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

研究总结 线粒体dna(Mtdna)突变会导致一系列神经退行性疾病，而对于这些疾病， 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)的突变，这是呼吸系统的复合体I 链条。与线粒体DNA突变引起的所有疾病一样，LHON的治疗总体上是不够的。 部分原因是将DNA输送到细胞器的障碍。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)载体来屏障，线粒体靶向该载体 将MTS序列附加到病毒衣壳上。修改后的载体将ND4基因直接输送到 线粒体对G11778A ND4突变小鼠视力丧失的逆转作用 LHON例，其余由ND1和ND6突变引起。我们现在将设计、修改和测试疗效和 通过传递编码基因的基因治疗这种线粒体疾病的临床相关载体的安全性 正常线粒体编码的人ND4亚单位的启动子和调控元件对受影响的细胞和 拯救携带NADH脱氢酶亚单位4突变的培养的人LHON细胞的组织 导致LHON，然后转基因小鼠和灵长类动物携带突变的ND4。我们开发的ND4小鼠 将含有突变的G11778A ND4的MTS AAV注射到啮齿类动物囊胚中，可导致视力丧失 出生后一年至失明，视神经头肿胀，视网膜萎缩变性 神经节细胞，这是LHON患者的特征特征。我们的目标是：(A)促进 通过构建适应野生型的临床级MTS AAV载体进行LHON的翻译研究 ND4LHON基因和调控元件在单个AAV盒中。(B)测试表情以抢救呼吸 在带有突变的G11778A ND4的小鼠和灵长类动物的胞质细胞和视觉系统中。(C)评估 MTS AAV载体玻璃体内注射对正常啮齿动物和灵长类动物的生物学效应 线粒体基因转移无不良反应。(D)开发单一AAV的IND应用程序 包含正常ND4，用于未来的I/II期临床试验，旨在恢复患者的视力 U10患者中最常见和最严重的LHON突变并防止其家庭成员视力丧失 成功完成本R24号文件后提出申请。

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.