Exploring the pathophysiology and treatment of LCHADD retinopathy

项目摘要 进行性视网膜病变伴视力丧失是长链3-羟基辅酶A的独特并发症 脱氢酶缺乏(LCHADD)和线粒体三功能蛋白缺乏(TFPD)，罕见遗传 脂肪酸氧化(FAO)紊乱。黄斑色素逐渐聚集，继而进行性恶化 几乎所有的LCHADD患者都会发生视网膜病变，从童年开始，并随着时间的推移而发展。这 最初表现为夜间视力下降，进展为色觉丧失，最终下降 中心视觉。而通过新生儿筛查和持续饮食治疗进行早期诊断可以减缓 进展，目前还没有针对视网膜病变的成功治疗，患有LCHADD/TFPD的儿童 继续遭受渐进性失明。LCHADD相关性视网膜病变需要新的治疗方法，但 目前还缺乏合适的实验模型。我们最近建立了两个LCHADD-视网膜病变模型； 小鼠敲击导致LCHADD的HADHA基因常见突变C.1528G和GT；C，并培养 从患者成纤维细胞来源的诱导多能干细胞(IPSC)分化为RPE样细胞。 LCHADD相关性视网膜病变的病理生理学尚未完全了解，但始于 视网膜色素上皮(RPE)丢失。两种潜在的分子机制包括：由于 粮农组织降低以支持正常的RPE功能，或部分氧化脂肪积累的选择性毒性 酸性代谢物。血中LCHADD特异性3-羟基肉碱浓度较低的儿童 保留的视网膜功能意味着视网膜损伤可能是通过有毒脂肪的堆积来调节的 酸性中间体。如果是这样，那么针对肌肉或肝脏等外周器官的基因治疗可能 降低循环中的有毒代谢产物，防止视网膜退化，并解决其他与LCHADD相关的问题 并发症。或者，视网膜保存可能需要直接在RPE中恢复LCHAD活性， 而循环中的酰肉碱只是视网膜中部分粮农组织的一个标志。我们将使用我们的2个型号的 LCHADD-视网膜病变，以测试哪种方法可以防止RPE功能障碍和视网膜变性。 LCHADD-RPE表现出氧化棕榈酸酯的能力降低，积累的酰肉碱和中性 与野生型(WT)RPE相比，RPE对过氧化氢氧化应激的敏感性增加。我们建议 一系列实验以测试与LCHADD相关的RPE改变，如脂质处理和对 不同粮农组织条件下的过氧化氢处理与WT RPE的比较。此外，LCHADD-MICE具有 与WT小鼠相比，视觉表现和视网膜电信号(ERG)反应降低。我们的目标是 LCHADD小鼠视力、视网膜结构和功能的测定及肝素对其的影响 与视网膜导向基因治疗方法相比。这些实验的结果将扩大我们的 了解LCHAD的特异性病理，为翻译治疗LCHADD提供基础 视网膜病变，可能与视网膜基因疗法，这是一种新的治疗方式，OHSU是全国领先的。

Project Summary Progressive retinopathy with vision loss is a unique complication of Long-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency (LCHADD) and mitochondrial Trifunctional Protein Deficiency (TFPD), rare genetic fatty acid oxidation (FAO) disorders. Gradual macular pigment clumping, followed by progressive deterioration of the retina occurs in almost all LCHADD patients beginning in childhood and progressing over time. This manifests initially as decreased night vision, progresses to loss of color vision, and ultimately to decreased central vision. While early diagnosis through newborn screening and sustained dietary therapy can slow progression, no successful treatment directed at retinopathy currently exists, and children with LCHADD/TFPD continue to suffer progressive blindness. Novel treatments for LCHADD-associated retinopathy are needed but appropriate experimental models have been lacking. We recently created 2 models of LCHADD-retinopathy; a murine knockin of c.1528G>C, the common mutation in the HADHA gene that causes LCHADD, and cultured RPE-like cells differentiated from induced pluripotent stem cells (iPSC) derived from patients' fibroblasts. The pathophysiology of LCHADD-associated retinopathy is not completely understood but begins with loss of retinal pigment epithelium (RPE). Two potential molecular mechanisms include; energy deficit due to decreased FAO to support normal RPE functions, or selective toxicity of accumulating partially oxidized fatty acid metabolites. Children with lower blood concentrations of LCHADD-specific 3-hydroxy-acylcarnitines have preserved retinal function implying that retinal damage may be mediated through accumulation of toxic fatty acid intermediates. If so, then gene therapy directed toward a peripheral organ such as muscle or liver could lower circulating toxic metabolites, prevent retinal degeneration, and address other LCHADD-associated complications. Alternatively, retinal preservation may require the restoration of LCHAD activity directly in RPE, and circulating acylcarnitines are simply a marker of partial FAO in the retina. We will use our 2 models of LCHADD-retinopathy to test which approach will prevent RPE dysfunction and retinal degeneration. LCHADD-RPE exhibit decreased ability to oxidize palmitate, accumulated acylcarnitines and neutral lipids, and increased susceptibility to H2O2 oxidative stress in comparison to wild type (WT) RPE. We propose a series of experiments to test LCHADD-associated RPE alterations such as lipid processing and sensitivity to H2O2 treatment under different FAO conditions compared to WT RPE. Additionally, the LCHADD-mouse has decreased visual performance and electroretinogram (ERG) responses compared to WT mice. Our goal is to characterize the visual acuity, retinal structure and function of LCHADD mice and to test the effects of hepatic versus retinal directed gene therapy approaches. The outcome of these experiments will expand our understanding of LCHAD-specific pathology and provide the basis for a translational effort to treat LCHADD retinopathy, potentially with retinal gene therapy, a new treatment modality of which OHSU is a national leader.