NON-NUCLEASE BASED GENE EDITING FOR HUTCHINSON-GILFORD PROGERIA

项目摘要 人们对基因编辑作为治疗人类遗传性疾病的一种手段非常感兴趣，例如哈钦森- 吉尔福德早衰综合征（HGPS）。许多努力都集中在靶向核酸酶上， CRISPR/Cas9，因为定点DNA损伤强烈促进同源重组（HR）。但是，在这方面， 靶向核酸酶的临床应用受到基因组中脱靶切割风险的挑战， 会导致致癌。作为替代方案，我们已经表明，化学修饰的三链体形成肽 核酸（TFP）和供体DNA（含有校正的碱基）通过聚（乳酸- 共-羟基乙酸（PLGA）纳米颗粒进入人β-地中海贫血小鼠模型产生了几乎完全 改善疾病，造血干细胞中临床相关的β-珠蛋白基因校正频率 细胞（HSC）高达7%。TFP可以以序列特异性的方式结合双链体DNA，从而刺激DNA的合成。 DNA修复和重组。小鼠表现出贫血的缓解，RBC形态的改善， 以及脾肿大和髓外造血的逆转， 与基于核酸酶的方法相比，这是该技术的一个关键优势。另一个关键 其优点在于，组分可以化学合成并配制成纳米颗粒， IV给药。在拟议的工作中，我们将测试是否可以将相同的技术应用于 编辑LMNA点突变的效率相同。在这里，我们的中心假设是建立的可行性， 一种新的微创和创新的治疗HGPS疾病的范例：应用进一步 用于LMNA突变定点编辑的核酸化学和纳米颗粒技术的进展 在体内通过简单的IV输注进行，具有高效和低毒性。我们将追求两个具体目标：目标1） 开发新一代化学修饰的PNA以促进LMNA突变位点的基因编辑 目的2）通过PLGA的简单IV输注来测试体内LMNA突变位点处的基因编辑效率 NP.这项工作将为HGPS的新型基因编辑疗法奠定基础，该疗法具有高效， 与现有的基于核酸酶的方法相比，脱靶效应的风险低得多。

Project Summary There is substantial interest in gene editing as a means to treat human genetic disorders such as Hutchinson- Gilford Progeria Syndrome (HGPS). Much effort has been focused on targeted nucleases such as CRISPR/Cas9, since site-directed DNA damage strongly promotes homologous recombination (HR). However, clinical application of targeted nucleases is challenged by the risk of off-target cleavage in the genome, which can lead to carcinogenesis. As an alternative, we have shown that chemically modified triplex-forming peptide nucleic acids (TFPs) and donor DNAs (containing corrected base) delivered intravenously (IV) via poly(lactic- co-glycolic) acid (PLGA) nanoparticles into a mouse model of human β-thalassemia produced almost complete amelioration of the disease, with clinically relevant β-globin gene correction frequencies in hematopoietic stem cells (HSCs) of up to 7%. TFPs can bind to duplex DNA in a sequence-specific manner and thereby stimulate DNA repair and recombination. The mice showed alleviation of anemia, improvement in RBC morphologies, and reversal of splenomegaly and extramedullary hematopoiesis with extremely low off-target effects in the genome compared to nuclease-based approaches, a key advantage of this technology. The other key advantage is that the components can be synthesized chemically and formulated into nanoparticles for simple IV administration. In the proposed work, we will test whether the same technology can be applied with the same efficiency for editing LMNA point mutation. Herein, our central hypothesis is to establish the feasibility of a new minimally invasive and innovative therapeutic paradigm for HGPS disease: application of further advances in nucleic acid chemistry and nanoparticle technology for the site-directed editing of LMNA mutation in vivo by facile IV infusion with high efficiency and low toxicity. We will pursue two specific aims; Aim 1) Development of new generation chemically modified PNAs to boost gene editing at the LMNA mutation site and in Aim 2) To test the gene editing efficiency at LMNA mutation site in vivo by simple IV infusion of PLGA NP. This work will lay the foundation for a novel gene editing therapy for HGPS that has a high efficiency and much lower risk of off-target effects compared to existing nuclease based approaches.