Applicability, efficacy and safety of targeted vehicle mediated in vivo base editing in a mouse model of hereditary hemochromatosis type I

血色素沉着病是白人人群中最常见的遗传性代谢性疾病之一，主要源于HFE基因的纯合C282Y突变。基因c.845位置的G > A过渡导致HFE蛋白错误折叠，最终导致其在细胞膜上缺失。这就导致了铁在肠道内的持续吸收，以及铁在肝脏、胰腺和心脏等不同器官中的积累。受影响的患者可能表现为肝硬化、糖尿病以及心肌病。目前最先进的治疗血色素沉着症包括终身静脉切开术和螯合剂如去铁胺的应用。在携带人类突变的小鼠模型（129-HFE tm 1.1Nca）中，我们的目标是通过应用靶向ABE碱基编辑RNA在体内纠正突变。在这项提议中，我们将开发先进的瞬时ABE碱基编辑器疗法，基于mRNA/gRNA分子结合到脂质纳米颗粒（LNP）或病毒样颗粒（VLP）中来纠正C282Y点突变。最初，我们将通过分析肝酶和细胞因子水平进行剂量发现和毒性研究。然后，我们将进行长达12个月的长期实验，以证明疗效和遗传毒性。治疗方法的有效性将通过DNA测序测量的碱基转化率和血色素沉着症相关血液参数的正常化来分析。碱基编辑器疗法的遗传毒性将在全球范围内进行分析，通过寻找DNA和RNA水平上的改变，这些改变可能发生在目标序列以外的其他区域(例如：“目标”)。总的来说，我们的实验将为遗传性血色素沉着症的安全有效的治疗提供基础，并为其他遗传性肝病的治疗提供蓝图。

Hemochromatosis is one of the most common inherited metabolic diseases among white populations and predominantly originates from a homozygous C282Y mutation in the HFE gene. The G > A transition at position c.845 of the gene causes misfolding of the HFE protein, ultimately resulting in its absence at the cell membrane.This consequently leads to continuous iron uptake in the intestine and accumulation of iron in various organs such as liver, pancreas and heart. Affected patients may exhibit liver cirrhosis, diabetes mellitus as well as cardiomyopathy. The current state-of-the-art treatment of hemochromatosis includes life-long phlebotomy and the application of chelating agents such as deferoxamine. In a mouse model (129-HFE tm 1.1Nca), which carries the human mutation, we aim to correct the mutation in vivo by application of a targeted ABE base editor RNA. In this proposal we will develop advanced transient ABE base editor therapies either based on mRNA/gRNA molecules incorporated into lipid nanoparticles (LNP) or into virus-like particles (VLP) to correct the C282Y point mutation. Initially, we will perform dose finding and toxicity studies by analyzing liver enzymes and cytokine levels. Then, we will perform long-term experiments for up to 12 months to demonstrate efficacy and genotoxicity. Efficacy of the therapeutic approach will be analysed by the rate of base conversion as measured by DNA sequencing and the normalisation of hemochromatosis relevant blood parameters. Genotoxicity of the base editor therapy will analysed on a global scale by searching for alterations at DNA and RNA levels, which could occur in other regions than the target sequence (e.g. "off targets"). Overall, our experiments will provide the basis for a safe and efficient therapy for hereditary hemochromatosis and a blueprint for the treatment of other hereditary liver diseases.