Transgenic Mitomice Models for Treatment of Blinding Diseases

描述（由申请人提供）：虽然突变的线粒体（mt）DNA与Leber遗传性视神经病变（LHON）的失明密切相关，但尚未开发具有突变的mtDNA复合物I亚基的真正动物模型，该模型将能够探测视神经病变的发病机制并测试潜在的治疗途径。由于将DNA输送到线粒体中存在障碍，因此不可能制作线粒体突变引起的人类疾病的动物模型。我们通过将线粒体靶向序列（MTS）附加到腺相关病毒（AAV）的病毒蛋白衣壳上以将突变的人ND4基因（负责所有LHON病例的一半）引导到鼠线粒体中来绕过这一障碍。当由人线粒体启动子驱动时，突变型人ND 4在鼠线粒体中的表达诱导视力丧失，伴随视网膜中的神经节细胞及其轴突（包括视神经）的进行性死亡，从而重现人LHON病症的特征。我们追求这个项目的动机是为无法治愈的导致失明的线粒体疾病开发基因疗法。为此，我们必须（1）开发一种合理的疗法，MTS AAV将正常基因递送到受影响的线粒体，以及（2）创建具有突变的复合物I亚基基因的动物模型，该突变的复合物I亚基基因重现人类疾病，以测试潜在的治疗方法和类似人类疾病阶段的视神经病变的发病机制。为了产生转基因小鼠，我们通过显微注射到小鼠的胚泡中将含有突变的人ND 4等位基因的MTS AAV递送到胚胎干细胞，产生具有进行性视网膜神经节细胞（RGC）死亡和视神经病变的后代，其具有在出生后一年稳定下降至噪声水平的模式视网膜电图（PERG）振幅。为了监测活动物中的线粒体基因表达，我们将线粒体编码的红色荧光蛋白（mCherry）添加到含有突变型人ND4的MTS AAV构建体中，所述突变型人ND4可以通过激光扫描检眼镜检查（LSO）在活后代中以及迄今为止在其后代的三代中可视化。我们的具体目标从理解突变型NADH脱氢酶亚基ND 4在小鼠中的后果到开发表达负责LHON的另外两种突变型人类复合物I亚基（ND 1和ND 6）的额外转基因系，这是合乎逻辑的进展。

DESCRIPTION (provided by applicant): While mutated mitochondrial (mt)DNA is firmly linked to the blindness of Leber's hereditary optic neuropathy (LHON), a bona fide animal model with mutated mtDNA complex I subunits that would enable probing the pathogenesis of the optic neuropathy and testing of potential avenues for therapy has yet to be developed. It had not been possible to produce animal models of human diseases caused by mitochondrial mutations due to the barrier in delivering DNA into mitochondria. We circumvented this barrier by appending to the adenoassociated virus (AAV) a mitochondrial targeting sequence (MTS) to the viral protein capsid to direct the mutant human ND4 gene (responsible for half of all LHON cases) into murine mitochondria. When driven by a human mitochondrial promoter, expression of mutant human ND4 in murine mitochondria induced visual loss with a progressive demise of ganglion cells in the retina and their axons comprising the optic nerve, thus recapitulating the hallmarks of the human LHON disorder. Our motivation in pursuing this project is to develop gene therapy for untreatable mitochondrial diseases that lead to blindness. For this, we have to (1) develop a plausible therapy, an MTS AAV to deliver normal genes to affected mitochondria and (2) create animal models with mutated complex I subunit genes that recapitulate the human disorder in which to test potential treatments and the pathogenesis of optic neuropathy at stages that resemble the human disease. To generate a transgenic mouse we delivered the MTS AAV containing the mutant human ND4 allele to embryonic stem cells by microinjection into the blastocyst of the mouse, generating offspring with progressive retinal ganglion cell (RGC) demise and optic neuropathy with pattern electroretinogram (PERG) amplitudes declining steadily to noise levels one year after birth. To monitor mitochondrial gene expression in live animals we added a mitochondrial encoded red fluorescent protein (mCherry) to the MTS AAV construct containing mutant human ND4 that could be visualized by laser scanning ophthalmoscopy (LSO) in live offspring and so far in three generations of their progeny. Our Specific Aims logically progress from understanding the consequences of mutant NADH dehydrogenase subunit ND4 in mice to developing additional transgenic lines expressing the two other mutant human complex I subunits (ND1 and ND6) responsible for LHON.