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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。

基本信息

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
项目类别：
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

项目摘要

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