery platform, these approaches generally require considerable synthetic work and sophisticated installation processes, and suffer from incompatibility and cytotoxicity. In this proposal, I propose to develop a synthetic biology-enabled protein nanoparticle system to deliver CRISPR/Cas9-sgRNA complexes for targeted gene editing. The general scheme is based on the use of artificial, amphiphilic proteins that can form lipid-like micelles. Cas9 proteins with necessary directing domains such as cell penetration peptides will be fused to amphiphilic proteins, and the target sgRNAs that bind to Cas9 in situ will be co-expressed in the same vector. On harvesting and purification, the amphiphilic proteins will readily form functional micelle-like nano-particle for therapeutic use. As a proof of concept, tumor cells expressing EGFP will be used to examine gene disruption activity of the particle, and animal experiments will be performed in tumor-bearing nude mice to study the biodistribution, pharmacokinetics and anticancer efficacy. Overall, the concept described will provide a novel approach to the production of biocompatible RNA nano-medicines, harnessing the power of synthetic biology. They are especially attractive due to the convenience of generating the desired cargo-vehicle complex in a one-step manner inside a producing strain, enabling readily installation and modification of the desired functional moiety through molecular cloning.