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Abstract：
Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.
摘要：
Bietti结晶性视网膜营养不良（BCD）是一种常染色体隐性遗传性脉络膜视网膜变性疾病，未经批准的治疗药物。它是由CYP4V2基因突变引起的，大约80％的BCD患者在外显子7至11中携带突变。这里,我们在HEK293T细胞中应用CRISPR/Cas9介导的基于同源非依赖性靶向整合（HITI）的基因编辑疗法，BCD患者来源的iPSCs,和人源化Cyp4v3小鼠模型(h-Cyp4v3mut/mut)使用两个rAAV2/8载体通过视网膜下施用。我们发现sgRNA引导的Cas9在CYP4V2基因的内含子6上产生双链切割，HITI供体插入携带的序列，内含子6的一部分，外显子7-11，以及进入DNA断裂的终止密码子，实现精确集成，在体外和体内有效的转录和翻译。基于HITI的编辑可恢复BCD患者iPSC-RPE细胞的活力，改善了形态，h-Cyp4v3mut/mut小鼠中RPE和光感受器的数量和代谢。这些结果表明，对于那些在外显子7至11中携带突变的BCD患者，基于HITI的编辑可能是一种有前途的治疗策略，一次注射将获得终身疗效。
