Mito-Targeted AAV to treat Leber Hereditary Optic Neuropathy caused by ND4 mutations

研究综述 线粒体DNA（mtDNA）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Leber遗传性视神经病变（LHON）， NADH脱氢酶亚基基因（ND 1、ND 4或ND 6）的突变，其是呼吸系统的复合物I， 链LHON的治疗与所有由突变的mtDNA引起的疾病一样是不够的， 部分原因是将DNA运送到细胞器中的障碍。我们已经成功地突破了这一点 通过开发一种开创性的腺相关病毒（AAV）载体， 序列（MTS）附加到病毒衣壳。经修饰的载体将ND 4基因直接递送至 线粒体用于逆转具有突变的G11778 A ND 4的小鼠的视力丧失， LHON病例，其余由突变的ND 1和ND 6引起。我们现在将设计，修改和测试功效， 用于通过递送编码线粒体疾病的基因治疗该线粒体疾病的临床相关载体的安全性 - 正常的经尿道编码的人ND 4亚基的启动子和调节元件， 用于拯救具有NADH脱氢酶亚基4突变的培养的人LHON细胞的组织 导致LHON，然后是ND 4突变的转基因小鼠和灵长类动物。我们开发的ND 4小鼠 将含有突变的G11778 A ND 4的MTS AAV注射到啮齿动物胚泡中， 至出生后一年失明，视神经乳头肿胀，继而视网膜萎缩变性， 神经节细胞，这是LHON患者的特征性标志。我们的目标是：（A）促进 通过开发容纳野生型LHON的临床级MTS AAV载体进行LHON的翻译研究 ND 4 LHON基因和调控元件在单个AAV盒中。(B)测试表情以拯救呼吸 在胞质杂交细胞和视觉系统的小鼠和灵长类动物与突变的G11778 A ND 4。(C)评价 玻璃体内递送MTS AAV载体在正常啮齿动物和灵长类动物中的生物学效应， 线粒体基因转移无副作用。(D)为单个AAV开发IND申请 含有正常ND 4的用于未来I/II期临床试验，旨在恢复患有 最常见和最严重的LHON突变，并防止其家庭成员的视力丧失， 成功完成此R24后的申请。

Research Summary Mutations in mitochondrial DNA (mtDNA) lead to a spectrum of neurodegenerative diseases for which no effective treatment exists. The most common of these is Leber hereditary optic neuropathy (LHON) caused by mutations in NADH dehydrogenase subunit genes (ND1, ND4 or ND6), which is complex I of the respiratory chain. Therapies for LHON in common with all disorders caused by mutated mtDNA are inadequate, in large part because of the barrier in delivering DNA into the organelle. We have successfully broken through this barrier by developing a pioneering adeno-associated virus (AAV) vector to which a mitochondrial targeting sequence (MTS) was appended to the viral capsid. The modified vector delivered the ND4 gene directly to the mitochondria for reversal of visual loss in mice with mutated G11778A ND4 responsible for more than half of all LHON cases, the rest caused by mutated ND1 and ND6. 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 promoter and regulatory elements of a normal mitochondrially encoded human ND4 subunit to affected cells and tissues for rescue of cultured human LHON cells harboring the NADH dehydrogenase subunit 4 mutation causing LHON and then transgenic mice and primates with mutated ND4. The ND4 mouse we developed by injection of a MTS AAV containing mutated G11778A ND4 into the rodent blastocyst has visual loss progressing to blindness a year after birth, optic nerve head swelling followed by atrophy and degeneration of retinal ganglion cells, which are the characteristic hallmarks of LHON patients. Our goals are: (A) To facilitate translational studies for LHON by developing a clinical grade MTS AAV vector that accommodates the wild-type ND4 LHON gene and regulatory elements in a single AAV cassette. (B) To test expression to rescue respiration in cybrid cells and the visual system of mice and primates with mutated G11778A ND4. (C) To evaluate biological effects of intravitreal delivery of MTS AAV vectors in normal rodents and primates that result in mitochondrial gene transfer without adverse effects. (D) To develop an IND application for a single AAV containing normal ND4 for a future phase I/II clinical trial designed to restore the vision of patients with the commonest and most severe LHON mutation and prevent visual loss in their family members in a U10 application to follow successful completion of this R24.