Optimization of Polymeric Nanoparticles Encapsulating Peptide Nucleic Acids for In Utero Gene Editing of the Fetal Brain.

封装肽核酸的聚合物纳米颗粒的优化，用于胎儿大脑的子宫内基因编辑。

• 批准号: 10468654
• 负责人: Anna Y Lynn
• 金额: $ 3.16万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-16 至 2024-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468654
• 关键词: Address; Adult; Affect; Affinity; Amniotic Fluid; Area; Base Sequence; Binding; Biodistribution; Brain; CRISPR/Cas technology; Cells; Characteristics; Childhood; Clinical Trials; Cognitive; Confocal Microscopy; DNA; DNA Sequence Alteration; Data; Development; Disease; Disorder of neurometabolic regulation; Embryo; Encapsulated; Engineering; Epilepsy; Exposure to; FDA approved; Fetal Development; Fetus; Flow Cytometry; Fluorescent Dyes; Formulation; Genes; Genetic; Gestational Age; Glycolates; Green Fluorescent Proteins; Harvest; Hemoglobin; Human; Immune response; Injections; Intervention; Kinetics; Knowledge; Libraries; Life Expectancy; Link; Methods; Mitotic; Modification; Molecular; Motor; Mucopolysaccharidosis I H; Mus; Mutation; Neurodevelopmental Disorder; Neurologic Symptoms; Neurons; Newborn Infant; Nucleic Acids; Pathologic Processes; Penetration; Peptide Nucleic Acids; Polyethylene Glycols; Polymerase Chain Reaction; Polymers; Population; Preventive treatment; Reporter; Resistance; Rett Syndrome; Risk; Route; Safety; Sensory; Site; Structure of omphalomesenteric vein; Surface; Symptoms; Therapeutic; Therapeutic Agents; Time; Training; Viral Vector; Work; autism spectrum disorder; base; beta Globin; brain tissue; clinically translatable; curative treatments; developmental disease; digital; disease phenotype; fetal; fetal medicine; functional group; gene therapy; immunogenicity; improved; in utero; in vivo; infancy; innovation; intravenous injection; mouse model; nanoparticle; nervous system disorder; neurodevelopment; neurogenetics; novel; nuclease; poly(lactic acid); polyglycerol; pregnant; prenatal; prevent; preventive intervention; relating to nervous system; skills; social; standard of care; stem cells; synthetic nucleic acid; targeted agent; targeted delivery; targeted treatment; translational applications; uptake

PROJECT SUMMARY: Neurogenetic developmental diseases (NDD) such as Rett syndrome are severe neurological disorders that affect thousands of newborn infants each year. As the current standard of care for most NDDs is focused on symptom mitigation, it is imperative that a preventative intervention is developed. The genetic mutations associated with NDDs have been shown to cause abnormal neurodevelopment from very early stages in fetal development. In utero gene therapy (IUGT) holds the promise of a treatment that could intervene early on in this pathologic process and prevent the development of NDDs. IUGT is advantageous because the plasticity and developmental immaturity of the fetus offers a window of opportunity that may be exceptionally receptive to the therapeutic agents. Previous work in gene editing has largely focused on CRISPR- Cas9 and viral vectors, which are associated with concerns of safety including off-target effects and immunogenicity. To address these concerns, we propose that polymeric nanoparticles (NPs) encapsulating gene editing peptide nucleic acids (PNAs) are a superior alternative. NPs made of FDA approved polymers have excellent safety profiles, evidenced by their approval for clinical trials, and offer extended release and targeting through surface modifications. Because PNAs are non-nuclease based, highly stable, and bind strongly, they offer a method of inducing site-specific gene editing with a decreased risk of off-target editing effects. PNA-NPs have been shown to be safe agents for gene editing in multiple translational applications. We hypothesize that in utero delivery of PNA-NPs will result in safe, highly efficient gene editing of the fetal brain. In my first aim, I will characterize the cellular and spatial biodistribution of NPs to the fetal brain after in utero delivery through the amniotic fluid and the vitelline vein. I will create a library of NPs made of different polymers and different sizes loaded with fluorescent dye. After administration of these NPs in utero to time dated pregnant mice, I will harvest fetal brains and use confocal microscopy and flow cytometry for analysis. For my second aim, I will demonstrate the feasibility of and optimize methods for in vivo gene editing in the fetal brain after in utero delivery of PNA-NPs. In Sub Aim 2a, I will formulate NPs encapsulating a PNA sequence developed by our lab to introduce a beta-globin mutation. These PNA-NPs will be used to treat differentiated, post-mitotic neuronal Lund Human Mesencephalic cells (LUHMES) and gene editing will be evaluated using droplet digital polymerase chain reaction (ddPCR). In Sub Aim 2b, I will formulate NPs based on the optimized characteristics identified in Aim 1. These NPs will encapsulate a PNA that allows expression of Green Fluorescent Protein (GFP) after successful gene editing. I will administer these NPs to GFP reporter fetuses in utero and analyze the gene editing in the brain using confocal microscopy and flow cytometry. In demonstrating the safety and efficacy of PNA-NPs as gene editing agents targeted to the fetal brain, our project will result in an important advance in the potential of clinically translatable IUGT.

