Mitochondrial DNA Delivery for LHON (Leber's Hereditary Optic Neuropathy)

描述(申请人提供)：项目摘要/摘要：自Leber遗传性视神经病变(LHON)被描述以来的130年里，一直没有找到有效的治疗方法。一代又一代的年轻人仍然失明，通常是永久性的。线粒体病因学的证明将注意力引向了寻找利用线粒体机制的治疗方法，如抗氧化剂和核基因疗法，以产生目的地为线粒体的治疗性蛋白质。然而，还没有成功的治疗方法被开发出来。Gencia公司开发了一种名为Protofect(蛋白质介导的转染法)的技术，该技术能够在体外和体内传递全长线粒体DNA(MtDNA)。Protofect是一种DNA结合的非病毒递送载体，由允许线粒体转基因的工程重组蛋白组成。在腹腔注射后，它将mtDNA输送到包括脑、肌肉和肝脏在内的多个组织中的线粒体，并实现了所传递DNA的强劲线粒体表达。这一阶段应用的目标是通过向受影响的细胞补充正常的线粒体DNA来证明原发感染作为治疗LHON的可行性。该提案将决定：(I)全长mtDNA能否被输送到视网膜神经节细胞(RGCs)，这是LHON的主要损伤部位；(Ii)以最小的细胞毒性实现最佳输送所需的条件；以及(Iii)可在体内转基因的RGCs的百分比。将具有新的RFLP或产生标记蛋白(绿色荧光蛋白，GFP)的全长mtDNA与原感染载体蛋白复合，并将其输送到RGCs的体外模型-大鼠细胞系RGC-5。线粒体DNA向细胞的传递将通过跟随GFP荧光和引入的RFLP的聚合酶链式反应来观察。TUNEL染色将被用作细胞毒性的衡量标准。在体外确定mtDNA的最佳剂量后，将通过玻璃体内注射与载体蛋白络合的mtDNA进行类似的实验。线粒体DNA的传递将通过视网膜的GFP荧光和视神经的RFLP PCR进行跟踪。该项目目标的成功实现将为对该技术在第二阶段应用中的有效性和安全性进行广泛调查奠定基础。第二阶段申请的目标将是进行必要的临床前研究，以便向FDA和生物制品评估和研究中心(CBER)申请IND治疗LHON。项目说明：在此框架下实现的目标 该提案将显示原发感染作为治疗Leber遗传性视神经病变的可行性，这是一种由mtDNA突变引起的失明形式，并为治疗其他线粒体疾病提供了基础。这对公共卫生的潜在影响是巨大的。尽管大多数个体遗传性线粒体疾病的发病率很低，但作为一个群体，它们仅在美国就估计有50万人受到影响。

DESCRIPTION (provided by applicant): Project Summary/Abstract: In the 130 years since Leber's Hereditary Optic Neuropathy (LHON) was described, no effective treatments for the disease have been found. Generations of young adults still lose sight, usually permanently. The demonstration of mitochondrial etiology has directed attention to finding treatments utilizing mitochondrial mechanisms, such as antioxidants and nuclear gene therapies to produce therapeutic proteins destined for the mitochondria. Nevertheless, no successful therapies have been developed. Gencia Corporation has developed a technology termed Protofection (Protein-Mediated Transfection) which is able to deliver full-length mitochondrial DNA (mtDNA) both in vitro and in vivo. Protofection is a DNA-binding, non-viral delivery vector consisting of an engineered recombinant protein that allows for transfection of mitochondria. After intraperitoneal injection, it delivers mtDNA to mitochondria in multiple tissues, including brain, muscle, and liver, and achieves robust mitochondrial expression of delivered DNA. The goal of this Phase I application is to show the feasibility of protofection as a treatment for LHON by supplementation of normal mitochondrial DNA to affected cells. The proposal will determine: (i) whether full- length mtDNA can be delivered to retinal ganglion cells (RGCs), the primary site of damage in LHON; (ii) the conditions necessary to achieve optimal delivery with minimal cytotoxicity; and (iii) the percentage of RGCs that can be transfected in vivo. Full-length mtDNA, engineered to have a new RFLP or to produce a marker protein (Green Fluorescent Protein, GFP), will be complexed with the protofection vector protein and delivered to the in vitro model of RGCs, the rat cell line RGC-5. The delivery of mtDNA to cells will be observed by following GFP fluorescence and by PCR for the introduced RFLP. TUNEL staining will be used as a measure of cellular toxicity. After establishing optimal dosage of mtDNA in vitro, analogous experiments will be performed in vivo through intravitreal injection of mtDNA complexed with the vector protein. The delivery of mtDNA will be tracked by GFP fluorescence in the retina and by RFLP PCR on the optic nerve. Successful achievement of the project goal will provide the basis for an extensive investigation into the efficacy and safety of this technology in a Phase II application. The goal of a Phase II application would be to conduct pre-clinical studies necessary for an IND application to the FDA and the Center for Biologics Evaluation and Research (CBER) for the treatment of LHON. Project Narrative: The aims carried out under this proposal will show feasibility for protofection as a treatment for Leber's hereditary optic neuropathy, a form of blindness caused by mutations in mtDNA and also provide a basis for treating other mitochondrial diseases. The potential impact on public health is significant. Although the incidence of most individual inherited mitochondrial diseases is low, taken as a group, they afflict an estimated 500,000 people in the U.S. alone.