近红外光控CRISPR-Cas9基因编辑递送系统的构建及其对杜氏肌营养不良症的精准治疗研究

Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR-Cas9) has shown great application potentials towards gene therapy, the lack of delivery systems and approaches to control off-target effects has become a major bottleneck for the clinical translation of CRISPR-Cas9 genome editing technologies. This project aims to develop near-infrared (NIR)-responsive polymeric micelles that can synergistically deliver upconversion nanoparticles (UCNPs), Cas9 protein as well as photocaged single-guided RNA (sgRNA). As the ultraviolet light can photolyze the delivery carrier and uncage the sgRNA, the proposed delivery system is able to respond NIR light of deep tissue penetration, which is converted to local ultraviolet light by UCNPs, so as to simultaneously control the release profile of Cas9/sgRNA and mediate the ‘ON’ and ‘OFF’ of genome editing system, thereby precisely controlling the genome editing activity in a spatiotemporal manner. We propose to elucidate the relationship between the carrier chemical structure and genome editing efficiency as well as cytotoxicity in order to optimize the delivery system of CRISPR/Cas9 with high efficiency and low cytotoxicity. In addition, the relationship between light control and off-target effect will also be elucidated. Finally, an approach for the precision therapy of duchenne muscular dystrophy and its safety and efficacy will be demonstrated through the efficient delivery of genome editing tools and the remote NIR control and activation we have developed. Thus, the proposed project provides a new strategy and fundamental insights for the in vivo precision therapy of CRISPR/Cas9 genome editing from the drug delivery point of view.

CRISPR/Cas9基因编辑系统已在基因治疗领域展现出极大的应用前景，然而其体内递送系统的匮乏和脱靶效率的不可控已成为制约CRISPR/Cas9基因编辑走向临床的瓶颈问题。本研究拟通过设计构建具有深部组织穿透的近红外光响应型聚合物胶束载体，用于协同递送上转换纳米粒、Cas9蛋白及光笼化介导RNA(sgRNA)。通过所递送的上转换纳米粒将外源性近红外光转换为局部紫外光，来光解载体以及光笼化sgRNA，以此实现Cas9及sgRNA的可控释放以及基因编辑的可控开关。研究聚合物胶束载体的理化性质与编辑效率及细胞毒性的关系，优化体内高效低毒的CRISPR/Cas9基因编辑递送系统, 并阐明光开关与脱靶效率的关系。通过对基因编辑元件的体内高效递送，近红外光的控制释放和远程激活，实现对杜氏肌营养不良症的精准治疗。此研究从给药系统角度为CRISPR/Cas9基因编辑系统实现体内精准治疗提供新思路。

CRISPR/Cas9基因编辑系统已在基因治疗领域展现出极大的应用前景，然而递送载体效率低、脱靶效应显著及空间特异性差，是限制CRISPR/Cas9基因组编辑系统在生物医学领域广泛应用的瓶颈问题。为解决这些问题，项目负责人团队构建了一种近红外二区（NIR-II）光控基因编辑递送系统。该系统由阳离子聚合物修饰的金纳米棒材料APC，含热敏激活的HSP70启动子启动的CRISPR质粒构成。通过APC将HSP70启动的CRISPR质粒递送进细胞，在NIR-II光的控制下，金纳米棒吸收光能转换成局部的热能，通过HSP70启动子启动CRISPR/Cas9的转录和表达。阳离子聚合物修饰的金纳米棒不仅能有效地运送质粒，同时也能高效地实现近红外光-热能的转换，实现近红外二区光控基因编辑和基因的转录调控，在HEK293等多种细胞上的转染效率达到了85%以上。静脉注射半乳糖化的APC可靶向聚集肝脏，因而通过NIR-II光控基因编辑能保护小鼠免受急性肝损伤，并在其他组织基因组的相同位点几乎未发现有脱靶效应。同时，通过光控的方法，有效地减弱了Cas9的持续表达，因而在脱靶效应显著降低。这些研究表明，通过近红外光控来调控Cas9基因表达，并联合靶向递送手段，不仅有助于提高靶组织的特异性编辑，而且大大降低了非靶组织的非特异性编辑。这种利用非病毒载体将基因编辑系统递送至靶器官，同时通过光控达到靶器官特异性转录启动的“双保险”方式，不仅有效实现了体内的精准基因编辑，同时也为安全基因编辑治疗提供了新思路。