光控CRISPR/Cas13系统靶向抑制TET3预防激素性股骨头坏死的研究

激素性股骨头坏死（SAOFH）是一种致残率极高的常见骨科疾病，缺乏有效预防方法。研究表明TET3在SAOFH发病起始发挥重要作用，但精准调控TET3预防SAOFH的具体方法尚不明确。申请人前期构建了一种光控CRISPR/Cas13系统，能够在RNA水平时空特异性调控生命活动，为预防SAOFH提供了新的思路。本项目拟在前期基础上，首先设计并优化新型光控CRISPR/Cas13 RNA敲低系统“PACK”，利用细胞及分子生物学技术，深入探究其RNA敲低效率、时空精准度、脱靶效应及生物安全性。进一步合成兼具高效基因转染效率和光转化性能的功能化上转换纳米颗粒，用于PACK体系的高效递送及远程激活，从而在体内外特异性敲低TET3表达，抑制激素致骨细胞凋亡并实现SAOFH的精准预防。本项目旨在阐明光控CRISPR/Cas13系统精准预防SAOFH的重要意义，有望为SAOFH的早期干预提供新方法。

Steroid-associated osteonecrosis of the femoral head (SAOFH) is a common orthopedic disease with a high rate of disability, but the effective prevention method is still lacking. It has been demonstrated that TET3 is a crucial pathogenic gene of SAOFH, however, the method of precisely targeting TET3 to prevent SAOFH is warranted. Technically, we engineered a photoactivatable CRISPR/Cas13 system, which enables the spatiotemporal control of biological process at RNA level, providing the possibility of early prevention SAOFH. This study intends to design and optimize a photoactivatable CRISPR/Cas13 RNA knockdown system (PACK) at first. The efficiency, spatiotemporal specificity, off targeting effects, and biocompatibility of the PACK system will be explored with cellular/molecular biology techniques. Furthermore, upconversion nanoparticles with high gene transfection and photoconversion efficiency will be prepared for delivery and remote manipulation of the PACK system. Specific knockdown of TET3 expression upon activation and its effects both in vivo and in vitro will be tested accordingly. This study aims to achieve specific inhibition of glucocorticoid-induced osteocyte apoptosis, which might shed a light on the precise prevention of SAOFH with photoactivatable CRISPR/Cas13 system.