Modeling monocyte and macrophage based gene therapy for neuroAIDS

项目概要/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-1。 相关性痴呆（HAD），一种中枢神经系统（CNS）的原发性疾病，影响约20%的 艾滋病毒感染者。目前HAD的治疗受到许多抗逆转录病毒药物疗效差的阻碍， 血脑屏障（BBB）。因此，需要HAD的新疗法。循环血液 已知单核细胞衍生的巨噬细胞（MDM）在正常情况下迁移穿过BBB并进入CNS。 在正常和某些情况下;一些随后成熟为长寿的组织驻留大脑 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递送到CNS中， 侵入性和非手术方式。我们实验室和其他人以前的研究表明，MDM （人和小鼠）可以使用缺陷型慢病毒载体（DLV）在体外进行遗传修饰， 不利地影响其生物学特性。此外，我们已经表明，小鼠的原始血液 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， 通过短暂破坏BBB可以提高CNS摄取这些细胞的效率。我们有 最近显示分泌的抗HIV-达特单链抗体（scFv）和可溶性肿瘤坏死因子 来自DLV转导的MDM的sTNFR受体（sTNFR）可以有效地阻断条件性神经毒性作用。 来自HIV-1感染细胞或相应重组蛋白的培养基。在这个项目中，我们将建立一个基因， 用载体高效（>50%）稳定转导原代小鼠MDM的转移方法， 编码分泌型scFv和sTNFR。然后，我们将进行研究，以确定是否吸收这些 可以使用暂时破坏BBB来增强将遗传修饰的细胞导入小鼠脑中，并且 我们将评估外周输注本发明药物后CNS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的原发性小鼠MDM是否 或抗HIV-达特scFv可以改善两种充分研究的小鼠模型系统中的疾病进展， 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见解 用于对抗神经艾滋病和其他神经系统疾病。

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.