Development, Ageing and Disease, UCL Institute of Ophthalmology, London EC1V 9EL, UK.
Ocular Genomics and Therapeutics, The Francis Crick Institute, London NW1 1AT, UK.
Int J Mol Sci. 2023 Oct 16;24(20):15225. doi: 10.3390/ijms242015225.
Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) patient-derived fibroblasts and (2) zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.
脉络膜视网膜变性（CHM）是一种 X 连锁的脉络膜视网膜营养不良，导致进行性视网膜变性，最终导致成年后期失明。它是由编码 Rab 逃逸蛋白 1（REP1）的基因突变引起的，REP1 在 Rab 蛋白的异戊二烯化中起着至关重要的作用，确保了正确的细胞内运输。基因增强是一种很有前途的治疗策略，目前有几个使用腺相关病毒（AAV）载体治疗 CHM 的已完成和正在进行的临床试验。然而，后期试验未能显示出显著的功能改善，并对使用病毒可能引起的炎症事件提出了安全方面的担忧。因此，需要替代的非病毒疗法。基于染色体外支架/基质附着区（S/MAR）的质粒载体被构建，其中包含人类 编码序列、GFP 报告基因和普遍启动子（pS/MAR-CHM）。这些载体在两种脉络膜视网膜变性疾病模型系统中进行了评估：（1）患者来源的成纤维细胞和（2）斑马鱼，通过 Western blot 检测 REP1 蛋白表达，体外异戊二烯化测定评估prenylation 功能的恢复。使用视网膜免疫组织化学检测注射斑马鱼视网膜中的载体表达和光感受器形态。pS/MAR-CHM 载体在 患者成纤维细胞中产生持久的 REP1 表达，并显示出prenylation 功能的显著恢复，达到 75%，表明纠正了与 CHM 相关的潜在生化缺陷。此外，在单细胞期用 pS/MAR-CHM 微注射斑马鱼时，检测到 GFP 和人 REP1 的表达。注射的 斑马鱼显示出存活率、prenylation 功能的提高和视网膜光感受器形态的改善。非病毒 S/MAR 载体有望成为一种潜在的基因增强策略，而无需使用免疫原性病毒成分，这可能适用于许多遗传性视网膜疾病基因。
