Nanocarriers Designed to Deliver Nucleic Acids to Brain

旨在将核酸输送到大脑的纳米载体

基本信息

批准号：
9770565
负责人：
JUAN R SANCHEZ-RAMOS
金额：
$ 35.05万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9770565
关键词：
Applications Grants Behavior Behavioral Blood - brain barrier anatomy Brain Brain Diseases Brain region Caliber Cell Culture Techniques Cells Cerebral cortex Chelating Agents Chitosan Chronic Clinical Corpus striatum structure Culture Media DNA delivery Data Development Diphosphates Dose Drug Delivery Systems Electron Microscopy Extracellular Fluid Fibroblasts Fluorescence Fluorescence Microscopy Formulation Gene Silencing Genes Goals Green Fluorescent Proteins Hour Huntington Disease Image Incubated Infusion procedures Injections Intranasal Administration Intrathecal Space Label Life Magnetic Resonance Imaging Manganese Measures Messenger RNA Metals Methods Microscopy Motor Activity Mouse Cell Line Mus Neurodegenerative Disorders Neurons Nose Nucleic Acids Olfactory Mucosa Operative Surgical Procedures Pathologic Patients Pharmaceutical Preparations Plasmids Play Process Research Proposals Residual state Role Series Signal Transduction Small Interfering RNA System Technology Testing Therapeutic Therapeutic Agents Time Transfection Transgenic Mice Transgenic Organisms Viral Vector Western Blotting base clinically significant comparative crosslink cytotoxicity design ds-DNA exosome experimental study extracellular gene therapy in vivo light scattering mRNA Expression mouse model nanocarrier nanoparticle novel therapeutics nucleic acid-based therapeutics olfactory bulb primary endpoint protein expression public health relevance red fluorescent protein

项目摘要

DESCRIPTION (provided by applicant): Gene therapy of brain diseases is hampered by the requirement for invasive stereotaxic injection into brain, or infusion of therapeutic agents into te intrathecal space. The surgical approach is not optimal for neurodegenerative diseases that require treatments throughout life. This obstacle has given impetus to the design of a new nose-to-brain delivery system for nucleic acids or drugs that cannot pass the blood-brain-barrier. The overall goal of this project is to refine and test a nanocarrier system consisting of chitosan-based, manganese-containing nanoparticles (mNPs) loaded with therapeutic nucleic acids: small interfering RNA (siRNA) and dsDNA. The experiments are designed to assess the mechanisms and extent to which the nanocarriers transport their payload from the olfactory mucosa to olfactory bulb and other brain regions to suppress marker genes in mouse models of rapid onset Huntington's Disease. Specific Aim 1: Testing optimized nanocarriers loaded with siRNA against a marker gene expressed in transgenic "green" mice that constitutively express green fluorescent protein (GFP) in brain neurons. mNPs will be packaged with siRNA directed against GFP. Primary end-points will be a) the extent and time-course of dissemination of mNPs from olfactory bulb to other regions of brain assessed by MRI T1-weighted imaging; b) GFP mRNA and GFP protein expression by comparative quantitiative PCR and Western blot analysis, respectively. Specific Aim 2: Study of gene-silencing in a mouse model of HD. siRNA directed against htt will be loaded into NPs and administered intra-nasally or microinjected directly into striatum in rapid-onset mouse models of Huntington's Disease. Primary-end points will be a) extent and time-course of dissemination of mNPs from olfactory bulb (or from striatum) to other regions of brain assessed by microbore MRI, b) quantitative analysis of htt mRNA expression by quantitative PCR and htt protein expression by Western blot in various brain regions, c) effects on behavior and locomotor activity. Specific Aim 3 a) Iterative optimization of the nanocarrier system in cell cultures expressing a marker gene guided by results from concurrent in vivo experiments in Aim 1 and b) studies of cellular extrusion of exosomes containing mNPs as an hypothesized mechanism for cell-to-cell dissemination. Specific Aim 4: Testing capacity of mNPs to deliver DNA (gene encoding a red fluorescent protein) in vivo. mNPs will be loaded with plasmid dsDNA encoding red fluorescent protein (RFP) and intranasally instilled into normal C57BL6 mice. End-points will be the same as those in Aim 2. Clinical Significance: The ability to dose patients chronically and non-invasively via intra-nasal administration of nanocarriers of gene-silencing agents or other large therapeutic molecules will have a dramatic impact in the therapeutics of brain disorders.

描述（由适用提供）：脑疾病的基因治疗受到侵入性立体定位注射到大脑的要求，或者将治疗剂注入到胸前空间中。手术方法对于需要一生需要治疗的神经退行性疾病并不是最佳的。这种障碍为无法通过血脑屏障的核酸或药物的新型脑之间递送系统的设计推动了动力。该项目的总体目标是完善和测试由含有壳聚糖的，含锰的纳米颗粒（MNP）组成的纳米载体系统，该纳米颗粒（MNP）装有治疗性核酸：小型干扰RNA（siRNA）和DSDNA。该实验旨在评估纳米载体从嗅觉粘膜传递有效载荷到嗅球和其他大脑区域的机制和程度，以抑制亨廷顿氏病小鼠模型中的标记基因。具体目标1：测试用siRNA载荷的优化纳米载体对在脑神经元中组成型表达绿色荧光蛋白（GFP）的转基因“绿色”小鼠中表达的标记基因。 MNP将用针对GFP的siRNA包装。主要的终点将是a）通过MRI T1加权成像评估的MNP从嗅球传播到其他大脑区域的MNP的程度和时间。 b）分别通过比较定量PCR和Western印迹分析的GFP mRNA和GFP蛋白表达。特定目的2：在HD小鼠模型中的基因沉默研究。针对HTT的siRNA将被加载到NP中，并在亨廷顿氏病快速发作的小鼠模型中直接施用非词内或直接注射到纹状体中。原发点将是A）MNP从嗅球（或从纹状体）传播到通过微生物MRI评估的大脑区域的MNP的范围和时间，b）通过定量PCR和HTT蛋白通过Western Blot在各种大脑区域中对HTT mRNA表达的定量分析，c）在各种大脑区域，c）对行为和locomotor and capry and Locomotor and cantimot and Locomotor and cantimot and locomotor and cantimotor and locomotor castion and locomotor and capry and locomotor and capry and locomotor and capry and locomotor castion。特定目标3 a）迭代优化在AIM 1和B）含有MNP的外泌体的细胞扩展的研究中，由体内实验的结果引导的细胞培养物中的纳米载体系统，其作为细胞对细胞传播的假设机制的研究。特定目标4：MNP在体内输送DNA（编码红色荧光蛋白的基因）的测试能力。 MNP将装有编码红色荧光蛋白（RFP）的质粒dsDNA，并将鼻内灌输到正常的C57BL6小鼠中。终点将与AIM 2中的终点相同。临床意义：通过鼻内施用基因偿难剂或其他大型治疗分子的纳米载体的慢性和非侵入性剂量的能力将对脑部疾病的治疗剂产生巨大影响。