DeVine Tiffany, Elizondo Gabriela, Gaston Garen, Babcock Shannon J, Matern Dietrich, Shchepinov Mikhail S, Pennesi Mark E, Harding Cary O, Gillingham Melanie B
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States.
Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, United States.
Invest Ophthalmol Vis Sci. 2024 Sep 3;65(11):22. doi: 10.1167/iovs.65.11.22.
Progressive choroid and retinal pigment epithelial (RPE) degeneration causing vision loss is a unique characteristic of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), a fatty acid oxidation disorder caused by a common c.1528G>C pathogenic variant in HADHA, the α subunit of the mitochondrial trifunctional protein (TFP). We established and characterized an induced pluripotent stem cell (iPSC)-derived RPE cell model from cultured skin fibroblasts of patients with LCHADD and tested whether addition of wildtype (WT) HAHDA could rescue the phenotypes identified in LCHADD-RPE.
We constructed an rAAV expression vector containing 3' 3xFLAG-tagged human HADHA cDNA under the transcriptional control of the cytomegalovirus (CMV) enhancer-chicken beta actin (CAG) promoter (CAG-HADHA-3XFLAG). LCHADD-RPE were cultured, matured, and transduced with either AAV-GFP (control) or AAV-HADHA-3XFLAG.
LCHADD-RPE express TFP subunits and accumulate 3-hydroxy-acylcarnitines, cannot oxidize palmitate, and release fewer ketones than WT-RPE. When LCHADD-RPE are exposed to docosahexaenoic acid (DHA), they have increased oxidative stress, lipid peroxidation, decreased viability, and are rescued by antioxidant agents potentially explaining the pathologic mechanism of RPE loss in LCHADD. Transduced LCHADD-RPE expressing a WT copy of TFPα incorporated TFPα-FLAG into the TFP complex in the mitochondria and accumulated significantly less 3-hydroxy-acylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress following DHA exposure than control.
iPSC-derived LCHADD-RPE are susceptible to lipid peroxidation mediated cell death and are rescued by exogenous HADHA delivered with rAAV. These results are promising for AAV-HADHA gene addition therapy as a possible treatment for chorioretinopathy in patients with LCHADD.
进行性脉络膜和视网膜色素上皮（RPE）变性导致视力丧失是长链3-羟基酰基辅酶A脱氢酶缺乏症（LCHADD）的一个独特特征，LCHADD是一种脂肪酸氧化障碍疾病，由线粒体三功能蛋白（TFP）的α亚基HADHA中常见的c.1528G>C致病性变异引起。我们从LCHADD患者的培养皮肤成纤维细胞中建立并鉴定了一种诱导多能干细胞（iPSC）衍生的RPE细胞模型，并测试添加野生型（WT）HAHDA是否能挽救LCHADD-RPE中发现的表型。
我们构建了一个rAAV表达载体，其包含在巨细胞病毒（CMV）增强子-鸡β-肌动蛋白（CAG）启动子（CAG-HADHA-3XFLAG）转录控制下的3' 3xFLAG标记的人HADHA cDNA。将LCHADD-RPE培养、成熟，并用AAV-GFP（对照）或AAV-HADHA-3XFLAG进行转导。
LCHADD-RPE表达TFP亚基并积累3-羟基酰基肉碱，不能氧化棕榈酸，且与野生型RPE相比释放的酮类更少。当LCHADD-RPE暴露于二十二碳六烯酸（DHA）时，它们的氧化应激增加、脂质过氧化增加、活力降低，并被抗氧化剂挽救，这可能解释了LCHADD中RPE丧失的病理机制。转导表达TFPα野生型拷贝的LCHADD-RPE将TFPα-FLAG整合到线粒体中的TFP复合物中，积累的3-羟基酰基肉碱显著减少，对棕榈酸反应释放更多酮类，并且在DHA暴露后比对照更耐氧化应激。
iPSC衍生的LCHADD-RPE易受脂质过氧化介导的细胞死亡影响，并通过rAAV递送的外源性HADHA得到挽救。这些结果对于AAV-HADHA基因添加疗法作为LCHADD患者脉络膜视网膜病变的一种可能治疗方法很有前景。
