Reference - Detail
RRC ID | 82184 |
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Author | Onishi A, Tsunekawa Y, Mandai M, Ishimaru A, Ohigashi Y, Sho J, Yasuda K, Suzuki K, Izpisua Belmonte JC, Matsuzaki F, Takahashi M. |
Title | Optimization of HITI-Mediated Gene Insertion for Rhodopsin and Peripherin-2 in Mouse Rod Photoreceptors: Targeting Dominant Retinitis Pigmentosa. |
Journal | Invest Ophthalmol Vis Sci |
Abstract |
PURPOSE:Among the genome-editing methods for repairing disease-causing mutations resulting in autosomal dominant retinitis pigmentosa, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. In this study, we aimed for the rapid optimization and validation of HITI-treatment gene constructs of this approach in developing HITI-treatment constructs for various causative target genes in mouse models of retinal degeneration. METHODS:We constructed the Cas9-driven HITI gene cassettes in plasmid vectors to treat the mouse Rho gene. A workflow utilizing in vivo electroporation was established to validate the efficacy of these constructs. Single-cell genotyping was conducted to evaluate allelic donor gene insertion. The therapeutic potency of HITI-treatment plasmid and adeno-associated virus (AAV) vectors was examined by section immunohistochemistry and optomotor response (OMR) in Rho+/P23H mutant mice. We also targeted mouse Prph2 to examine the workflow. RESULTS:The optimized HITI-treatment constructs for mouse Rho genes achieved gene insertion in 80% to 90% of transduced mouse rod photoreceptor cells. This construct effectively suppressed degeneration and induced visual restoration in mutant mice. HITI-treatment constructs for the Rhodopsin gene demonstrated efficacy in AAV vectors and are adaptable for the mouse Prph2 gene locus. CONCLUSIONS:The study showcases a workflow for the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance in inherited retinal dystrophy. These findings suggest the potential utility of this approach in developing HITI-treatment constructs for various target genes, advancing gene therapy products for diverse genetic disorders. |
Volume | 65(13) |
Pages | 38 |
Published | 2024-11-4 |
DOI | 10.1167/iovs.65.13.38 |
PII | 2802250 |
PMID | 39556087 |
PMC | PMC11578159 |
MeSH | Animals Dependovirus / genetics Disease Models, Animal* Electroporation Gene Editing / methods Genetic Therapy* / methods Genetic Vectors Mice Mice, Inbred C57BL Peripherins* / genetics Peripherins* / metabolism Plasmids / genetics Retinal Rod Photoreceptor Cells* / metabolism Retinitis Pigmentosa* / genetics Retinitis Pigmentosa* / metabolism Retinitis Pigmentosa* / therapy Rhodopsin* / genetics |
Resource | |
Human and Animal Cells | 293T(RCB2202) |