Investigating mitochondrial dysfunction in neurodegeneration using A Nanoparticle-based Synthetic Mitochondrial DNA (mtDNA) Transcription Regulator

项目总结 线粒体疾病是由编码结构线粒体蛋白的基因突变引起的 参与线粒体功能的蛋白质。线粒体功能异常是最常见的 遗传性神经障碍，使它们成为理想的治疗目标。对两者之间联系的不断增长的了解 线粒体基因转录异常与人类疾病迫切需要一种有效的方法 控制线粒体DNA(MtDNA)转录。为此，开发了一种调制mtDNA的方法 转录位点对于理解和治疗线粒体相关疾病至关重要。然而， DNA结合基序向线粒体传递效率低，难以激活线粒体基因 目前的技术限制了线粒体基因组编辑/基因操作。先进的监管技术 线粒体DNA的转录依赖于外源转录因子的传递，如线粒体 转录因子A(TFAM)、DNA寡聚体或DNA碱基编辑核酸酶。此外，将这些翻译为 进入治疗学的先进工具需要在将其定向输送到线粒体方面取得实质性进展， 由于大多数线粒体转录因子(TF)和碱基编辑工具在流通过程中面临重大障碍 在血液或其他生物液体中。因此，迫切需要开发新的方法来实现选择性和 有效地激活线粒体中的基因。 针对上述挑战，本提案的主要目标是开发一种基于纳米颗粒的 合成线粒体DNA(MtDNA)转录调节剂研究线粒体功能障碍 神经退行性变。模块化MitoScrip平台将由以下材料组装：i)超小荧光金 纳米团簇(NC)作为组装生物分子配体的支架；II)合成的PIP低聚物，如mtDNA 结合结构域(DBD)对线粒体转录进行定点调控；iii)线粒体穿透 多肽(MPPs)作为线粒体定位结构域；以及iv)线粒体转录因子基序 从TFAM作为激活域(AD)。通过这样做，我们的目标是构建一种人工/合成线粒体 可：i)有效靶向线粒体基因，无细胞毒性或免疫原性(例如，病毒载体) 载体；ii)选择性地与线粒体基因组中的任何靶DNA序列结合，以及iii)可控地 下调和上调线粒体基因，最终可以改变神经细胞的命运。 我们建议客观地检验我们的中心假设，并通过解决以下问题来实现我们的目标 具体目的：目的#1−构建靶向ND6的MitoScrip(ND6-MitoScrip)以调节线粒体中的ND6基因 有效；AIM#2−验证ND6-mitoScrip对ND6基因的过度表达和改善PD患者IPSC的生存- 总的来说，我们预计我们提出的研究将提供一种创新的、高效的、 以及开发线粒体DNA介导的神经疾病治疗干预的选择性方法。

PROJECT SUMMARY Mitochondrial diseases are caused by mutations in genes that encode structural mitochondrial proteins or proteins involved in mitochondrial function. Mitochondrial function abnormalities are among the most common genetic neurological disorders, making them an ideal therapeutic target. The growing knowledge of links between aberrant mitochondrial gene transcription and human diseases critically necessitates an effective approach to controlling mitochondrial DNA (mtDNA) transcription. To this end, developing a method to modulate mtDNA transcription sites specifically is vital for understanding and treating mitochondria-related diseases. However, the Inefficient delivery of DNA binding motifs into mitochondria and difficulty activating mitochondria genes with current technologies limit mitochondrial genome editing/gene manipulation. Advanced techniques for regulating mtDNA transcription have relied on the delivery of exogenous transcription factors, such as mitochondrial transcription factor A (TFAM), DNA oligomers, or DNA-base editing nucleases. Furthermore, translating these advanced tools into therapeutics would require substantial advances in their targeted delivery into mitochondria, as most mitochondrial transcription factors (TFs) and base editing tools face significant hurdles during circulation in blood or other biofluids. Therefore, there is an urgent need to develop novel methods to achieve selective and efficient gene activation in the mitochondria. Addressing the above challenges, the main goal of this proposal is to develop a nanoparticle-based synthetic mitochondrial DNA (mtDNA) transcription regulator to investigate mitochondrial dysfunction in neurodegeneration. The modular MitoScript platform will be assembled from: i) ultra-small fluorescent gold nanoclusters (NC) as scaffolds for assembly of biomolecular ligands; ii) synthetic PIP oligomers as mtDNA binding domains (DBDs) for site-specific mitochondrial transcription regulation; iii) mitochondria-penetrating peptides (MPPs) as mitochondrial localization domains; and iv) a mitochondrial transcription factor motif derived from TFAM as an activation domain (AD). By doing so, we aim to construct an artificial/synthetic mitochondrial TF that can: i) efficiently target mitochondria genes with no cytotoxic effects or immunogenic (e.g., viral vectors) carriers; ii) selectively bind to any target DNA sequences in the mitochondria genome, and iii) controllably downregulate and upregulate mitochondria genes that can eventually alter neural cell fates. We propose to objectively test our central hypothesis and achieve our objectives by addressing the following specific aims: AIM #1 − Construct ND6-targeting MitoScript (ND6-MitoScript) to regulate ND6 genes in mitochondria efficiently; AIM #2 − Validate ND6-MitoScript for ND6 gene overexpression and improved survival in PD patient iPSC- derived neurons; Collectively, we anticipate that our proposed studies will provide an innovative, highly effective, and selective method for developing therapeutic interventions for mtDNA-mediated neurological disorders.