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In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment.
- Source :
-
Nature communications [Nat Commun] 2024 May 06; Vol. 15 (1), pp. 3773. Date of Electronic Publication: 2024 May 06. - Publication Year :
- 2024
-
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-Cyp4v3 <superscript>mut/mut</superscript> ) 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-Cyp4v3 <superscript>mut/mut</superscript> 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.<br /> (© 2024. The Author(s).)
- Subjects :
- Humans
Animals
HEK293 Cells
Mice
Disease Models, Animal
Mutation
Retinal Pigment Epithelium metabolism
Retinal Pigment Epithelium pathology
Genetic Vectors genetics
Introns genetics
Exons genetics
Gene Editing methods
CRISPR-Cas Systems
Corneal Dystrophies, Hereditary genetics
Corneal Dystrophies, Hereditary therapy
Corneal Dystrophies, Hereditary pathology
Corneal Dystrophies, Hereditary metabolism
Induced Pluripotent Stem Cells metabolism
Genetic Therapy methods
Cytochrome P450 Family 4 genetics
Cytochrome P450 Family 4 metabolism
Retinal Diseases
Subjects
Details
- Language :
- English
- ISSN :
- 2041-1723
- Volume :
- 15
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Nature communications
- Publication Type :
- Academic Journal
- Accession number :
- 38710738
- Full Text :
- https://doi.org/10.1038/s41467-024-48092-9