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: 10671586
• 负责人: Anna Y Lynn
• 金额: $ 5.2万
• 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/10671586
• 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; 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; beta Globin; brain tissue; clinical translation; 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; nanopolymer; nervous system disorder; neural; neurodevelopment; neurogenetics; novel; nuclease; poly(lactic acid); polyglycerol; postmitotic; pregnant; prenatal; prevent; preventive intervention; skills; social; standard of care; stem; 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：雷特综合征等神经遗传发育疾病 (NDD) 很严重 每年影响数千名新生儿的神经系统疾病。按照目前的护理标准 大多数 NDD 的重点是缓解症状，因此必须制定预防性干预措施。这 与 NDD 相关的基因突变已被证明会导致神经发育异常 胎儿发育的早期阶段。子宫内基因治疗（IUGT）有望成为一种治疗方法 尽早干预这一病理过程并预防 NDD 的发展。 IUGT有优势 因为胎儿的可塑性和发育不成熟提供了一个可能的机会之窗 对治疗剂特别容易接受。之前的基因编辑工作主要集中在 CRISPR- Cas9 和病毒载体，与安全问题相关，包括脱靶效应和 免疫原性。为了解决这些问题，我们提出封装基因的聚合物纳米颗粒（NP） 编辑肽核酸 (PNA) 是一种更好的选择。由 FDA 批准的聚合物制成的 NP 卓越的安全性，通过临床试验的批准证明，并提供延长的释放和靶向 通过表面修饰。由于 PNA 是非核酸酶、高度稳定且结合力强，因此它们 提供了一种诱导位点特异性基因编辑的方法，同时降低了脱靶编辑效应的风险。纳米粒子 已被证明是多种翻译应用中基因编辑的安全剂。我们假设 PNA-NP 的子宫内递送将对胎儿大脑进行安全、高效的基因编辑。 在我的第一个目标中，我将描述纳米粒子在胎儿大脑中的细胞和空间生物分布。 通过羊水和卵黄静脉进行子宫分娩。我将创建一个由不同材料制成的 NP 库 聚合物和不同尺寸的负载荧光染料。在子宫内施用这些 NP 后的日期 怀孕的老鼠，我会收获胎儿大脑并使用共聚焦显微镜和流式细胞术进行分析。为了我的 第二个目标，我将展示胎儿大脑体内基因编辑的可行性和优化方法 PNA-NP 宫内分娩后。在子目标 2a 中，我将制定封装已开发的 PNA 序列的 NP 由我们的实验室引入β-珠蛋白突变。这些 PNA-NP 将用于治疗分化的、有丝分裂后的 神经元隆德人类中脑细胞（LUHMES）和基因编辑将使用液滴数字技术进行评估 聚合酶链反应（ddPCR）。在子目标2b中，我将根据优化后的特征制定NP 目标 1 中确定的。这些 NP 将封装允许表达绿色荧光蛋白的 PNA (GFP) 成功基因编辑后。我将在子宫内对 GFP 报告基因胎儿施用这些 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-NPs 作为针对胎儿大脑的基因编辑剂，我们的项目将在 临床可转化的IUGT的潜力。