MOLECULAR MODULATION OF HSV VECTOR CNS INTERACTIONS

HSV 载体 CNS 相互作用的分子调节

• 批准号: 6128291
• 负责人: JOHN A OLSCHOWKA
• 金额: $ 29.86万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-20 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6128291
• 关键词: alkaline phosphatase; brain; chemoattractants; corpus striatum; helper virus; herpes simplex virus 1; host organism interaction; immunocytochemistry; laboratory mouse; neuroimmunomodulation; plasmids; reporter genes; transfection /expression vector

DESCRIPTION(adapted from applicant's abstract): With development and application of direct gene transfer systems for the potential treatment of neurodegenerative disease, several considerations related to vector safety and gene product expression quickly become apparent. The current proposal will examine whether the innate andl/or cellular arms of the irnmune system are activated following placement of a transgene within the rnouse CNS? Two specific aims will be addressed. The first specific aim seeks to deterrnine the immunopathology within the mouse striatum for 3 HSV-1 based vectors: 1) lHSV-lL plasmid based amplicon generated using a replication deficient helper virus; 2) a plasmid-based HSV-1 amplicon produced in a helper virus-free systen about. In both cases, the vector will express lacZ as a novel reporter gene. 3) A third plasmid based HSV-1 amplicon produced in a helper virus-free system and expressing mouse intestinal alkaline phosphatase (iAP) will be exarnined. The use of iAP as the reporte:r gene will allow for easy histochemical localization of a non CNS protein similar to 1acZ but one that should not be immunogenic. The effects of pre-immunization with HSV will also be examined. Mouse brains will be analyzed using both molecular analysis (quantitative PGR, RT-PCR), immunocytochernistry and enzyme assay to correlate t]he inflammatory :response to the viral o:r transgene proteins to transgene expressior. Analyses will examie pro-inflammatory and inhibitory cytokines about chemokines, adhesion molecules, blood-brain barrier irtegrity and transgene expression and survival. The second specific aim will examine the role of the innate immune response in li aboutting transfection efficiency. The innate immnune response (consisting of non-specific phagocytic neutrophils and monocytes/maGrophages and driven by the alternative complement pathway) will be examined following injection of the HSViAP amplicon. The response will be altered in several ways. First, the selective rernoval of either neutrophils or circulating monocytes/macrophages will be accomplished using specific antibodies to be each cell type injected immediately before during and after infusion of the arnplicon vecto: Secondly, we will attempt to inhibit adhesion and limit diapedesis utrophils/monocytes/macrophages into the transfection area using specific antibodies to the adhesion molecules P-selectin and ICAM- 1. Third, we wil1 attempt to prevent or limit induction of pro-inflammatory cytokines, chemokines and adhesion molecules co-expressing the inhilbitory cytokine IL-10 in our HSVi AP vector. Finally, the role of complement in the innate immune response will be tested by injecting recombinant Crry-Ig ip an attempt to block the role of chemoattractants and activators of irnmune cells. The effectiveness of each manipulation will be assayed with the same methods used in Specific Aim 1.

描述（根据申请人的摘要改编）：开发和 直接基因转移系统在潜在处理中的应用 神经退行性疾病，与载体安全性有关的几个考虑因素和 基因产物表达迅速变得明显。当前的建议将 检查iNmune系统的先天和蜂窝臂是 将转基因放置在Rnouse CNS中后激活？二 将解决具体目标。第一个特定目的旨在威慑 基于3 HSV-1的载体的小鼠纹状体内的免疫病理学：1）LHSV-LL 基于质粒的扩增子使用不足的辅助病毒复制产生； 2） 在无辅助病毒中产生的基于质粒的HSV-1扩增子。在 两种情况下，矢量都将表达LACZ为新的记者基因。 3）三分之一 在无辅助病毒系统中产生的基于质粒的基于质粒的HSV-1扩增子和 表达小鼠肠道碱性磷酸酶（IAP）的表达。这 使用IAP作为报告：R基因将允许简单的组织化学定位 类似于1ACZ的非CNS蛋白，但不应是免疫原性的。 还将检查使用HSV前免疫的影响。鼠标大脑 将使用两个分子分析（定量PGR，RT-PCR）进行分析， 免疫细胞化学和酶测定以相关的炎症：反应 到病毒o：r转基因蛋白到转基因表达。分析将 考试促炎和抑制性细胞因子有关趋化因子，粘附 分子，血脑屏障的生物细胞和转基因表达和存活。 第二个特定目的将检查先天免疫反应在 关于转染效率。先天的不可死性响应（包括 非特异性吞噬性嗜中性粒细胞和单核细胞/杂噬细胞 注射后将检查替代补体途径） HSVIAP扩增子。响应将通过多种方式改变。首先， 选择性的嗜中性粒细胞或循环单核细胞/巨噬细胞 将使用特定抗体作为注入的每种细胞类型来完成 立即在Arnplicon Vecto注入之前和之后：其次， 我们将尝试抑制粘附并限制尿布 使用特定 粘附分子P-选择素和ICAM-1。第三，我们Wil1的抗体 尝试预防或限制促炎性细胞因子，趋化因子的诱导 和粘附分子在我们的HSVI中共表达了不合理的细胞因子IL-10 AP向量。最后，补体在先天免疫反应中的作用将 可以通过注射重组crry-ig ip来测试 iNmune细胞的化学诱使剂和活化剂。每个人的有效性 操作将通过特定目标1中使用的相同方法进行测定。