Modification of AdenoAssociated Virus to Deliver DNA Directly to Mitochondria

线粒体DNA突变导致一系列神经退行性疾病， 有有效的治疗方法。其中最常见的是利伯氏遗传性视神经病变 （LHON）由NADH脱氢酶的亚基基因（ND 1、ND 4或ND 6）中的突变引起， 它是呼吸链的复合体I。该应用程序建立在 目前的赠款，开创了腺相关病毒（AAV）载体，其中线粒体 将靶向序列（MTS）附加到病毒包膜上。经过改造的载体 NADH脱氢酶亚基4（ND 4）基因直接作用于线粒体， 也注射了突变的G11778 A ND 4的小鼠的视力丧失导致所有LHON的一半， 例我们现在将设计、修改和测试临床相关载体的有效性和安全性 用于通过递送编码正常人的基因来治疗这种线粒体疾病 ND 1、ND 4和ND 6亚基对受影响的细胞和组织的作用;用于拯救培养的人LHON 携带导致LHON的三种突变中的每一种的细胞以及转基因小鼠， 通过将含有突变的人ND 4的MTS AAV注射到胚泡中而开发。这 小鼠在出生一年后视力丧失进展为失明，视神经头肿胀 其次是神经节细胞的萎缩和变性，这是神经节细胞的特征性标志。 LHON患者我们的目标是：（1）通过开发LHON， MTS AAV载体将ND 1、ND 4和ND 6亚基基因中的每一个直接递送至细胞。 线粒体和测试表达以拯救具有100%突变的ND 1的胞质杂交细胞中的呼吸， ND 4或ND 6。我们还开发了一个单一的AAV盒，容纳所有三个基因， 需要一个IND申请FDA批准(2)评价玻璃体内注射的生物学效应 MTS AAV载体在正常小鼠中的递送导致线粒体基因转移，而不 不良影响(3)拯救转基因LHON中的视力丧失和视神经变性 我们在与人类疾病密切平行的阶段进行治疗， RGC损失和视神经萎缩，使用LHON转基因小鼠中的病毒滴度， 实验结果是无副作用的抢救。我们希望能确定 长期拯救小鼠视神经病变，因此这种方法可以在I/II期试验中进行测试。 临床旨在恢复我们的患者的视力与所有三个常见的LHON 突变。

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.