文献类型： 外文期刊

作者： Chou, Shih-Jie ¹ ; Yang, Peng ⁴ ; Ban, Qian ⁵ ; Yang, Yi-Ping ⁶ ; Wang, Mong-Lien ³ ; Chien, Chian-Shiu ¹ ; Chen, Shih ¹ ;

作者机构： 1.Taipei Vet Gen Hosp, Div Basic Res, Dept Med Res, Taipei 112, Taiwan

2.Taipei Vet Gen Hosp, Dept Ophthalmol, Taipei 112, Taiwan

3.Natl Yang Ming Univ, Sch Med, Inst Pharmacol, Taipei 112, Taiwan

4.Univ Calif Los Angeles, Calif NanoSyst Inst CNSI, Dept Mol & Med Pharmacol, Crump Inst Mol Imaging CIMI, Los Angeles, CA 90095 USA

5.Anhui Univ, Sch Life Sci, Ctr Stem Cell & Translat Med, Hefei 230601, Peoples R China

6.Taipei Vet Gen Hosp, Dept Med Res, Taipei 112, Taiwan

7.Natl Yang Ming Univ, Sch Med, Taipei 112, Taiwan

8.Natl Yang Ming Univ, Sch Pharmaceut Sci, Taipei 112, Taiwan

9.Natl Yang Ming Univ, Inst Food Safety & Hlth Risk Assessment, Taipei 112, Taiwan

10.Shandong First Med Univ, Hosp 1, Shandong Prov Qianfoshan Hosp, Jinan 250014, Peoples R China

11.Anhui Acad Agr Sci, Inst Anim Husb & Vet Med, Hefei 230031, Peoples R China

12.Fudan Univ, Dept Macromol Sci, State Key Lab Mol Engn Polymers, Shanghai 200433, Peoples R China

13.Univ Calif Los Angeles, Calif NanoSyst Inst CNSI, Dept Mat Sci & Engn, Dept Chem & Biochem,Dept Bioengn, Los Angeles, CA 90095 USA

14.Univ Calif Los Angeles, Childrens Discovery & Innovat Inst, Eli & Edythe Broad Ctr Regenerat Med & Stem Cell, Calif NanoSyst Inst CNSI,Dept Pediat,David Geffen, Los Angeles, CA 90095 USA

关键词： codelivery; CRISPR; Cas9; gene therapy; retina; supramolecular nanoparticles; X-linked juvenile retinoschisis

期刊名称：ADVANCED SCIENCE （ 影响因子：16.806； 五年影响因子：17.835 ）

ISSN：

年卷期： 2020 年 7 卷 10 期

页码：

收录情况： SCI

摘要： The homology-independent targeted integration (HITI) strategy enables effective CRISPR/Cas9-mediated knockin of therapeutic genes in nondividing cells in vivo, promising general therapeutic solutions for treating genetic diseases like X-linked juvenile retinoschisis. Herein, supramolecular nanoparticle (SMNP) vectors are used for codelivery of two DNA plasmids-CRISPR-Cas9 genome-editing system and a therapeutic gene, Retinoschisin 1 (RS1)-enabling clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) knockin of the RS1 gene with HITI. Through small-scale combinatorial screenings, two SMNP vectors, with Cas9 and single guide RNA (sgRNA)-plasmid in one and Donor-RS1 and green fluorescent protein (GFP)-plasmid in the other, with optimal delivery performances are identified. These SMNP vectors are then employed for CRISPR/Cas9 knockin of RS1/GFP genes into the mouse Rosa26 safe-harbor site in vitro and in vivo. The in vivo study involves intravitreally injecting the two SMNP vectors into the mouse eyes, followed by repeated ocular imaging by fundus camera and optical coherence tomography, and pathological and molecular analyses of the harvested retina tissues. Mice ocular organs retain their anatomical integrity, a single-copy 3.0-kb RS1/GFP gene is precisely integrated into the Rosa26 site in the retinas, and the integrated RS1/GFP gene is expressed in the retinas, demonstrating CRISPR/Cas9 knockin of RS1/GFP gene.