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-羟酰辅酶A的独特并发症 脱氢酶缺乏症（LCHADD）和线粒体三功能蛋白缺乏症（TFPD），罕见的遗传性 脂肪酸氧化（FAO）病症。逐渐的黄斑色素结块，随后进行性恶化 几乎所有的LCHADD患者在儿童期就开始发生视网膜的损伤，并随着时间的推移而进展。这 最初表现为夜间视力下降，发展为色觉丧失，最终表现为夜间视力下降。 中央视觉虽然通过新生儿筛查和持续的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，LCHADD/TFPD儿童 继续遭受渐进性失明。需要新的治疗LCHADD相关视网膜病变的方法， 缺乏合适的实验模型。我们最近创建了2个LCHADD视网膜病变模型; 小鼠敲入c.1528G>C（HADHA基因中导致LCHADD的常见突变）， RPE样细胞从源自患者成纤维细胞的诱导多能干细胞（iPSC）分化。 LCHADD相关视网膜病变的病理生理学尚未完全了解，但开始于 视网膜色素上皮（RPE）的丧失。两种潜在的分子机制包括： 减少FAO以支持正常的RPE功能，或积累部分氧化脂肪酸的选择性毒性， 酸性代谢物。血液中LCHADD特异性3-羟基酰基肉毒碱浓度较低的儿童， 视网膜功能保留，这意味着视网膜损伤可能是通过有毒脂肪酸的积累介导的。 酸性中间体如果是这样的话，那么针对外周器官如肌肉或肝脏的基因治疗可以 降低循环毒性代谢物，预防视网膜变性，并解决其他LCHADD相关 并发症或者，视网膜保存可能需要直接在RPE中恢复LCHAD活性， 而循环中的酰基肉毒碱只是视网膜中部分FAO的标志物。我们将使用我们的两个模型， LCHADD-视网膜病变，以测试哪种方法将防止RPE功能障碍和视网膜变性。 LCHADD-RPE表现出降低的氧化棕榈酸酯、积累的酰基肉毒碱和中性肉毒碱的能力。 脂质，并且与野生型（WT）RPE相比对H2 O2氧化应激的易感性增加。我们提出 一系列实验来测试LCHADD相关的RPE改变，如脂质加工和对 与WT RPE相比，在不同FAO条件下H2 O2处理。此外，LCHADD小鼠具有 与WT小鼠相比，视觉表现和视网膜电图（ERG）反应降低。我们的目标是 表征LCHADD小鼠的视力、视网膜结构和功能，并测试肝细胞因子的作用。 与视网膜定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特异性病理学，并为治疗LCHADD的转化努力提供基础 视网膜病变，可能与视网膜基因治疗，一个新的治疗方式，其中OHSU是一个国家的领导者。

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.