PNA Nanoparticles for Gene Editing In Vivo

用于体内基因编辑的 PNA 纳米颗粒

基本信息

批准号：
10414795
负责人：
PETER M GLAZER
金额：
$ 40.42万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-05 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10414795
关键词：
Adopted Adoption Affinity Anemia Benchmarking Binding Binding Sites Biological Assay CRISPR/Cas technology Cell Survival Cells Chemicals Chemistry Chromatin Chromosomes Clinical Collaborations Communication Communities Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator DNA DNA Binding DNA Damage DNA Repair DNA Repair Pathway DNA strand break Data Development Disease Disease model Event Extramedullary Hematopoiesis Follow-Up Studies Formulation Foundations Frequencies Gene Frequency Genes Genetic Diseases Genetic Recombination Genome Genome engineering Genomics Glycolates Goals Hematopoietic stem cells Hemoglobin concentration result Hereditary Disease Human Human Genetics Human Genome Injections Intravenous Intravenous infusion procedures Lead Measures Mediating Mendelian disorder Methods Morphology Mucopolysaccharidosis I H Mus Mutation Nature Nylons Oligonucleotides Peptide Nucleic Acids Phenotype Polymers Positioning Attribute Production Property Publishing Purines Reagent Reporter Research Personnel Risk Sickle Cell Anemia Site Splenomegaly Structure Technology Testing Thalassemia Toxic effect Translations Vertebral column Work XPA gene base beta Globin beta Thalassemia clinical application clinical development clinically relevant cost deep sequencing design dimer gene correction genome editing homologous recombination human model improved in utero in vivo inflammatory marker inhibitor interest knockout gene minimally invasive monomer mouse model nanoparticle next generation novel nuclease nucleic acid-based therapeutics nucleobase scale up somatic cell gene editing synthetic nucleic acid targeted nucleases therapeutic gene tool zinc finger nuclease

项目摘要

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.

人们对基因编辑作为治疗人类遗传疾病的潜在手段产生了浓厚的兴趣，例如地中海贫血和镰状细胞病的研究主要集中在靶向核酸酶上。 CRISPR/Cas9 和锌指核酸酶 (ZFN)，基于表明定点 DNA 损伤的研究强烈促进同源重组（HR）然而，靶向核酸酶的临床应用是。作为替代方案，最近在工作中面临着基因组脱靶切割事件风险的挑战。 Ly、Saltzman 和 Glazer 实验室发表在《自然通讯》上的论文表明，γ-取代的三链体形成肽核酸 (PNA) 和供体 DNA 通过聚乳酸-乙醇酸静脉内 (IV) 输送酸（PLGA）纳米粒子（NPs）进入人类β-地中海贫血小鼠模型中几乎完全产生通过造血干中临床相关的β-珠蛋白基因校正频率改善疾病细胞 (HSC) 高达 7% 小鼠表现出贫血减轻、红细胞形态改善以及逆转脾肿大和髓外造血，脱靶效应极低基因组，该技术的另一个关键优势是组件可以。化学合成并配制成纳米颗粒，用于简单的静脉注射。 γPNA 复杂且昂贵，并且无法商业化，限制了其能力研究人员利用这项技术，符合 RFA-RM-18-024，“扩展人类基因组”。 Engineering Repertoire”，这项由 Ly、Saltzman 和 Glazer 提出的多 PI 提案旨在推进基于 PNA/NP 的技术通过简化和扩大 PNA 合成、结合下一代 PNA 化学来进行基因编辑通过战略性地利用细胞 DNA 来增强结合亲和力、提高选择性并增强效力具体目标是：(1) 扩大 PNA 生产并增强 DNA 结合，以便加快用于治疗性基因编辑的 PNA 的转化，并促进广泛采用我们将设计一种对映选择性策略来扩大单体的生产，我们将合成并测试具有修饰核碱基的 γPNA，以实现改进的 DNA 结合特性并 (2)制定操纵策略基于有希望的初步结果，DNA 修复可提高 PNA 介导的基因编辑的效率 (3) 提供强大的检测平台来评估进展从目标 1-2 开始，并将这种方法推广到多个基因，我们将继续利用简单的小鼠和基因。我们期望通过基于细胞的检测来校正 IVS2-654 地中海贫血突变的人类 β-珠蛋白基因。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。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Surface Topography of Polyethylene Glycol Shell Nanoparticles Formed from Bottlebrush Block Copolymers Controls Interactions with Proteins and Cells.

由 Bottlebrush 嵌段共聚物形成的聚乙二醇壳纳米颗粒的表面形貌控制与蛋白质和细胞的相互作用。

DOI：
10.1021/acsnano.1c04835
发表时间：
2021-10-11
期刊：
ACS nano
影响因子：
17.1
作者：
J. Grundler;Kwangsoo Shin;H. Suh;Mingjiang Zhong;W. Saltzman
通讯作者：
W. Saltzman

In vivo correction of cystic fibrosis mediated by PNA nanoparticles.

PNA 纳米粒子介导的囊性纤维化的体内校正。

DOI：
发表时间：
2022-10-07
期刊：
影响因子：
13.6
作者：
Piotrowski;Barone, Christina;Lin, Chun;Deng, Yanxiang;Wu, Douglas;Binns, Thomas C;Xu, Emily;Ricciardi, Adele S;Putman, Rachael;Garrison, Alannah;Nguyen, Richard;Gupta, Anisha;Fan, Rong;Glazer, Peter M;Saltzman, W Mark
通讯作者：
Saltzman, W Mark