Modification of AdenoAssociated Virus to deliver DNA directly to Mitochondria

修饰腺相关病毒以将 DNA 直接递送至线粒体

• 批准号: 7686732
• 负责人: John Guy
• 金额: $ 38.01万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7686732
• 关键词: Affect; Animal Model; Antibodies; Apoptosis; Blindness; Capsid; Cell Nucleus; Cell Respiration; Cells; Cessation of life; Characteristics; Complex; Cultured Cells; DNA; Data; Dependovirus; Disease; Epitopes; Eye; Eyelid structure; Fluorescence; Galactose; Gene Delivery; Gene Expression; Generations; Genes; Glucose; Goals; Human; Immunochemistry; Lead; Leber' s Hereditary Optic Neuropathy; Link; Magnetic Resonance Imaging; Measurement; Measures; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Modification; Mus; Mutate; Mutation; Neurodegenerative Disorders; Ophthalmopareses; Optic Atrophy; Optic Disk; Optic Nerve; Parents; Patients; Plasmids; Production; Ptosis; Research Personnel; Respiration; Respiratory Chain; Retina; Retinal Ganglion Cells; Structure; Swelling; Testing; Time; Tissues; Transmission Electron Microscopy; Viral; Virion; Virus; Visual system structure; cell growth; design; disability; effective therapy; experience; ganglion cell; gene therapy; improved; in vivo; mouse model; mutant; new technology; optic nerve disorder; orbit muscle; premature; transmission process; vector

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.

描述（由申请人提供）：线粒体DNA中的突变导致不存在治疗的神经退行性疾病。它们几乎涉及包括视神经，视网膜，眼外肌肉或眼睑的眼部结构。其中最常见的是Leber遗传性视神经病神经病（LHON）（在一半的情况下）是由呼吸链中复合物I的突变的ND4亚基基因引起的。我们的目标是通过递送编码正常ND4亚基的基因来开发这种线粒体疾病的基因疗法。我们建议设计，修改和测试与腺相关的病毒（AAV），将线粒体靶向序列附加到病毒膜上，以传递其有效载荷DNA（正常的ND4亚基基因），直接将其直接送至培养的Lhon细胞和我们的动物模型的线粒体中，以营救培养的Lhon细胞和LHON动物模型。我们的小鼠模型显示了视神经头肿胀，随后是视网膜神经节细胞的视神经萎缩和变性，这是LHON患者的特征。我们通过修改突变的人ND4基因以在细胞核中的表达来开发这种LHON模型，然后以靶向序列将其引导到线粒体。我们的具体目的是：1）（a）将AAV病毒座引导到培养细胞的线粒体上，并测试有效载荷DNA的存在和表达，然后（b）测试该策略以挽救LHON细胞有缺陷的呼吸。 2）测试该策略以将人ND4基因传递到LHON模型眼中，以营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，我们提供了广泛的初步数据，表明我们的第一代载体针对线粒体，在培养的细胞和鼠视觉系统中表达，并拯救了培养的Lhon细胞。此处开发的新技术几乎可以提供任何线粒体基因，因此可以提供不仅治疗视觉丧失，眼科和pTosis的平台，还可以提供无数的残疾，包括由突变的线粒体DNA引起的疾病患者所经历的早期死亡。我们将与其他目标是治疗这些疾病的其他群体共享矢量。