LHON Gene Therapy Vector Prevents Visual Loss and Optic Neuropathy Induced by G11778A Mutant Mitochondrial DNA: Biodistribution and Toxicology Profile
LHON Gene Therapy Vector Prevents Visual Loss and Optic Neuropathy Induced by G11778A Mutant Mitochondrial DNA: Biodistribution and Toxicology Profile
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DOI：
10.1167/iovs.14-15388
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发表时间：
2014-12-01
影响因子：
4.4
通讯作者：
Guy, John
中科院分区：
文献类型：
--
作者：
Koilkonda, Rajeshwari;Yu, Hong;Guy, John
目的.证明同种异体人ND 4用于治疗携带G11778 A线粒体突变的Leber遗传性视神经病变（LHON）小鼠模型的安全性和有效性。我们通过玻璃体内注射突变体（G11778 A）人ND 4 DNA在小鼠中诱导LHON，所述突变体（G11778 A）人ND 4 DNA负责LHON的大多数情况，其使用我们将线粒体靶向序列附加到VP 2衣壳的AAV 2载体导向线粒体。然后，我们尝试使用含有异源表达的合成核编码G11778 G ND 4基因的测试品（ScAAV 2-P1 ND 4v 2）来挽救视力丧失。对照小鼠未注射或接受AAV 2-GFP或AAV 2-mCherry。在注射后2周进行RT-PCR和共聚焦显微镜检查。进行图形视网膜电图（PERG）、光谱域光学相干断层扫描（SD-OCT）、组织学和透射电子显微镜（TEM）检查。对于毒理学和生物分布研究，将供试品以不同剂量玻璃体内给予大鼠和恒河猴。突变型和野生型ND 4在视网膜神经节细胞（RGC）的线粒体中有效表达。通过连续PERG评估的视觉功能和通过连续SD-OCT评估的视网膜结构显示出供试品的显著挽救作用。组织学和超微结构分析证实，RGC的损失和轴突的死亡被ScAAV 2-P1 ND 4v 2阻止。大鼠和非人灵长类动物生物分布研究表明，载体从注射眼外扩散到脾脏和淋巴结的程度很小。包括眼睛在内的组织和器官的组织学与未感染和盐水注射的眼睛相当。异位表达的野生型ND 4防止G11778 A线粒体DNA诱导的表型，其毒理学特征可用于I期临床试验的测试。
PURPOSE. To demonstrate safety and efficacy of allotopic human ND4 for treatment of a Leber's hereditary optic neuropathy (LHON) mouse model harboring the G11778A mitochondrial mutation.METHODS. We induced LHON in mice by intravitreal injection of mutant (G11778A) human ND4 DNA, responsible for most cases of LHON, that was directed to mitochondria using an AAV2 vector to which we appended a mitochondrial targeting sequence to the VP2 capsid. We then attempted rescue of visual loss using our test article (ScAAV2-P1ND4v2) containing a synthetic nuclear encoded G11778G ND4 gene that was allotopically expressed. Control mice either were uninjected or received AAV2-GFP or AAV2-mCherry. We performed RT-PCR and confocal microscopy at 2 weeks post injection. Pattern electroretinograms (PERGs), spectral-domain optical coherence tomography (SD-OCT), histology, and transmission electron microscopy (TEM) were performed. For toxicology and biodistribution studies, the test article was administered intravitreally to rats and rhesus macaques at different doses.RESULTS. Mutant and wild-type ND4 were efficiently expressed in the mitochondria of retinal ganglion cells (RGCs). Visual function assessed by serial PERGs and retinal structure by serial SD-OCT showed a significant rescue by the test article. Histology and ultrastructural analysis confirmed that loss of RGCs and demise of axons was prevented by ScAAV2-P1ND4v2. Rat and nonhuman primate biodistribution studies showed that vector spread outside the injected eye into spleen and lymph nodes was minimal. Histopathology of tissues and organs including the eyes was comparable to that of uninfected and saline-injected eyes.CONCLUSIONS. Allotopically expressed wild-type ND4 prevents the phenotype induced by G11778A mitochondrial DNA with a toxicology profile acceptable for testing in a phase I clinical trial.