Modification of AdenoAssociated Virus to deliver DNA directly to Mitochondria

描述（由申请人提供）：线粒体DNA突变导致一系列神经退行性疾病，目前尚无治疗方法。几乎所有这些都涉及到眼部结构，包括视神经、视网膜、眼外肌或眼睑。其中最常见的是Leber遗传性视神经病变（LHON），由呼吸链中复合物I的ND4亚基基因突变引起（半数病例）。我们的目标是通过将编码正常ND4亚基的基因传递到受影响的细胞和组织中来开发这种线粒体疾病的基因治疗。我们建议设计、修饰和测试一种腺相关病毒（AAV），该病毒在病毒包膜上附加线粒体靶向序列，将其有效载荷DNA（正常ND4亚基基因）直接递送到线粒体，以拯救培养的LHON细胞，然后再拯救我们的LHON动物模型。我们的小鼠模型显示视神经头肿胀，视神经萎缩和视网膜神经节细胞变性，这是LHON患者的特征。我们通过修饰突变的人ND4基因，使其在细胞核中表达，然后用靶向序列将其引导到线粒体中，从而建立了LHON-模型。我们的具体目标是：1)(a)将AAV病毒粒子引导到培养细胞的线粒体，并检测有效载荷DNA的存在和表达，然后(b)测试该策略对LHON细胞呼吸缺陷的拯救作用。2)将人ND4基因转染lhon模型眼，以挽救视网膜神经节细胞变性和视神经病变。为了证明我们项目的可行性，我们提供了大量的初步数据，表明我们的第一代载体针对线粒体，在培养细胞和小鼠视觉系统中表达，并且可以拯救培养的LHON细胞。这里开发的新技术几乎可以传递任何线粒体基因，因此可以提供一个平台，不仅可以治疗视力丧失、眼瘫和上睑下垂，还可以治疗由线粒体DNA突变引起的疾病患者所经历的无数残疾，包括过早死亡。我们将与其他以治疗这些疾病为目标的团体分享这一载体。

DESCRIPTION (provided by applicant): Mutations in mitochondrial DNA lead to a spectrum of neurodegenerative diseases for which no treatment exists. Almost all of them involve ocular structures that include the optic nerve, retina, extraocular muscles or eyelids. The most common of these is Leber Hereditary Optic Neuropathy (LHON) caused (in half the cases) by a mutated ND4 subunit gene of complex I in the respiratory chain. Our goal is to develop gene therapy for this mitochondrial disease by delivery of genes encoding the normal ND4 subunit to affected cells and tissues. We propose to design, modify and test an adeno-associated virus (AAV) to which a mitochondrial targeting sequence is appended to the viral envelope for delivery of its payload of DNA (a normal ND4 subunit gene), directly to the mitochondrion for rescue of cultured LHON cells and then of our animal model of LHON. Our mouse model shows optic nerve head swelling followed by optic atrophy and degeneration of retinal ganglion cells, which are characteristics of LHON patients. We developed this LHON- model by modifying the mutant human ND4 gene for expression in the nucleus then directed it to the mitochondria with a targeting sequence. Our Specific Aims are: 1) To (a) direct the AAV virion to mitochondria of cultured cells and test for the presence and expression of the payload DNA and then (b) test this strategy for the rescue of defective respiration of LHON cells. 2) To test this strategy for delivery of the human ND4 gene to the LHON-model eye for rescue of retinal ganglion cell degeneration and optic neuropathy. As proof of the feasibility of our project we provide extensive preliminary data showing that our first generation vector is targeted to mitochondria, is expressed in cultured cells as well as the murine visual system, and that it rescues cultured LHON cells. The novel technology developed here may deliver virtually any mitochondrial gene, and thus may provide the platform to treat not only visual loss, ophthalmoparesis and ptosis, but also the myriad of disabilities including premature death experienced by patients with diseases caused by mutated mitochondrial DNA. We will share the vector with other groups whose goal it is to treat these disorders.