Engineered Mitochondria for Therapeutic Donation and Mitochondrial Genome Editing

项目摘要/摘要 线粒体基因组的大约16,000个核苷酸在人类健康中扮演着非常重要的角色。这 基因密集的环状基因组包含、复制、转录和调控独立于我们的核 基因组。它的含量对我们体内几乎每个细胞的功能都是至关重要的，所以当一个核苷酸发生突变时 或者丢失，细胞中的生理过程就会被破坏。线粒体基因组的突变会导致疾病 常与中枢神经系统、心脏和肌肉的退化有关。五千人中就有一人 遗传了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 组织特异性和复杂的诊断。此外，线粒体基因组积累了突变 在一个人的一生中，它们被认为在像帕金森氏症这样的退行性疾病中发挥作用 甚至是正常的细胞衰老。 与核基因组不同，在活细胞中重写DNA的新兴技术使 惊人的实验洞察力，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。线粒体的内膜是不透水的 多核苷酸，所以基于模板的修复或用新DNA增强是遥不可及的。这意味着我们可以 只管理，永远不会真正治愈患有线粒体突变所致疾病的人 DNA这也意味着我们不能通过实验将精确的突变引入线粒体dna进行测试。 在受控条件下特定变化的效果。 这项提案中包含的研究旨在克服这些限制。我们将测试 设计线粒体，使线粒体群体能够作为一种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 移植于退行性疾病的模型，如Leber遗传性视神经病变。我们还将 修改移植的线粒体的基因组以引入或修复突变，从而允许表型 在受控实验框架下进行分析。这项工作的完成将产生新的治疗机会 难治疾病和研究对所有细胞至关重要的生物系统的新分子范例。

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.