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

项目摘要：Rett综合征等神经发育疾病(NDD)很严重 每年影响数以千计的新生儿的神经系统疾病。作为目前护理的标准 大多数NDDS侧重于缓解症状，制定预防性干预措施势在必行。这个 与NDDS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。宫内基因治疗(IUGT)有望提供一种治疗方法， 早期干预这一病理过程，防止NDDS的发展。IUGT具有优势 因为胎儿的可塑性和发育不成熟提供了一扇机会之窗 对治疗剂的接受度极高。以前的基因编辑工作主要集中在CRISPR- Cas9和病毒载体，它们与安全问题相关，包括靶外效应和 免疫原性。为了解决这些问题，我们建议将包裹基因的聚合物纳米颗粒(Nps) 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NPS具有 极佳的安全性，临床试验的批准证明了这一点，并提供了延长释放和靶向 通过表面修饰。因为PNA不是基于核酸酶的，高度稳定，结合很强，所以它们 提供了一种诱导位点特异性基因编辑的方法，同时降低了非靶标编辑效果的风险。PNA-NPs 已被证明是在多种翻译应用中进行基因编辑的安全试剂。我们假设 在宫内交付PNA-NPs将导致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NPs在胎脑中的细胞和空间生物分布。 通过羊水和卵黄静脉分娩。我将创建一个由不同的NPs组成的图书馆 聚合物和不同大小的负载荧光染料。在子宫内给药后至日期 怀孕小鼠，我将采集胎脑，用共聚焦显微镜和流式细胞仪进行分析。为了我的 第二个目标，我将论证在胎脑中进行体内基因编辑的可行性和优化方法。 PNA-NPs宫内分娩后。在次级目标2a中，我将制定封装所开发的PNA序列的NPs 我们实验室引入了一种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Lund人中脑细胞(LUHMES)和基因编辑将使用Drop Digital进行评估 聚合酶链式反应(DdPCR)。在子目标2b中，我将根据优化后的特征来制定NP 在AIM 1中确定。这些NPs将包裹允许表达绿色荧光蛋白的PNA (GFP)在成功编辑基因后。我将这些NPs注射到GFP记者胎儿的宫内，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的过程中 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 临床可翻译IUGT的潜力。

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.