CRISPR/Cas9介导的视网膜色素变性在体基因治疗的研究

Retinitis pigmentosa (RP), an inherited monogenic disease, leads to blindness and exerts great effect on the quality of life in patients. However, effective treatments for RP are currently unavailable. CRISPR/Cas9 is an effective gene editing tool and can modify genes in vivo. Our preliminary experiments showed that tyrosine-capsid mutant AAV2/8 (Y733F) mediated CRISPR/Cas9 could edit Rosa26 and Pde6b genes in vivo in the retina and induce site-directed mutation of Pde6b in in vitro cultured retina. On basis of that, we hypothesize that CRISPR/Cas9 can treat RP in vivo. Firstly, we will determine whether CRISPR/Cas9 has toxic effect on the structure and function of the retina, and then optimize the parameters of in vivo editing by CRISPR/Cas9. Finally, we will investigate in vivo correction of the mutation of Pde6b in rd10 mouse model by AAV2/8(Y733F) delivered paired sgRNA and Cas9n. This study will provide a novel insight into the gene therapy of RP and has the significance of theory and clinical application.

视网膜色素变性(Retinitis pigmentosa, RP)是一种致盲性单基因遗传疾病，给患者的生活质量造成了极大的影响，目前仍无有效的治疗方法。CRISPR/Cas9作为一种高效的基因编辑工具可实现在体基因修饰。预试验结果显示AAV2/8(Y733F)介导的CRISPR/Cas9可在小鼠视网膜中对Rosa26和Pde6b基因进行在体编辑，并在离体小鼠视网膜中诱发Pde6b基因的定点突变。基于以上结果，我们提出CRISPR/Cas9可对RP进行在体基因治疗。首先，我们将探讨CRISPR/Cas9是否会对视网膜的结构和功能产生影响及优化在体编辑体系；其次，将以rd10小鼠为模型，探讨AAV2/8(Y733F)介导的成对sgRNA与Cas9n在体矫正rd10小鼠Pde6b基因的突变。本研究为RP的基因治疗提供新的思路，具有重要的理论和临床应用价值。

视网膜色素变性（retinitis pigmentosa, RP）是一类不可治愈的遗传性视网膜退行性疾病，给患者的生活质量造成了极大影响。基因治疗作为一种极具前景的治疗方式，给RP患者带来了新的希望。CRISPR/Cas9 作为一种简单高效的基因编辑工具，可在不区分遗传方式的前提下对突变基因进行在体修饰。在本研究中，AAV2/8(Y733F)介导的Cas9过表达未对视网膜的形态结构产生显著性影响。在此基础上完成了在体编辑视网膜Pde6b基因，并在离体培养的视网膜中实现了Pde6b的定点突变。然而，成对sgRNA介导的在体定点矫正pde6b突变未能够实现，需要进一步的探索。本研究为Cas9编辑体系在RP基因治疗中的应用提供了一定的理论基础。