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: 9767197
• 负责人: Hong Yu
• 金额: $ 36.81万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767197
• 关键词: ATP Synthesis Pathway; Adverse effects; Affect; Alleles; American; Animals; Atrophic; Autopsy; Biological; Birth; Blindness; Capsid Proteins; Categories; Cell Line; Cell physiology; Cells; Characteristics; Childhood; Clinical; Clinical Trials; Complex; Cytochrome c Reductase; DNA; DNA delivery; Defect; Dependovirus; Development; Disease; Electroretinography; Epitopes; Eye; Gene Delivery; Gene Expression; Gene Transfer; Genes; Genetic Code; Goals; Grant; Heart; Homologous Gene; Human; Injections; Knowledge; Laser Scanning Confocal Microscopy; Lead; Leber' s Hereditary Optic Neuropathy; Length; Link; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modification; Molecular; Mus; Mutate; Mutation; NADH dehydrogenase (ubiquinone); Nerve Degeneration; Neuro-Ocular System; Neurodegenerative Disorders; Noise; Nucleotides; Optic Atrophy; Optic Disk; Optic Nerve; Organelles; Oxidative Phosphorylation; Pathogenesis; Patients; Pattern; Phase; Preclinical Testing; Prevention; Proteins; Rare Diseases; Recovery; Respiration; Respiratory Chain; Retina; Rodent; Safety; Swelling; Technology; Testing; Tissues; Toxic effect; Transgenic Mice; Transgenic Organisms; Treatment Efficacy; Vertebral column; Viral; Virion; Virus; Vision; Visual; Work; adeno-associated viral vector; blastocyst; clinically relevant; design; effective therapy; efficacy testing; emerging adult; experimental study; ganglion cell; gene therapy; human disease; mutant; new technology; non-invasive imaging; optic nerve disorder; prevent; promoter; success; transgene expression; translational study; vector; visual optics

Mutations in mitochondrial DNA lead to a spectrum of neurodegenerative diseases for which no effective treatment exists. The most common of these is Leber's hereditary optic neuropathy (LHON) caused by mutations in subunit genes (ND1, ND4 or ND6) of NADH dehydrogenase, which is complex I of the respiratory chain. This application builds on the successes of the current grant that pioneered an adeno-associated virus (AAV) vector to which a mitochondrial targeting sequence (MTS) was appended to the viral envelope. The modified vector delivered the NADH dehydrogenase subunit 4 (ND4) gene directly to the mitochondria for prevention of visual loss in mice also injected with a mutated G11778A ND4 responsible for half of all LHON cases. We will now design, modify and test the efficacy and safety of a clinically relevant vector for treatment of this mitochondrial disease by delivery of genes encoding the normal human ND1, ND4 and ND6 subunits to affected cells and tissues; for rescue of cultured human LHON cells harboring each of the three mutations causing LHON and also a transgenic mouse we developed by injection of the MTS AAV containing mutant human ND4 into the blastocyst. This mouse has visual loss progressing to blindness a year after birth, optic nerve head swelling followed by atrophy and degeneration of ganglion cells, which are the characteristic hallmarks of LHON patients. Our Aims are: (1) To facilitate translational studies for LHON by developing MTS AAV vectors to deliver each of the ND1, ND4 and ND6 subunit genes directly to the mitochondria and test expression to rescue respiration in cybrid cells with100% mutated ND1, ND4 or ND6. We also develop a single AAV cassette that accommodates all three genes that would require a single IND for FDA approval. (2) To evaluate biological effects of intravitreal delivery of MTS AAV vectors in normal mice that result in mitochondrial gene transfer without adverse effects.(3) To rescue visual loss and optic nerve degeneration in transgenic LHON mice, we carry out treatment at stages that closely parallel the human disease before and after RGC loss and optic atrophy, using viral titers in LHON transgenic mice that in our current experiments resulted in rescue without adverse effects. We hope to identify the conditions for long-term rescue of optic neuropathy in mice, so that this approach can be tested in a phase I/II clinical designed to restore the vision of our patients with all three of the common LHON mutations.

线粒体DNA突变会导致一系列神经退行性疾病，对这些疾病来说， 有效的治疗方法是存在的。其中最常见的是Leber遗传性视神经病变 (LHON)由NADH脱氢酶亚单位基因(ND1、ND4或ND6)突变引起， 这是呼吸链的复合体I。这个应用程序建立在 目前的赠款开创腺相关病毒(AAV)载体，线粒体到该载体上 将靶向序列(MTS)附加到病毒包膜上。修改后的向量传递 NADH脱氢酶亚单位4(ND4)基因直接连接到线粒体以预防 同时注射突变的G11778A ND4导致一半LHON的小鼠视力丧失 案子。我们现在将设计、修改和测试一种临床相关载体的有效性和安全性 通过传递编码正常人类的基因来治疗这种线粒体疾病 ND1、ND4和ND6亚单位与受影响的细胞和组织；拯救培养的人LHON 携带导致LHON的三种突变的细胞以及一只转基因小鼠 通过将含有突变的人ND4的MTS AAV注射到囊胚中而开发的。这 小鼠出生一年后视力丧失进展至失明，视神经头部肿胀 其次是神经节细胞萎缩和变性，这是 LHON患者。我们的目标是：(1)促进LHON的翻译研究 MTS AAV载体将ND1、ND4和ND6亚基基因分别直接传递到 线粒体和检测表达以挽救100%突变的ND1细胞的呼吸， ND4或ND6。我们还开发了一种单一的AAV盒式磁带，它容纳了所有三种基因 将需要一个单一的IND来获得FDA的批准。(2)评价玻璃体内植入的生物学效应 在正常小鼠体内传递MTS AAV载体导致线粒体基因转移而不需要 不良反应。(3)挽救转基因LHON的视力损失和视神经变性 小鼠，我们进行的治疗阶段与人类疾病的前后阶段非常相似 RGC丢失和视神经萎缩，使用病毒滴度在LHON转基因小鼠中，在我们目前的 实验结果表明，救援没有产生不良影响。我们希望确定以下条件： 长期抢救小鼠视神经病变，以便这种方法可以在I/II阶段进行测试 临床设计用于恢复我们所有三种常见LHON患者的视力 突变。

项目成果

Comparison of different gene-therapy methods to treat Leber hereditary optic neuropathy in a mouse model.

• DOI: 10.3389/fnins.2023.1119724
• 发表时间: 2023
• 期刊: FRONTIERS IN NEUROSCIENCE
• 影响因子: 4.3
• 作者: Velmurugan, Sindhu;Chou, Tsung-Han;Eastwood, Jeremy D.;Porciatti, Vittorio;Liu, Yuan;Hauswirth, William W.;Guy, John;Yu, Hong
• 通讯作者: Yu, Hong