Dual Supramolecular Nanoparticle Vectors Enable CRISPR/Cas9-Mediated Knockin of Retinoschisin 1 Gene-A Potential Nonviral Therapeutic Solution for X-Linked Juvenile Retinoschisis
Dual Supramolecular Nanoparticle Vectors Enable CRISPR/Cas9-Mediated Knockin of Retinoschisin 1 Gene-A Potential Nonviral Therapeutic Solution for X-Linked Juvenile Retinoschisis
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DOI：
10.1002/advs.201903432
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发表时间：
2020-05-01
期刊：
影响因子：
15.1
通讯作者：
Chiou, Shih-Hwa
中科院分区：
文献类型：
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作者：
Chou, Shih-Jie;Yang, Peng;Chiou, Shih-Hwa
同源非依赖的靶向整合(HITI)策略使CRISPR/Cas9介导的治疗基因在体内未分裂的细胞中有效敲打，有望成为治疗X连锁青少年视网膜劈裂等遗传性疾病的通用治疗方案。在这里，超分子纳米颗粒(SMNP)载体被用于共传递两个DNA质粒-CRISPR-Cas9基因组编辑系统和治疗基因Retinoschisin 1(RS1)-使RS1基因的簇状间隔短回文重复序列(CRISPR)相关蛋白9(CRISPR/Cas9)能够与Hiti一起敲打。通过小规模的组合筛选，筛选出了两种递送性能最好的SMNP载体，一种是Cas9和单引导RNA(SgRNA)载体，另一种是供体RS1和绿色荧光蛋白(GFP)载体。然后利用这些SMNP载体在体外和体内将RS1/GFP基因的CRISPR/CAS9敲击到小鼠ROSA26的安全港位置。体内研究包括将两个SMNP载体玻璃体内注射到小鼠眼内，然后用眼底相机和光学相干断层扫描重复眼部成像，并对采集的视网膜组织进行病理和分子分析。小鼠眼部器官保持其解剖结构的完整性，RS1/GFP基因被精确整合到视网膜的rosa26位点，整合的RS1/GFP基因在视网膜中表达，证实了RS1/GFP基因的CRISPR/Cas9敲打。
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.