RRC ID 82184
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)