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

开发并评估鼻内递送的纳米制剂 siBeclin1 消除大脑中 HIV 的功效

• 批准号: 9893032
• 负责人: NAZIRA EL-HAGE
• 金额: $ 22.46万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-13 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893032
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Alanine; Alanine Transaminase; Anti-Retroviral Agents; Antibodies; Area; Astrocytes; Attenuated; Autophagocytosis; Basal Ganglia; Behavior Therapy; Behavioral; Biodistribution; Biological Assay; Blood; Blood Urea Nitrogen; Body Weight; Brain; CD34 gene; Calcium Binding; Calcium ion; Cardiac; Cell Nucleus; Cells; Chemistry; DNA; Development; Disease; Drug Kinetics; Enzyme-Linked Immunosorbent Assay; Female; Freezing; Funding Opportunities; Future; Glial Fibrillary Acidic Protein; Gliosis; HIV; HIV Infections; HIV antiretroviral; HIV therapy; HIV-1; Hand Strength; Health; Heart; Hematoxylin and Eosin Staining Method; Hepatotoxicity; Hippocampus (Brain); Histologic; Histology; Immune response; Immunofluorescence Immunologic; Individual; Inflammatory; Inflammatory Response; Intravenous; Kidney; Kinetics; Label; Learning; Liquid Chromatography; Liquid substance; Liver; Lung; Mannose; Measures; Membrane; Methodology; Microglia; Microtubule-Associated Protein 2; Microtubule-Associated Proteins; Morphology; Motor; Mus; National Institute of Mental Health; Neuraxis; Neurons; Nitrogen; Organ; Pathologic; Pathology; Pathway interactions; Perfusion; Pharmaceutical Preparations; Pharmacotherapy; Polymerase Chain Reaction; Proteins; Public Health; Puncture procedure; Quantitative Reverse Transcriptase PCR; RNA; Regimen; Research; Research Priority; Reverse Transcription; Safety; Sensory; Short-Term Memory; Small Interfering RNA; Stains; Testing; Therapeutic; Therapeutic Agents; Time; Toxic effect; Treatment Efficacy; Tube; Viral; Virus Replication; Western Blotting; antiretroviral therapy; base; brain cell; chemokine; cytokine; cytotoxicity; design; dosage; glial activation; humanized mouse; insight; macrophage; male; man; memory process; morris water maze; nanoformulation; nephrotoxicity; neuropathology; next generation; nonhuman primate; novel; novel therapeutic intervention; object recognition; particle; preservation; response; spatial memory; tandem mass spectrometry

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.

项目概要 开发并评估鼻内递送的纳米制剂 siBeclin1 消除大脑中 HIV 的功效。 总体假设是，针对自噬途径的小干扰 (si) RNA 可以充当协同作用。 具有抗逆转录病毒药物的治疗剂，可消除中枢神经系统 (CNS) HIV 储存库和病毒相关病毒 血管周围巨噬细胞、小胶质细胞 [1,2] 和星形胶质细胞 [3] 的炎症反应。 siBeclin1 siRNA-聚乙烯亚胺 (PEI) 复合物 [4] 促进鼻内递送至大脑 [4]。 纳米配方的 siBeclin1 可暂时减少宿主蛋白 Beclin1 的表达，将测试其在 消除人源化小鼠脑细胞 HIV 储存库，有效鼻内递送（图 1），部署甘露糖。 将采用装饰颗粒（图 2）和病毒复制的定量测量（图 3）。 目标 1 的子目标 (a 和 b)，我们将定量测量 siBeclin1 在大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) 和液相色谱法检测肺、心脏、肝脏和肾脏 - 由于细胞毒性是许多人遇到的问题，因此采用串联质谱 (LC-MS/MS) 方法。 抗逆转录病毒疗法，在目标 1c 中，将通过组织学评估由于大脑细胞毒性引起的形态变化 使用苏木精和伊红 (H&E) 和尼氏染色来检测神经元损伤。 神经元核 (NeuN) 或微管相关蛋白 2 (MAP2) 的免疫组织化学标记，以评估 大脑中存活的神经元细胞标记物、离子钙结合接头分子 1 (Iba-1) 或 CD68。 特异于小胶质细胞和特异于星形胶质细胞的神经胶质纤维酸性蛋白 (GFAP) 将用于检测神经胶质细胞 炎症反应将通过细胞因子和趋化因子膜抗体来测量。 阵列并通过比色夹心酶联免疫吸附 (ELISA) 化学测定分析进行确认。 血液尿素氮和丙氨酸转氨酶活性水平将表明肾脏和肝脏的毒性， 在目标 2a 中，我们将确定纳米制剂 siBeclin1 对 CNS 不同方面的功效 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 PCR 的检测进行测量）；(2) 免疫反应的分泌（通过基于 ELISA 的测定进行测量）；(3) 神经胶质激活和 (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 神经节，因为这些区域受艾滋病毒影响最严重，并且对于基础大脑发育、学习和 记忆过程和感觉运动功能 在目标 2b 中，HIV 感染单独或单独引起的行为变化。 与抗逆转录病毒药物方案的组合±纳米配方的siBeclin1将使用新对象进行评估 识别测试作为短期记忆的指标，莫里斯水迷宫测量空间记忆。 运动测试将使用旋转杆、握力和单杠进行评估。