Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
Cell and Gene Therapy in Ophthalmology Laboratory, RIKEN Baton Zone Program, Kobe, Japan.
Invest Ophthalmol Vis Sci. 2024 Nov 4;65(13):38. doi: 10.1167/iovs.65.13.38.
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.
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.
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.
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.
在修复导致常染色体显性遗传视网膜色素变性的致病突变的基因组编辑方法中，同源非依赖性靶向整合（HITI）介导的致病基因正常形式的基因插入是有用的，因为它允许开发针对突变的治疗产品。在这项研究中，我们旨在快速优化和验证该方法的 HITI 治疗基因构建体，以在视网膜变性的小鼠模型中针对各种致病变异位点基因开发 HITI 治疗构建体。
我们构建了 Cas9 驱动的 HITI 基因盒，以质粒载体治疗小鼠 Rho 基因。建立了利用体内电穿孔的工作流程来验证这些构建体的功效。单细胞基因分型用于评估等位基因供体基因插入。通过对 Rho+/P23H 突变小鼠进行切片免疫组化和光动反应（OMR）评估，研究了 HITI 治疗质粒和腺相关病毒（AAV）载体的治疗效力。我们还针对小鼠 Prph2 进行了靶向研究，以检验该工作流程。
优化的用于小鼠 Rho 基因的 HITI 治疗构建体在 80%至 90%的转导的小鼠杆状光感受器细胞中实现了基因插入。该构建体有效抑制了突变小鼠的变性并诱导了视觉恢复。Rhodopsin 基因的 HITI 治疗构建体在 AAV 载体中具有疗效，并且适用于小鼠 Prph2 基因座。
该研究展示了一种快速优化和验证针对遗传性视网膜营养不良中显性遗传的高效 HITI 治疗基因构建体的工作流程。这些发现表明，该方法在开发针对各种靶基因的 HITI 治疗构建体方面具有潜在的应用价值，推进了针对多种遗传疾病的基因治疗产品的发展。
