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

项目摘要 开发和评估鼻内递送的纳米配方siBeclin 1消除大脑中HIV的功效。 总的假设是，靶向自噬途径的小干扰（si）RNA可以作为一种协同作用， 与抗逆转录病毒药物一起消除中枢神经系统（CNS）HIV储库和病毒相关的治疗剂 血管周围巨噬细胞、小胶质细胞[1，2]和星形胶质细胞[3]中的炎症反应。为此，我们将合成一种 siBeclin 1 siRNA-聚乙烯亚胺（PEI）聚合复合物[4]以促进鼻内递送至脑[4]。的 将测试瞬时减少宿主蛋白Beclin 1表达的纳米制剂siBeclin 1在以下方面的功效： 在人源化小鼠中消除脑细胞HIV储库。有效的鼻内递送（图1），甘露糖的部署 将使用修饰的颗粒（图2）和病毒复制的定量测量（图3）。前两 目标1的子目标（a &B），我们将定量测量siBeclin 1在脑中的药代动力学和生物分布， 通过逆转录聚合酶链反应（RT-PCR）和液相色谱法， 串联质谱法（LC-MS/MS）。由于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，将通过组织学评估脑细胞毒性引起的形态学变化 使用苏木精和伊红（H&E）和尼氏染色检测神经元损伤。其次是 神经元核（NeuN）或微管相关蛋白2（MAP 2）的免疫组织化学标记，以评估 大脑中存活的神经元细胞。细胞标志物，离子钙结合衔接分子1（Iba-1）或CD 68 特异于小胶质细胞和特异于星形胶质细胞的胶质细胞酸性蛋白（GFAP）将用于检测胶质细胞 激活（神经胶质增生）。炎症反应将通过基于细胞因子和趋化因子膜的抗体来测量 阵列，并通过比色夹心酶联免疫吸附测定（ELISA）确认。化学分析 血尿素氮和丙氨酸转氨酶活性的水平，将指示肾和肝的毒性， 分别在目标2a中，我们将确定纳米制剂siBeclin 1对CNS不同方面的功效。 HIV ±抗逆转录病毒药物诱导的病理学，包括（1）病毒复制（通过基于PCR的试验测量）;（2） 免疫应答的分泌（通过基于ELISA的测定测量）;（3）神经胶质活化和（4）神经元健康（测量 通过基于组织学和免疫组织化学的测定）。将特别强调海马体和 基底神经节是受艾滋病影响最严重的区域，对大脑发育、学习和 记忆过程和感觉运动功能。在目标2b中，HIV感染单独引起的行为变化或 将使用新对象评估与抗逆转录病毒药物方案±纳米制剂siBeclin 1的联合治疗 再认测试作为短期记忆的指标，Morris水迷宫测量空间记忆。感官 运动测试将通过使用旋转杆、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.