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

描述（由申请人提供）：项目概要/摘要：自Leber遗传性视神经病变（LHON）被描述以来的130年里，还没有发现有效的治疗方法。一代又一代的年轻人仍然失去视力，通常是永久性的。线粒体病因学的证明已经将注意力转向寻找利用线粒体机制的治疗，例如抗氧化剂和核基因疗法以产生用于线粒体的治疗性蛋白质。然而，尚未开发出成功的治疗方法。Gencia公司开发了一种称为Protofection（蛋白质介导的转染）的技术，该技术能够在体外和体内递送全长线粒体DNA（mtDNA）。原转染是一种DNA结合的非病毒递送载体，由允许转染线粒体的工程重组蛋白组成。腹膜内注射后，它将mtDNA递送到多个组织中的线粒体，包括脑、肌肉和肝脏，并实现递送DNA的稳健线粒体表达。该I期申请的目的是通过向受影响的细胞补充正常线粒体DNA来显示原转染作为LHON治疗的可行性。该提案将确定：（i）全长mtDNA是否可以被递送至视网膜神经节细胞（RGC）（LHON中的主要损伤部位）;（ii）以最小的细胞毒性实现最佳递送所必需的条件;和（iii）可以在体内转染的RGC的百分比。全长mtDNA，经工程改造具有新的RFLP或产生标记蛋白（绿色荧光蛋白，GFP），将与原转染载体蛋白复合并递送至RGC的体外模型，大鼠细胞系RGC-5。将通过跟踪GFP荧光和通过引入的RFLP的PCR来观察mtDNA向细胞的递送。TUNEL染色将用作细胞毒性的测量。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复合的mtDNA在体内进行类似的实验。mtDNA的递送将通过视网膜中的GFP荧光和视神经上的RFLP PCR来跟踪。项目目标的成功实现将为在第二阶段应用中对该技术的有效性和安全性进行广泛调查提供基础。II期申请的目标是进行向FDA和生物制品评价和研究中心（CBER）申请IND治疗LHON所需的临床前研究。项目说明：本项目的目标 该提案将显示protofection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，也为治疗其他线粒体疾病提供了基础。对公众健康的潜在影响是巨大的。虽然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，仅在美国就有大约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.