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.

项目概述：神经遗传性发育疾病（NDD），如Rett综合征是严重的 神经系统疾病每年影响成千上万的新生儿。作为目前的护理标准， 大多数NDD都集中在症状缓解上，因此必须制定预防性干预措施。的 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， 早期干预这一病理过程，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一个机会之窗， 对治疗剂的接受性非常好此前的基因编辑工作主要集中在CRISPR上。 Cas9和病毒载体，它们与安全性问题相关，包括脱靶效应， 免疫原性为了解决这些问题，我们提出，聚合物纳米颗粒（NPs）封装基因 编辑肽核酸（PNA）是上级的选择。由FDA批准的聚合物制成的NP具有 良好的安全性，证明了他们批准的临床试验，并提供延长释放和靶向 通过表面改性。因为PNA是基于非核酸酶的，高度稳定，并且结合强烈，所以它们 提供了一种诱导位点特异性基因编辑的方法，其具有降低的脱靶编辑效应的风险。PNA-NPs 已被证明是用于多种翻译应用中的基因编辑的安全试剂。我们假设 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，我将描述NPs在胎儿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内分娩。我将创建一个由不同材料制成的纳米粒子库， 聚合物和不同尺寸的荧光染料。在子宫内给予这些NP后至日期 怀孕的老鼠，我将收获胎儿的大脑，并使用共聚焦显微镜和流式细胞术进行分析。为我 第二个目标，我将证明在胎儿大脑中进行体内基因编辑的可行性和优化方法 在子宫内递送PNA-NP后。在子目标2a中，我将配制封装开发的PNA序列的NP 引入β-珠蛋白突变这些PNA-NPs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 聚合酶链反应（ddPCR）。在子目标2b中，我将根据优化的特性制定NP 在目标1中确定。这些纳米颗粒将封装允许表达绿色荧光蛋白的PNA (GFP)基因编辑成功后我将在子宫内将这些纳米颗粒给予GFP报告基因胎儿，并分析其在子宫内的表达。 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方面， PNA-NPs作为针对胎儿大脑的基因编辑剂，我们的项目将在以下方面取得重要进展： 临床上可翻译的IUGT的潜力。