Mitochondrial Gene Therapy Augments Mitochondrial Physiology in a Parkinson's Disease Cell Model
Mitochondrial Gene Therapy Augments Mitochondrial Physiology in a Parkinson's Disease Cell Model
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DOI：
10.1089/hum.2009.023
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发表时间：
2009-08-01
影响因子：
4.2
通讯作者：
Bennett, James P., Jr.
中科院分区：
文献类型：
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作者：
Keeney, Paula M.;Quigley, Caitlin K.;Bennett, James P., Jr.
帕金森病 (PD) 中的神经退行性变主要影响黑质中的多巴胺能神经元，其中与年龄相关的、越来越多的细胞失去可检测到的呼吸活动，这与完整线粒体 DNA (mtDNA) 的消耗有关。补充线粒体DNA可能会改善神经元生物能功能并防止进一步的细胞死亡。我们开发了一种技术（“ProtoFection”），该技术使用重组人线粒体转录因子 A (TFAM)，该重组人线粒体转录因子 A (TFAM) 经 N 端蛋白转导结构域 (PTD) 改造，随后添加 SOD2 线粒体定位信号 (MLS)，将 mtDNA 货物递送至活细胞的线粒体。MTD-TFAM (MTD=PTD+MLS="线粒体转导结构域") 结合 mtDNA，并将其快速跨质膜转运至线粒体。为了进行治疗原理验证，我们使用从市售人类基因组 DNA（gDNA；罗氏）生成的 mtDNA 在帕金森病 cybrid 细胞中测试了 ProtoFection 技术。单次暴露于 MTD-TFAM+mtDNA 复合物后 9 至 11 周，呼吸受损和 mtDNA 基因减少的 PD cybrid 细胞的 mtDNA 基因拷贝数增加至 24 倍，mtDNA 衍生的 RNA 增加至 35 倍，TFAM 和 ETC 蛋白、细胞呼吸和线粒体运动速度增加。没有或极少基础线粒体损伤的杂种细胞对治疗的反应减少或没有，这表明治疗选择性的可能性。将PD而非对照细胞杂种细胞暴露于单独的MTD-TFAM蛋白或MTD-TFAM+mtDNA复合物会增加PGC-1α的表达，表明线粒体生物发生的激活。用于线粒体基因治疗的 ProtoFection 技术有望改善受损 PD 神经元的生物能量功能，但需要进一步开发来定义其药效学并描绘其分子机制。与本研究中使用的混合 gDNA 相比，还不清楚用于生成 mtDNA 的单一供体 gDNA 是否是首选治疗方法。
Neurodegeneration in Parkinson's disease (PD) affects mainly dopaminergic neurons in the substantia nigra, where age-related, increasing percentages of cells lose detectable respiratory activity associated with depletion of intact mitochondrial DNA (mtDNA). Replenishment of mtDNA might improve neuronal bioenergetic function and prevent further cell death. We developed a technology ("ProtoFection'') that uses recombinant human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction domain (PTD) followed by the SOD2 mitochondrial localization signal (MLS) to deliver mtDNA cargo to the mitochondria of living cells. MTD-TFAM (MTD=PTD+MLS="mitochondrial transduction domain'') binds mtDNA and rapidly transports it across plasma membranes to mitochondria. For therapeutic proof-of-principle we tested ProtoFection technology in Parkinson's disease cybrid cells, using mtDNA generated from commercially available human genomic DNA (gDNA; Roche). Nine to 11 weeks after single exposures to MTD-TFAM+mtDNA complex, PD cybrid cells with impaired respiration and reduced mtDNA genes increased their mtDNA gene copy numbers up to 24-fold, mtDNA-derived RNAs up to 35-fold, TFAM and ETC proteins, cell respiration, and mitochondrial movement velocities. Cybrid cells with no or minimal basal mitochondrial impairments showed reduced or no responses to treatment, suggesting the possibility of therapeutic selectivity. Exposure of PD but not control cybrid cells to MTD-TFAM protein alone or MTD-TFAM+mtDNA complex increased expression of PGC-1 alpha, suggesting activation of mitochondrial biogenesis. ProtoFection technology for mitochondrial gene therapy holds promise for improving bioenergetic function in impaired PD neurons and needs additional development to define its pharmacodynamics and delineate its molecular mechanisms. It also is unclear whether single-donor gDNA for generating mtDNA would be a preferred therapeutic compared with the pooled gDNA used in this study.