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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要开发和评估纳米成型的sibeclin1的疗效，用于消除大脑中的HIV。总体假设是针对自噬途径的小型干扰（SI）RNA可以作为协同作用抗逆转录病毒药物的治疗剂可消除中枢神经系统（CNS）HIV储存剂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[3]中的炎症反应。为此，我们将合成一个 sibeclin1 siRNA-聚乙烯胺（PEI）多链体[4]，以促进鼻内递送到大脑[4]。这纳米制造的sibeclin1瞬时降低宿主蛋白beclin1的表达，将测试其有效消除人源化小鼠中的脑细胞HIV储量。有效的鼻内输送（图1），甘露糖的部署将进行装饰的颗粒（图2）和病毒复制的定量测量方法（图3）。在前两个 AIM 1的子目标（A＆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 - 肺，心脏，肝脏和肾脏串联质谱法（LC-MS/MS）方法。由于细胞毒性是许多人遇到的问题抗逆转录病毒疗法，在AIM 1C中，由于大脑的细胞毒性而引起的形态学变化将通过组织学评估使用苏木精和曙红（H＆E）和NISSL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（MAP2）的免疫组织化学标记以评估在大脑中存活的神经元细胞。细胞标记，电离钙结合衔接子分子1（IBA-1）或CD68 特异性的小胶质细胞和神经胶质原纤维酸性蛋白（GFAP）针对星形胶质细胞的特异性将用于检测神经胶质激活（神经胶质）。炎症反应将通过细胞因子和趋化因子膜抗体进行测量阵列并通过比色夹心酶连接的免疫吸附测定法（ELISA）确认。化学分析血液尿素氮和丙氨酸转氨酶活性的水平将表明儿童和肝脏的毒性，在AIM 2A中，我们将确定纳米成型SibeClin1在CNS不同方面的效率由HIV±抗逆转录病毒药物诱导的病理，包括（1）病毒复制（通过基于PCR的测定法测量）；（2）免疫调查的分泌（通过基于ELISA的测定法测量）；（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测定法）。特别重点将放在海马和基底神经节是这些地区受艾滋病毒疾病影响最大，对于大脑发育，学习和内存过程和感觉运动功能。在AIM 2B中，仅HIV感染引起的行为变化或将使用新物体评估与抗逆转录病毒药物方案±纳米成型SibeClin1的结合识别测试是短期记忆和莫里斯水迷宫的指标，以测量空间记忆。感官电机测试将通过使用Rota-Rod，Grip-strength and Mofortal Bar评估。