PNA Nanoparticles for Gene Editing In Vivo

人们对基因编辑作为治疗人类遗传疾病的潜在手段有很大的兴趣， 地中海贫血和镰状细胞病。许多努力都集中在靶向核酸酶上， CRISPR/Cas9和锌指核酸酶（ZFN），基于显示定点DNA损伤 强烈促进同源重组（HR）。然而，靶向核酸酶的临床应用受到限制。 受到基因组中脱靶切割事件风险的挑战。作为替代，在最近的工作中， 发表在《自然通讯》上，Ly，Saltzman和Glazer实验室已经表明，γ-取代的三链体- 形成肽核酸（PNA）和供体DNA，通过聚（乳酸-共-乙醇酸）静脉内（IV）递送 酸（PLGA）纳米颗粒（NPs）进入人β-地中海贫血小鼠模型产生几乎完全 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%。小鼠表现出贫血的缓解，RBC形态的改善， 脾肿大和髓外造血的逆转，在 基因组，这项技术的一个关键优势。另一个主要优点是，组件可以 化学合成并配制成纳米颗粒用于简单的IV给药。然而，合成 γ PNA是复杂且昂贵的，并且它们不是商业上可获得的，限制了γ PNA的能力。 研究人员利用这项技术。根据RFA-RM-18-024，“扩展人类基因组 Ly、Saltzman和Glazer的多PI提案旨在推进基于PNA/NP的 通过简化和扩大PNA合成的基因编辑，通过结合下一代PNA化学， 增强结合亲和力，增加选择性，增强效力，并通过战略性地利用细胞DNA 修复路径。具体目的是：（1）扩大PNA的生产并增加DNA结合，以 加快用于治疗性基因编辑的PNA的翻译，并使 技术.我们将设计一种对映选择性策略来扩大单体的生产，我们将 合成和测试具有修饰的核碱基的γ PNA，以实现改进的DNA结合特性， 克服了三链体形成的同嘌呤序列限制。(2)制定策略来操纵 DNA修复以提高PNA介导的基因编辑的效率，基于有希望的初步结果 一种新型DNA修复抑制剂(3)提供一个强大的检测平台，以评估 目的1-2，并将这种方法推广到多个基因。我们将继续利用简易鼠标-和 用于校正IVS 2 -654地中海贫血突变的人β-珠蛋白基因的基于细胞的测定。我们预计 这项工作为设计适用于许多人类基因编辑的更有效的PNA提供了基础。 遗传性疾病。

There is substantial interest in gene editing as a potential means to treat human genetic disorders such as thalassemia and sickle cell disease. Much effort has been focused on targeted nucleases such as CRISPR/Cas9 and zinc-finger nucleases (ZFNs), based on work showing that site-directed DNA damage strongly promotes homologous recombination (HR). However, clinical application of targeted nucleases is challenged by the risk of off-target cleavage events in the genome. As an alternative, in work recently published in Nature Communications, the Ly, Saltzman, and Glazer labs have shown that γ-substituted triplex- forming peptide nucleic acids (PNAs) and donor DNAs delivered intravenously (IV) via poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) 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%. 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, 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. However, synthesis of γPNAs is complicated and expensive, and they are not commercially available, limiting the ability of investigators to exploit this technology. In line with RFA-RM-18-024, “Expanding the Human Genome Engineering Repertoire”, this multi-PI proposal by Ly, Saltzman, and Glazer seeks to advance PNA/NP-based gene editing by simplifying and scaling up PNA synthesis, by incorporating next generation PNA chemistry to boost binding affinity, increase selectivity, and enhance potency, and by strategically exploiting cellular DNA repair pathways. The Specific Aims are: (1) To scale up PNA production and augment DNA binding, in order to expedite the translation of PNAs for therapeutic gene editing and enable widespread adoption of the technology. We will devise an enantioselective strategy for scaling up the production of monomers, and we will synthesize and test γPNAs with modified nucleobases to achieve improved DNA binding properties and to overcome the homopurine sequence restriction for triplex formation. (2) To develop strategies to manipulate DNA repair to enhance the efficiency of PNA-mediated gene editing, based on promising preliminary results with a novel DNA repair inhibitor. (3) To provide a robust platform of assays to evaluate the advancements from Aims 1-2 and to generalize this approach to multiple genes. We will continue to exploit facile mouse- and cell-based assays for correction of the human β-globin gene at the IVS2-654 thalassemia mutation. We expect this work to provide the basis for designing even more potent PNAs applicable to gene editing for many human genetic disorders.