Engineered Mitochondria for Therapeutic Donation and Mitochondrial Genome Editing

项目总结/摘要 线粒体基因组的约16000个核苷酸在人类健康中发挥着巨大的作用。这 基因密集的环状基因组是独立于我们的核基因组而被包含、复制、转录和调控的。 基因组它的内容对我们身体中几乎每个细胞的功能都至关重要，所以当一个核苷酸发生突变时， 或失去，细胞中的生理过程就会崩溃。线粒体基因组的突变会导致疾病 通常与中枢神经系统、心脏和肌肉的退化有关。五千分之一的人 遗传线粒体基因组的致病突变（例如Leber遗传性视神经病变），但 这可能低估了疾病的总负担，因为每200人中就有一人携带疑似 致病突变我们并不了解所有线粒体突变的后果，这些突变通常是 组织特异性和诊断复杂。而且，线粒体基因组积累突变 在个体的一生中，这被认为在帕金森氏症等退行性疾病中发挥作用 甚至是正常的细胞老化。 与核基因组不同，在核基因组中，在活细胞中重写DNA的新兴技术使 令人惊叹的实验见解，并将很快使精确的遗传医学，线粒体基因组， 几乎没有被科学家触及过。线粒体的内膜是不可渗透的， 因此，基于模板的修复或新DNA的扩增是遥不可及的。这意味着我们可以 我们只能管理，而不是真正治愈一个人的疾病，源于突变，他们的线粒体 DNA.这也意味着我们无法通过实验将精确的突变引入线粒体DNA来测试 在受控条件下特定变化的影响。 本提案所载的研究旨在克服这些限制。我们将测试 工程化线粒体以使得能够将线粒体群体主动移植到活细胞中， 治疗线粒体基因组疾病。我们将评估线粒体的治疗效果， 在退行性疾病如Leber遗传性视神经病变的模型中移植。我们还将 修改移植线粒体的基因组以引入或修复突变，从而允许表型突变。 在受控实验框架内进行分析。这项工作的完成将产生新的机会， 难治性疾病和一种新的分子范式，以研究对所有细胞都至关重要的生物系统。

Project Summary/Abstract The ~sixteen-thousand nucleotides of the mitochondrial genome play an outsized role in human health. This gene-dense, circular genome is contained, replicated, transcribed, and regulated independently from our nuclear genome. Its contents are critical to the function of nearly every cell in our body, so when a nucleotide is mutated or lost, physiological processes in cells break down. Mutations to the mitochondrial genome cause diseases often associated with degeneration of central nervous system, heart, and muscle. One in ~five-thousand people inherits a disease-causing mutation to the mitochondrial genome (e.g. Leber’s hereditary optic neuropathy), but that likely underestimates the total burden of disease, as one in ~two-hundred people carries a suspected pathogenic mutation. We do not understand the ramifications of all mitochondrial mutations, which are often tissue-specific and complex to diagnose. Moreover, the mitochondrial genome accumulates mutations throughout the life of an individual, which are thought to play a role in degenerative diseases like Parkinson’s and even normal cellular aging. Unlike the nuclear genome, where emerging technologies to re-write DNA in living cells have enabled breathtaking experimental insights and will soon enable precision genetic medicine, the mitochondrial genome has remained nearly untouched by scientists. The inner membrane of the mitochondria is impermeable to polynucleotides, so template-based repair or augmentation with new DNA is out of reach. This means we can only manage, and never truly cure an individual with a disease that stems from a mutation to their mitochondrial DNA. It also means that we cannot experimentally introduce precise mutations to the mitochondrial DNA to test the effect of particular changes under controlled conditions. The research contained in this proposal is aimed at overcoming these limitations. We will test means of engineering mitochondria to enable active transplantation of mitochondrial populations into living cells as a therapeutic in diseases of the mitochondrial genome. We will assess therapeutic efficacy of mitochondrial transplantation in models of degenerative diseases such as Leber’s hereditary optic neuropathy. We will also modify the genome of the transplanted mitochondria to introduce or fix mutations, allowing for phenotypic analysis in a controlled experimental framework. Completion of this work will yield new opportunities to treat intractable disease and a new molecular paradigm to investigate a biological system that is critical to all cells.