Nanocarriers Designed to Deliver Nucleic Acids to Brain

 描述(申请人提供)：脑部疾病的基因治疗因侵入性脑内立体定向注射或鞘内注射治疗剂的要求而受阻。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的。这一障碍推动了一种新的鼻脑递送系统的设计，这种递送系统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提炼和测试一个纳米载体系统，该系统由壳聚糖为基础的含锰纳米颗粒(MNPs)组成，其中携带治疗性核酸：小干扰RNA(SiRNA)和dsDNA。这些实验旨在评估纳米载体将其有效载荷从嗅黏膜转移到嗅球和其他大脑区域，以抑制快速发病的亨廷顿病小鼠模型中标记基因的机制和程度。具体目标1：测试携带siRNA的优化纳米载体与在转基因“绿色”小鼠中表达的标记基因的对比，该转基因“绿色”小鼠在大脑神经元中结构性地表达绿色荧光蛋白(GFP)。MNPs将与针对GFP的siRNA一起包装。主要终点将是a)MRI T1加权成像评估mNPs从嗅球向脑内其他区域扩散的程度和时间进程；b)比较定量PCR和Western印迹分析GFP mRNA和GFP蛋白的表达。特定目的2：HD小鼠模型中基因沉默的研究。针对HTT的siRNA将被装载到NPs中，并在快速发病的亨廷顿病小鼠模型中直接鼻内或显微注射到纹状体内。主要终点将是a)微孔MRI评估mNPs从嗅球(或纹状体)扩散到脑其他区域的程度和时间过程，b)定量PCR定量分析HTT mRNA的表达和Western印迹分析hTt蛋白在不同脑区的表达，c)对行为和运动活动的影响。具体目标3 a)迭代优化 细胞培养中表达标记基因的纳米载体系统由目标1和b)中同时进行的体内实验的结果指导，目的1和b)研究含有mNPs的外切体的细胞排泄作为细胞间传播的假设机制。具体目标4：在体内测试mNPs运送DNA(编码红色荧光蛋白的基因)的能力。MNPs将装载编码红色荧光蛋白(RFP)的质粒dsDNA，并经鼻注入正常C57BL6小鼠。终点将与AIM 2中的相同。临床意义：通过鼻腔给药纳米载体基因沉默试剂或其他大的治疗分子给患者慢性和非侵入性给药的能力将对大脑疾病的治疗产生巨大影响。

 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.