Developing next generation synthetic nucleic acid analogous for sickle cell disease gene editing

项目摘要 人们对基因编辑作为治疗人类遗传性疾病（如镰状细胞病）的一种手段非常感兴趣。 疾病（SCD）。许多努力已经集中在靶向核酸酶如CRISPR/Cas9上，因为定点核酸酶可以在靶向核酸酶上进行。 DNA损伤强烈促进同源重组（HR）。然而，针对性的临床应用 核酸酶受到基因组中脱靶切割的风险的挑战，脱靶切割可导致致癌作用。作为 另一种选择是，我们已经表明，化学修饰的三链体形成肽核酸（TFPs）和供体 通过聚乳酸-羟基乙酸共聚物（PLGA）静脉内（IV）递送的DNA（含校正碱基） 将纳米颗粒植入人β-地中海贫血小鼠模型中，几乎完全改善了β-地中海贫血的症状。 疾病，造血干细胞（HSC）中临床相关的β-珠蛋白基因校正频率高达 至7%。TFP可以以序列特异性的方式结合双链体DNA，从而刺激DNA修复， 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，基因组脱靶效应极低， 到基于核酸酶的方法，这是这项技术的一个关键优势。另一个关键优势是， 这些成分可以化学合成并配制成纳米颗粒用于简单的IV给药。在 在拟议的工作中，我们将测试是否可以以相同的效率应用于密码子 6的β-珠蛋白基因，镰状细胞病突变的网站。在这里，我们的中心假设是建立 镰状细胞病新型微创创新治疗模式的可行性：应用 核酸化学和纳米颗粒技术在SCD定点编辑方面的进一步进展 通过高效低毒的简便静脉输注，在体内对β-珠蛋白基因进行突变。该项目将 通过核酸化学的进步，最终帮助将SCD的基因疗法转化为临床实践 和药物输送。我们将追求目标1）开发新一代化学改性的PNA， 在SCD突变位点进行基因编辑。该方法的有效性将在镰状细胞病中进行评估。 小鼠模型我们还将探索基于PNA的基因编辑的机制。目标2）确定新的 基于纳米治疗的策略，通过使药物能够进入骨髓来将试剂递送至HSC 简单静脉输注NP后这项工作将为SCD的新型基因编辑疗法奠定基础， 与现有的基于核酸酶的方法相比，具有高效率和低得多的脱靶效应风险。

Project Summary There is substantial interest in gene editing as a means to treat human genetic disorders such as sickle cell disease (SCD). 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 codon 6 of the β-globin gene, the site of the sickle cell disease mutation. Herein, our central hypothesis is to establish the feasibility of a new minimally invasive and innovative therapeutic paradigm for sickle cell disease: application of further advances in nucleic acid chemistry and nanoparticle technology for the site-directed editing of SCD mutation in the β-globin gene in vivo by facile IV infusion with high efficiency and low toxicity. This project will eventually help translate gene therapies for SCD to clinical practice through advances in nucleic acid chemistry and drug delivery. We will pursue Aim 1) Development of new generation chemically modified PNAs to boost gene editing at the SCD mutation site. The efficacy of the approach will be evaluated in a sickle cell disease mouse model. We will also explore the mechanism of PNA based gene editing. In Aim 2) Identify novel nanotherapeutics based strategies to deliver reagent to HSCs by enabling penetrance into the bone marrow following simple IV infusion of NP. This work will lay the foundation for a novel gene editing therapy for SCD that has a high efficiency and much lower risk of off-target effects compared to existing nuclease based approaches.