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：
9804726
负责人：
PETER M GLAZER
金额：
$ 42.11万
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/9804726
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/antagonist 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.

基因编辑是治疗人类遗传疾病的潜在手段，例如地中海贫血和镰状细胞疾病。大量精力集中在有针对性的核武器上，例如 CRISPR/CAS9和锌指核（ZFN），基于表明该位置定向DNA损伤的工作强烈促进同源重组（HR）。但是，靶向核武器的临床应用是受到基因组中脱靶裂解事件的风险的挑战。作为替代方案，最近在工作 LY，Saltzman和Glazer Labs发表在《自然通信》上，表明γ-取代的三重形成肽核酸（PNA）和供体DNA，通过聚（乳酸 - 糖酸）静脉内传递（IV）酸（PLGA）纳米颗粒（NP）进入人β-丘脑贫血的小鼠模型，几乎完整疾病的改善，具有临床相关的β-珠蛋白基因校正频率细胞（HSC）高达7％。小鼠表现出缓解贫血，RBC形态的改善和脾肿大和外肺造血的逆转，脱靶效应极低基因组，这项技术的关键优势。另一个关键优势是组件可以是合成的合成，并配制成纳米颗粒，以进行简单的静脉注射。但是，合成 γPNA是复杂且昂贵的，并且它们无法商业上使用，从而限制了调查人员利用这项技术。与RFA-RM-18-024一致，“扩展人类基因组 LY，Saltzman和Glazer的这项多PI的工程曲目”，试图推进基于PNA/NP 通过简化和扩展PNA合成，通过将下一代PNA化学结合到提高结合亲和力，提高选择性并提高效力，并通过策略性利用细胞DNA 维修途径。具体目的是：（1）扩展PNA的产生和增强DNA结合，以便加快对PNA进行治疗基因编辑的翻译，并可以采用宽度技术。我们将制定一种对映射策略来扩展单体的生产，我们将用改良的核碱基合成和测试γPNA，以提高DNA结合特性，并与克服三重形成的同尿序列限制。（2）制定操纵策略基于有希望的初步结果，DNA修复以提高PNA介导的基因编辑的效率带有新型的DNA修复抑制剂。（3）提供一个强大的暗杀平台来评估进步从目标1-2概括到多个基因。我们将继续利用便捷的鼠标 - 和在IVS2-654 thalassyamia突变上校正人β-蛋白基因基因的基于细胞的测定。我们期望这项工作为设计更多适用于许多人的基因编辑的潜在PNA提供了基础遗传疾病。