PNA Nanoparticles for Gene Editing In Vivo

人们对基因编辑作为一种潜在的治疗人类遗传性疾病的手段非常感兴趣，例如 地中海贫血和镰状细胞病。很多工作都集中在靶向核酸酶上，例如 CRISPR/Cas9和锌指核酸酶(ZFN)，基于研究表明定点DNA损伤 强烈促进同源重组(HR)。然而，靶向核酸酶的临床应用是 受到基因组中偏离目标的切割事件的风险的挑战。作为另一种选择，最近在工作中 发表在《自然通讯》上的Ly、Saltzman和Glazer实验室已经表明，γ取代的三链- 聚乳酸-羟基醇酸形成肽核酸及其供体DNA的静脉给药(IV) 酸性(PLGA)纳米粒(NPs)进入人β-地中海贫血小鼠模型制作几乎完全 通过临床相关的β-珠蛋白基因纠正频率在造血干细胞中改善疾病 高达7%的细胞(HSCs)。小鼠表现出贫血的缓解、红细胞形态的改善和 逆转脾肿大和髓外造血，极低的非靶向效应 基因组，这项技术的一个关键优势。另一个关键优势是组件可以 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。然而，合成的 γPNA复杂且昂贵，而且无法在商业上获得，从而限制了 调查人员利用这项技术。根据RFA-RM-18-024，“扩展人类基因组 这个由Ly、Saltzman和Glazer提出的多PI提案旨在推进基于PNA/NP的 通过简化和放大PNA合成进行基因编辑，通过将新一代PNA化学结合到 通过战略性地利用细胞DNA，提高结合亲和力、增加选择性和增强效力 修复小路。具体目标是：(1)扩大PNA生产和增加DNA结合，以便 加快PNA的翻译以进行治疗性基因编辑，并使广泛采用 技术我们将制定一种对映选择性战略，以扩大单体的生产，我们将 合成和测试具有修饰碱基的γ核酸以实现更好的DNA结合性能和 克服同型嘌呤序列对三链形成的限制。(2)制定操纵战略 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 使用一种新的DNA修复抑制剂。(3)提供强大的分析平台，以评估进展 从目标1到2，并将该方法推广到多基因。我们将继续利用灵活的鼠标-和 人β-珠蛋白基因在IVS2-654地中海贫血突变中的细胞校正分析。我们预计 这项工作为设计更强大的PNA提供了基础，适用于许多人类的基因编辑 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.