Related Experiment Video
Updated: Jun 7, 2025
10:10
Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research
Published on: November 2, 2018
9.2K
Optimization of HITI-Mediated Gene Insertion for Rhodopsin and Peripherin-2 in Mouse Rod Photoreceptors: Targeting
Akishi Onishi1,2,3,4,5, Yuji Tsunekawa6,7, Michiko Mandai1,3,5
1Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
Investigative Ophthalmology & Visual Science
|November 18, 2024
Summary
Homology-independent targeted integration (HITI) gene therapy effectively restored vision in mouse models of inherited retinal disease. This approach rapidly optimizes gene constructs for treating dominant-negative mutations, advancing gene therapy for genetic disorders.
Area of Science:
- Genetics
- Molecular Biology
- Ophthalmology
Background:
- Autosomal dominant retinitis pigmentosa is caused by mutations leading to photoreceptor degeneration.
- Current gene therapies often target specific mutations, limiting their applicability.
- Homology-independent targeted integration (HITI) offers a mutation-agnostic approach for gene insertion.
Purpose of the Study:
- To rapidly optimize and validate HITI-treatment gene constructs for autosomal dominant retinitis pigmentosa.
- To develop mutation-agnostic therapeutic products for inherited retinal dystrophies.
- To assess the efficacy of HITI in mouse models targeting Rho and Prph2 genes.
Main Methods:
- Constructed Cas9-driven HITI gene cassettes in plasmid vectors for the mouse Rho gene.
- Established an in vivo electroporation workflow for construct validation.
- Evaluated gene insertion via single-cell genotyping and therapeutic potency using immunohistochemistry and optomotor response (OMR) in Rho+/P23H mutant mice.
Main Results:
- Optimized HITI constructs achieved 80-90% gene insertion in mouse rod photoreceptor cells.
- Demonstrated suppression of degeneration and visual restoration in mutant mice.
- Showcased adaptability of HITI constructs for Rhodopsin and Prph2 genes using AAV vectors.
Conclusions:
- Developed a workflow for rapid optimization and validation of HITI gene constructs.
- Demonstrated high efficacy of HITI for treating dominant-negative inherited retinal dystrophy.
- Highlighted the potential of HITI for developing gene therapy products for various genetic disorders.
