Nanocarriers Designed to Deliver Nucleic Acids to Brain

旨在将核酸输送到大脑的纳米载体

• 批准号: 9147011
• 负责人: JUAN R SANCHEZ-RAMOS
• 金额: $ 44.85万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9147011
• 关键词: Applications Grants; Behavior; Behavioral; Blood - brain barrier anatomy; Brain; Brain Diseases; Brain region; Caliber; Cell Culture Techniques; Cells; Cerebral cortex; Chitosan; Clinical; Corpus striatum structure; Culture Media; DNA; Data; Development; Diphosphates; Dose; Drug Delivery Systems; Electron Microscopy; Extracellular Fluid; Fibroblasts; Fluorescence; Fluorescence Microscopy; Formulation; Gene Silencing; Genes; Goals; Green Fluorescent Proteins; Health; Hour; Huntington Disease; Image; Incubated; Infusion procedures; Injection of therapeutic agent; Intrathecal Space; Label; Life; Magnetic Resonance Imaging; Manganese; Measures; Messenger RNA; Metals; Methods; Motor Activity; Mouse Cell Line; Mus; Neurodegenerative Disorders; Neurons; Nose; Nucleic Acids; Olfactory Mucosa; Operative Surgical Procedures; 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; Weight; Western Blotting; base; clinically significant; comparative; crosslink; cytotoxicity; design; ds-DNA; exosome; extracellular; gene therapy; in vivo; light scattering; mRNA Expression; mouse model; nanocarrier; nanoparticle; novel therapeutics; olfactory bulb; protein expression; red fluorescent protein; research study

 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（由申请人提供）：脑部疾病的基因治疗因需要侵入性立体定位注射到脑部或将治疗剂输注到鞘内空间而受到阻碍。对于需要终生治疗的神经退行性疾病来说，手术方法并不是最佳选择。这一障碍推动了一种新的鼻到脑输送系统的设计，用于无法通过血脑屏障的核酸或药物。该项目的总体目标是完善和测试一种纳米载体系统，该系统由基于壳聚糖的含锰纳米颗粒（mNP）组成，负载有治疗性核酸：小干扰RNA（siRNA）和双链DNA。这些实验旨在评估纳米载体将其有效负载从嗅粘膜运输到嗅球和其他大脑区域以抑制快速发病亨廷顿病小鼠模型中的标记基因的机制和程度。具体目标 1：针对在转基因“绿色”小鼠中表达的标记基因，测试装载有 siRNA 的优化纳米载体，这些小鼠在脑神经元中组成型表达绿色荧光蛋白 (GFP)。 mNP 将与针对 GFP 的 siRNA 一起包装。主要终点是 a) 通过 MRI T1 加权成像评估 mNP 从嗅球传播到大脑其他区域的程度和时间过程； b) 分别通过比较定量 PCR 和蛋白质印迹分析检测 GFP mRNA 和 GFP 蛋白表达。具体目标 2：HD 小鼠模型中基因沉默的研究。针对 htt 的 siRNA 将被装载到 NP 中，并通过鼻内给药或直接显微注射到亨廷顿病快速发病小鼠模型的纹状体中。主要终点是a)通过微孔MRI评估mNP从嗅球（或纹状体）传播到大脑其他区域的程度和时间过程，b)通过定量PCR对htt mRNA表达进行定量分析，通过蛋白质印迹对不同大脑区域的htt蛋白表达进行定量分析，c)对行为和运动活动的影响。具体目标 3 a) 迭代优化 细胞培养物中的纳米载体系统表达标记基因，该标记基因由目标 1 和 b) 中同时进行的体内实验结果引导，对含有 mNP 的外泌体的细胞挤出进行研究，作为细胞间传播的假设机制。具体目标 4：测试 mNP 在体内递送 DNA（编码红色荧光蛋白的基因）的能力。 mNP 将装载编码红色荧光蛋白 (RFP) 的质粒 dsDNA，并鼻内滴注到正常 C57BL6 小鼠中。终点将与目标 2 中的终点相同。 临床意义：通过鼻内施用基因沉默剂纳米载体或其他大治疗分子对患者进行长期非侵入性给药的能力将对脑部疾病的治疗产生巨大影响。