In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment

Xiang Meng, Ruixuan Jia, Xinping Zhao, Fan Zhang, Shaohong Chen, Shicheng Yu, Xiaozhen Liu, Hongliang Dou, Xuefeng Feng, Jinlu Zhang, Ni Wang, Boling Xu and Liping Yang ()
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Xiang Meng: Third Hospital, Peking University
Ruixuan Jia: Third Hospital, Peking University
Xinping Zhao: LTD
Fan Zhang: LTD
Shaohong Chen: LTD
Shicheng Yu: Third Hospital, Peking University
Xiaozhen Liu: Third Hospital, Peking University
Hongliang Dou: Third Hospital, Peking University
Xuefeng Feng: Third Hospital, Peking University
Jinlu Zhang: LTD
Ni Wang: LTD
Boling Xu: Third Hospital, Peking University
Liping Yang: Third Hospital, Peking University

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-48092-9

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