Develop and evaluate efficacy of nanoformulated siBeclin1 delivered intranasally to eliminate HIV in brain

项目总结 开发和评估鼻腔给药的纳米制剂siBeclin1消除大脑中艾滋病毒的效果。 总体假设是，针对自噬途径的小干扰(Si)rna可以起到协同作用。 与抗逆转录病毒药物联合治疗消除中枢神经系统(CNS)艾滋病毒储存库和病毒相关 血管周围巨噬细胞、小胶质细胞[1，2]和星形胶质细胞的炎症反应[3]。为此，我们将合成一个 SiBeclin1 siRNA-聚乙烯亚胺(PEI)复合体[4]，以促进鼻腔给药到大脑[4]。这个 纳米配方的siBeclin1可以暂时减少宿主蛋白Beclin1的表达，它将在 消除人源化小鼠的脑细胞艾滋病毒储备库。高效鼻腔给药(图1)，甘露糖的部署 将采用装饰颗粒(图2)和病毒复制的定量测量(图3)。在前两个 目标1的子目标(a和b)，我们将定量测量siBeclin1在脑内的药代动力学和生物分布， 肺、心、肝、肾组织经逆转录聚合酶链式反应(RT-PCR)和高效液相色谱(LC)检测. 串联质谱仪(LC-MS/MS)方法。因为细胞毒性是许多人遇到的问题 抗逆转录病毒治疗，在目标1c中，将通过组织学来评估脑组织中细胞毒性引起的形态变化。 苏木精-伊红(H&E)和尼氏染色检测神经元损伤。紧接着是 神经元核(NeuN)或微管相关蛋白2(MAP2)的免疫组织化学标记 大脑中幸存的神经细胞。细胞标志物，离子钙结合接头分子1(Iba-1)或CD68 小胶质细胞特异性和星形胶质细胞特异性胶质纤维酸性蛋白(GFAP)将用于检测胶质细胞 激活(胶质细胞增多症)。炎症反应将通过细胞因子和趋化素膜抗体进行测量 经比色夹心酶联免疫吸附试验(ELISA)证实。关于化学分析的几点思考 血尿素氮和丙氨酸转氨酶活性的水平，将表明肾脏和肝脏的毒性， 分别进行了分析。在目标2a中，我们将确定纳米配方siBeclin1对中枢神经系统不同方面的疗效 HIV±抗逆转录病毒药物引起的病理包括(1)病毒复制(通过基于聚合酶链式反应的检测)；(2) 免疫反应的分泌(通过基于ELISA法测定)；(3)胶质细胞的激活和(4)神经元的健康(测定) 通过组织学和免疫组织化学方法)。将特别强调海马体和 基底节，因为这些区域是受艾滋病毒影响最大的区域，对大脑发育、学习和 记忆过程和感觉运动功能。在目标2b中，单独或间接感染艾滋病毒引起的行为变化 将使用新对象评估与抗逆转录病毒药物方案±纳米配方siBeclin1的结合 再认测验作为短时记忆的指标，用Morris水迷宫测量空间记忆。感官 马达测试将使用ROTA杆、握力和单杠进行评估。

PROJECT SUMMARY Develop and Evaluate the Efficacy of Nanoformulated siBeclin1 Delivered Intranasally to Eliminate HIV in the Brain. The overall hypothesis is that small interfering (si) RNAs targeting the autophagy pathway can act as a synergistic therapeutic agent with antiretroviral drugs to eliminate central nervous system (CNS) HIV reservoirs and viral associated inflammatory responses in perivascular macrophages, microglia [1,2] and astrocytes [3]. To this end, we will synthesize a siBeclin1 siRNA- polyethylenimine (PEI) polyplex [4] to facilitate intranasal delivery to the brain [4]. The nanoformulated siBeclin1 which transiently diminishes expression of host protein Beclin1, will be tested for its efficacy in eliminating brain cell HIV reservoirs in humanized mice. Efficient intranasal delivery (Figure 1), deployment of mannose decorated particles (Figure 2) and quantitative measures of viral replication will be employed (Figure 3). In the first two sub aims (a &b) of Aim 1, we will quantitatively measure the pharmacokinetics and bio-distribution of siBeclin1 in brain, lung, heart, liver and kidney by reverse transcription polymerase chain reaction (RT-PCR) and liquid chromatography- tandem mass spectrometry (LC-MS/MS) methodologies. Since cell toxicity is a problem encountered with many antiretroviral therapies, in Aim 1c, morphological changes due to cytotoxicity in the brain will be assessed by histology using Hematoxylin and eosin (H&E) and Nissl staining to detect for neuronal damages. Followed by immunohistochemical labeling of neuronal nuclei (NeuN) or microtubule-associated protein 2 (MAP2) to assess for the surviving neuronal cells in the brain. The cell marker, ionized calcium binding adaptor molecule 1 (Iba-1) or CD68 specific for microglia and glial fibrillary acidic protein (GFAP) specific for astrocytes will be used to detect Glial activation (gliosis). Inflammatory responses will be measured by cytokine and chemokine membrane-based antibody arrays and confirmed by colorimetric sandwich enzyme-linked immunosorbent assay (ELISA). Chemistry analysis on the levels of blood urea nitrogen and alanine transaminase activity, will indicate toxicity of the kidneys and liver, respectively. In Aim 2a, we will determine the efficacy of the nanoformulated siBeclin1 on the different aspects of CNS pathology induced by HIV ± antiretroviral drugs including (1) viral replication (measured by PCR-based assays); (2) secretion of immune responses (measured by ELISA-based assays); (3) glial activation and (4) neuronal health (measured by histological and immunohistochemical based assays). Special emphasis will be placed on the hippocampus and the basal ganglia as these regions are most affected by HIV disease and are critical for brain development, learning and memory processes and sensory motor function. In aim 2b, behavioral changes elicited by HIV infection alone or in combination with antiretroviral drug regimen ± nanoformulated siBeclin1 will be assessed using the novel object recognition test as an indicator of short-term memory and the Morris water maze to measure spatial memory. The sensory motor test will be assessed by using the Rota-rod, grip-strength and horizontal bar.