Modeling monocyte and macrophage based gene therapy for neuroAIDS

项目摘要/摘要 人类免疫缺陷病毒1型(HIV)感染脑巨噬细胞和小胶质细胞，并导致HIV- 关联性痴呆(HAD)，一种原发的中枢神经系统(CNS)疾病，影响约20%的 感染艾滋病毒的人。目前对HAD的治疗受到许多抗逆转录病毒药物效果不佳的阻碍 跨越血脑屏障(BBB)的药物。因此，需要治疗HAD的新疗法。循环血液 已知单核细胞来源的巨噬细胞(MDM)可通过血脑屏障迁移并进入中枢神经系统。 在正常和特定情况下都是如此；其中一些后来成熟为长期驻留在组织中的大脑 巨噬细胞和小胶质细胞。这项研究的假设是，有可能利用自然资源 血液MDM的归巢/迁移特性，以便将神经保护因子输送到中枢神经系统，在非 侵入性和非手术方式。我们实验室和其他实验室之前的研究表明，MDM (人和小鼠)可以使用有缺陷的慢病毒载体(DLV)在体外进行基因改造，而不需要 对它们的生物学特性产生不利影响。此外，我们已经证明了小鼠的主要血液 经DLV基因修饰的单核细胞和骨髓来源的巨噬细胞可以进入大脑，并且 通过短暂破坏血脑屏障可以提高这些细胞摄取中枢神经系统的效率。我们有 新近发现分泌的抗HIV-Tat单链抗体(ScFv)和可溶性肿瘤坏死因子 DLV转导MDM的受体(STNFR)可有效阻断条件性神经毒性作用 来自HIV-1感染细胞的培养液或相应的重组蛋白。在这个项目中，我们将建立一个基因 一种高效(50%)稳定转导原代小鼠多药耐药载体的转移方法 编码可分泌的scFv和sTNFR。然后我们将进行研究，以确定是否吸收了这些 通过暂时阻断血脑屏障，可以增强进入小鼠大脑的转基因细胞，并且 我们将评估外周输注后中枢神经系统内基因表达的时程和稳定性 转基因MDM。最后，我们将确定稳定表达TNFR的小鼠原始MDM 或者抗HIV-Tat单链抗体可以在两个研究良好的小鼠模型系统中改善疾病进展 神经艾滋病。这项研究的成功完成有望为新方法提供重要的洞察力 用于抗击神经艾滋病和其他神经疾病。

Project Summary/Abstract Human immunodeficiency virus type 1 (HIV) infects brain macrophages and microglia and causes HIV- associated dementia (HAD), a primary disorder of the central nervous system (CNS) that affects about 20% of HIV-infected individuals. Current treatment of HAD is hampered by the poor efficiency of many antiretroviral drugs to cross the blood-brain barrier (BBB). Hence, new therapies for HAD are needed. Circulating blood monocyte-derived macrophages (MDM) are known to migrate across the BBB and to enter the CNS under both normal and certain circumstances; some subsequently maturing into long-lived tissue-resident brain macrophages and microglia. The hypothesis of this research is that it may be possible to exploit the natural homing/migratory properties of blood MDM in order to deliver neuroprotective factors into the CNS, in a non- invasive and non-surgical manner. Previous studies from our laboratory and others, have shown that MDM (human and mouse) can be genetically modified in vitro using defective lentiviral vectors (DLV) without adversely affecting their biological properties. Furthermore, we have shown that primary mouse blood monocytes and bone marrow-derived macrophages genetically modified by DLV can enter the brain, and that the efficiency of CNS uptake of these cells can be enhanced through transient disruption of the BBB. We have recently shown that secreted anti-HIV-Tat single chain antibodies (scFv) and soluble tumor necrosis factor receptor (sTNFR) from DLV-transduced MDM can effectively block the neurotoxic effects of conditioned medium from HIV-1 infected cells or respective recombinant proteins. In this project, we will establish a gene transfer method for high efficiency (>50%), stable transduction of primary mouse MDM with vectors that encode secretable scFv and sTNFR. We will then conduct studies to determine whether uptake of these genetically modified cells into the mouse brain can be enhanced using temporary disruption of the BBB, and we will evaluate the time course and stability of intra-CNS gene expression following peripheral infusion of the genetically-modified MDM. Finally, we will determine whether primary mouse MDM that stably express TNFR or anti-HIV-Tat scFv can ameliorate disease progression in two well-studied murine model systems for neuroAIDS. Successful completion of this study is expected to provide significant insight into new approaches for combating neuroAIDS and other neurologic disorders.