Mitochondrial Genome Replacement Therapy (MGRT)

线粒体基因组替代疗法 (MGRT)

基本信息

批准号：
7670892
负责人：
SHAHARYAR M KHAN
金额：
$ 12.3万
依托单位：
GENCIA CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2009-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7670892
关键词：
Achievement Address Adult Affect Aging Alzheimer&apos s Disease Animal Model Animals Area Bacteriophage lambda Biochemical Biology Blindness Canis familiaris Cell Culture Techniques Cell Death Cell Line Cell Nucleus Cells Childhood Cloning DNA Development Diabetes Mellitus Disease Encephalopathies Engineering Evaluation Research Generations Genetic Engineering Genome Genome engineering Goals Housing Human Human Cloning In Vitro Incidence Individual Inherited Investigation Investigational Drugs Investigational New Drug Application Lactic Acidosis Leber&apos s Hereditary Optic Neuropathy Length Life Location Longevity Malignant Neoplasms Measurable Methods Mitochondria Mitochondrial DNA Mitochondrial Diseases Morbidity - disease rate Mus Mutate Mutation Nuclear Parkinson Disease Patients Phase Phenotype Primates Proteins Public Health Publishing Rattus Replacement Therapy Research Site Small Business Innovation Research Grant Stroke Technology Testing Therapeutic Time Tissues Transfection United States Variant Vision age related base deafness early childhood economic impact effective therapy human disease in vivo insight mitochondrial DNA mutation mitochondrial dysfunction mitochondrial genome mortality mutant new technology pre-clinical public health relevance research clinical testing restriction enzyme tool vector willingness young adult

项目摘要

DESCRIPTION (provided by applicant): In the past two decades mutations in the mitochondrial genome were found to be responsible for a significant burden of human disease. More recently mtDNA mutations were shown to accumulate over time and may be responsible for some phenotypes of aging. Whereas DNA transfections into the nuclear genome are commonplace and have yielded great insight into biology, no mitochondrial genome engineering methods have been published. Further complicating this methodological hurdle is the fact that multiple mitochondrial genomes can exist within a single mitochondrion and tens of hundreds of genomes within a single cell. What is required is a strategy that not only delivers DNA to mitochondria but also selectively removes and replaces the mitochondrial genomes in the cell. Gencia has developed Protofection(tm), a technology capable of delivering full-length mitochondrial DNA in vitro and in vivo, as well as a cloning method capable of producing full- length human mitochondrial DNA in a modified bacteriophage lambda vector and host. Through the use of protofection and a restriction-enzyme strategy, Gencia will prove the concept of fully replacing defective mtDNA as a therapy (or, Mitochondrial Genome Replacement Therapy - MGRT) for mitochondrial disease. In this Phase I SBIR, Gencia will develop MGRT for the treatment of a major cause of childhood blindness, LHON. MGRT will be used to treat a cybrid cell line derived from a LHON patient with wild-type cloned human mtDNA. This will result in a shift of the heteroplasmic ratios towards the healthy, wild-type engineered genome. The degree of correction of the LHON biochemical phenotype will be analyzed and correlated with the amount of engineered mtDNA delivered to cells. The specific proof-of-concept tasks are: (i) produce sufficient amounts of cGLP quality human and mouse cloned mtDNA as well as the protofection protein vector; (ii) introduce cloned human mtDNA expressing a restriction enzyme into a homoplasmic LHON cell line aiming for at least 90% content of delivered DNA, less than 10% cell death; and (iii) verify that this degree of replacement leads to measurable improvements in biochemical phenotypes. If successful, a Phase II SBIR will target testing of MGRT in animals to prove the ability to shift heteroplasmic ratios towards delivered mtDNA in vivo. While the goal of the project is to provide proof-of-concept of MGRT for LHON and to conduct important pre-clinical testing, insights and discoveries generated in this translational project should also validate MGRT as a platform technology for the treatment of other mitochondrial diseases. PUBLIC HEALTH RELEVANCE: The aims carried out under this proposal will show feasibility for MGRT (Mitochondrial Genome Replacement Therapy) as a treatment for Leber's Hereditary Optic Neuropathy (LHON), a form of blindness caused by mutations in mtDNA. In the 130 years since LHON was described, no effective treatments for the disease have been found. Generations of young adults still lose sight, usually permanently. Successful achievement of the aims will 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 US alone.

描述（由申请人提供）：在过去的二十年中，线粒体基因组中的突变被发现负责人类疾病的重大负担。最近显示，mtDNA突变会随着时间的推移积累，可能导致某些衰老表型。尽管DNA转染到核基因组是司空见惯的，并且对生物学产生了深刻的见解，但尚未发表过线粒体基因组工程方法。这种方法上的障碍的进一步复杂化是，多个线粒体基因组可以存在于单个细胞中的单个线粒体和数十基因组中。所需的是一种策略，不仅将DNA传递到线粒体，还可以选择性地去除并取代细胞中的线粒体基因组。 Gencia开发了Protofection（TM），这是一种能够在体外和体内输送全长线粒体DNA的技术，以及一种克隆方法，能够在改良的噬菌体lambda载体和宿主中产生全长的人形线粒体DNA。通过使用原理和限制性酶策略，Gencia将证明将有缺陷的mtDNA作为一种治疗（或，线粒体基因组替代疗法 - MGRT）的概念。在这一阶段I SBIR中，Gencia将开发MGRT来治疗儿童失明的主要原因，LHON。 MGRT将用于治疗源自具有野生型克隆人mtDNA的LHON患者的Cybrid细胞系。这将导致异质比向健康的野生型工程基因组转移。 LHON生化表型的校正程度将进行分析并与输送到细胞的工程mtDNA的量相关。具体的概念验证任务是：（i）产生足够量的CGLP质量人和小鼠克隆的mtDNA以及原构蛋白载体；（ii）将克隆的人mtDNA引入，将限制酶表达到同型LHON细胞系中，旨在至少90％的递送DNA含量，小于10％的细胞死亡；（iii）验证这种替代程度会导致可测量的生化表型改善。如果成功，II期SBIR将靶向动物中MGRT的测试，以证明在体内将异质比向递送的mtDNA转移的能力。尽管该项目的目的是为LHON提供MGRT的概念证明并进行重要的临床前测试，但该转化项目中产生的洞察力和发现也应验证MGRT作为治疗其他线粒体疾病的平台技术。公共卫生相关性：根据该提案进行的目的将显示MGRT（线粒体基因组替代疗法）的可行性，作为对Leber的遗传性视神经病变（LHON）的治疗，这是MTDNA突变引起的盲目形式。在描述了LHON以来的130年中，未发现该疾病的有效治疗方法。几代年轻人通常会永久失去视力。成功实现目标还将为治疗其他线粒体疾病提供基础。对公共卫生的潜在影响很大。尽管大多数个体遗传的线粒体疾病的发生率很低，但作为一个小组，他们仅在美国就遭受了大约500,000人的困扰。