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

项目摘要 基因编辑作为一种治疗人类遗传性疾病如镰状细胞的手段引起了极大的兴趣 疾病(SCD)。自从定点定位以来，许多工作都集中在靶向核酸酶上，如CRISPR/Cas9 DNA损伤强烈促进同源重组(HR)。然而，靶向的临床应用 核酸酶面临着基因组非靶标切割的风险，这可能导致癌症发生。AS 另一种选择，我们已经证明了化学修饰的三链形成肽核酸(TFP)和供体 DNA(含正确碱基)通过聚乳酸-羟基乙酸(PLGA)静脉注射(IV) 纳米粒进入人类β-地中海贫血小鼠模型后几乎完全改善了 疾病，与临床相关的β-珠蛋白基因校正频率在UP的造血干细胞(HSC)中 降至7%。TFP可以以序列特异性的方式与双链DNA结合，从而刺激DNA修复和 重组。小鼠表现出贫血的缓解，红细胞形态的改善，以及 脾肿大和髓外造血在基因组中的非靶向效应极低的比较 以核酸酶为基础的方法，这项技术的一个关键优势。另一个关键优势是 成分可以通过化学方法合成并配制成纳米颗粒，用于简单的静脉注射。在……里面 在提出的工作中，我们将测试相同的技术是否可以以相同的效率应用于密码子 6的β-珠蛋白基因，镰状细胞病突变的位置。在这里，我们的中心假设是建立 镰状细胞病微创创新治疗方案的可行性：应用 用于SCD定点编辑的核酸化学和纳米颗粒技术的进一步进展 β-珠蛋白基因体内高效低毒输注突变的研究这个项目将 最终通过核酸化学的进步帮助将SCD的基因治疗转化为临床实践 和药物输送。我们将追求目标1)开发新一代化学修饰的PNA以促进 SCD突变位点的基因编辑。该方法的有效性将在镰状细胞疾病中进行评估。 老鼠模型。我们还将探索基于PNA的基因编辑机制。在目标2)中识别小说 基于纳米疗法的策略，通过使试剂能够穿透骨髓将试剂输送到HSCs 在单纯静脉滴注NP后。这项工作将为治疗SCD的新的基因编辑疗法奠定基础 与现有的基于核酸酶的方法相比，具有更高的效率和更低的脱靶效应风险。

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.