项目摘要：神经遗传发育疾病（NDD），例如RETT综合征很严重 每年影响数千名新生婴儿的神经疾病。作为当前的护理标准 大多数NDD都集中在减轻症状上，必须开发预防性干预。这 与NDD有关 胎儿发育的早期阶段。在子宫基因疗法（IUGT）中，有可能接受治疗的承诺 在这种病理过程中进行干预，并防止NDD的发展。 IUGT是有利的 因为胎儿的可塑性和发育不成熟提供了可能是机会之窗 特别接受治疗剂。基因编辑的先前工作主要集中在CRISPR- CAS9和病毒矢量与安全的关注有关，包括脱靶效应和 免疫原性。为了解决这些问题，我们提出了封装基因的聚合物纳米颗粒（NPS） 编辑肽核酸（PNA）是一种优越的选择。由FDA批准的聚合物制成的NP具有 出色的安全概况，通过批准临床试验的批准证明，并提供扩展的发布和定位 通过表面修饰。由于PNA是基于非核酶，高度稳定和强烈结合的，因此 提供一种诱导特定地点基因编辑的方法，其脱靶编辑效果的风险降低。 PNA-NP 已证明在多个翻译应用中是基因编辑的安全代理。我们假设这一点 在子宫内递送PNA-NP将导致胎儿大脑的安全，高效的基因编辑。 在我的第一个目标中，我将表征NP在胎儿大脑之后的细胞和空间生物分布 子宫通过羊水和卵黄静脉的递送。我将创建一个由不同的NP库 聚合物和带有荧光染料的不同尺寸。在子宫内给予这些NP之后 怀孕的小鼠，我将收集胎儿大脑，并使用共聚焦显微镜和流式细胞仪进行分析。为我 第二个目的，我将证明并优化胎儿体内基因编辑方法的可行性 在子宫内递送PNA-NP之后。在Sub Aim 2a中，我将制定封装PNA序列的NP 由我们的实验室引入β-珠蛋白突变。这些PNA-NP将用于治疗差异化的有丝分裂后 神经元隆德人中脑细胞（LUHMES）和基因编辑将使用液滴数字评估 聚合酶链反应（DDPCR）。在Sub AIM 2B中，我将根据优化特征制定NP 在AIM 1中确定。这些NP将封装一个PNA，允许表达绿色荧光蛋白 （GFP）成功基因编辑后。我将向子宫内的GFP记者胎儿管理这些NP，并分析 使用共聚焦显微镜和流式细胞术在大脑中进行基因编辑。在证明安全性和有效性时 PNA-NP作为针对胎儿大脑的基因编辑剂的，我们的项目将导致重要的进步 临床上可翻译的IUGT的潜力。