Eradication of brain/CNS HIV reservoirs via anti-HIV gene-modified macrophages

项目总结 联合抗逆转录病毒疗法(CART)非常有效，但在中枢神经系统中效果较弱。 由于药物渗透性差，导致中枢神经系统(CNS)。此外，在中枢神经系统中感染艾滋病毒的细胞-被称为“中枢神经系统 主要由髓系细胞组成，如小胶质细胞和巨噬细胞，因此抗肿瘤。 所采用的艾滋病毒战略需要适应这些细胞类型。已经应用了各种方法 为了控制中枢神经系统中的病毒载量，例如，表达抗HIV嵌合抗原受体的T细胞(CAR-1) T)或广谱中和抗体(BNAb)--这些方法有效地针对HIV或HIV感染细胞 但对中枢神经系统储存库无效，因为细胞毒性T细胞不能攻击髓系 细胞生长良好，抗体对中枢神经系统的渗透率极低。因此，没有切实可行的办法。 足够成功地安全地消除了这些水库。 在这里，我们打算通过使用表达抗-CNS抗体的转基因巨噬细胞来净化中枢神经系统的储备库 艾滋病毒CARS(CAR-M)(目标1)和多向bNAb(bNAb-M)(目标2)。这两个都将在最新的 从血液来源分离的单核细胞作为中枢神经系统的载体和效应细胞。重要的是 当神经炎症发生时，单核细胞将被招募到中枢神经系统；艾滋病毒感染本身以及一些 抗逆转录病毒药物，如替诺福韦富马酸二异丙酯(TDF)或奈韦拉平(NVP)，是已知的 神经元性炎症。为了克服艾滋病毒准种的问题，我们将使用每个工具针对几个 不同的表位，即在同一细胞中表达2个或2个以上的抗HIV CAR(目标1)或在 针对HIV包膜蛋白上的3个独立表位的分子(目标2)。此外，为了保护 在基因修饰的单核细胞中，我们将同时表达两个抗HIV基因，这些免疫治疗 工具；a)C46，干扰艾滋病毒包膜和细胞膜之间的融合，从而阻止艾滋病毒感染 和b)针对艾滋病毒LTR的shRNA(Sh516)，它破坏所有艾滋病毒转录本，包括来自 HIV LTR.总之，我们的方法将确保CNS在基因修饰后的单核细胞中表达 多辆抗艾滋病毒汽车或多向bNAb，同时保护免受艾滋病毒感染和/或生产。这个 然后单核细胞会在中枢神经系统分化为巨噬细胞，并通过抗HIV-CAR攻击感染的细胞 (CAR-M)或分泌多向bNAbs(bNAb-M)诱导抗体依赖的细胞毒作用或 对感染细胞的吞噬作用(ADCC/ADCP)。在bNAb-M方法的情况下，分泌的bNAbs将 也适用于支持ADCC/ADCP的相邻髓系细胞。此外，bNAbs将中和新的 艾滋病毒感染。因此，我们的方法非常独特，如果成功，将开辟一条战胜艾滋病毒的新途径 中南地区的水库。

PROJECT SUMMARY Combination antiretroviral therapy (cART) is highly effective, but it is weakened in the central nervous system (CNS) due to poor drug penetration. In addition, cells infected by HIV in the CNS - termed “CNS reservoirs” - are mainly constituted by myeloid lineage cells, such as microglia cells and macrophages, thus anti- HIV strategies employed are needed to be adapted to these cell types. Various approaches have been applied for controlling viral load in the CNS - for example, T cells expressing anti-HIV chimeric antigen receptor (CAR- T) or broadly neutralizing antibodies (bNAb) – and these approaches effectively target HIV or HIV-infected cells systemically but are ineffective against the CNS reservoirs since cytotoxic-T cells cannot attack myeloid-lineage cells well and antibody penetration into the CNS is extremely low. As such, there is no practical approach successful enough to eliminate these reservoirs safely. We here intend to purge the CNS reservoirs via using genetically modified macrophages expressing anti- HIV CARs (CAR-M) (Aim 1) and multidirectional bNAb (bNAb-M) (Aim 2). Both will be expressed in freshly isolated monocytes from blood sources as a carrier vehicle to the CNS and an effector cell. Importantly, monocytes will be recruited to the CNS when neuroinflammation occurs; HIV infection itself as well as some antiretroviral drugs such as Tenofovir disoproxil fumarate (TDF) or nevirapine (NVP), are known mediators of neuronal inflammation. To overcome the issue of HIV quasispecies, we will use each tool to target several different epitopes, i.e., express 2 or more anti-HIV CARs in the same cells (Aim 1) or multidirectional bNAbs in one molecule which target 3 independent epitopes on the HIV envelope protein (Aim 2). In addition, to protect the gene-modified monocytes, we will simultaneously express two anti-HIV genes with these immunotherapeutic tools; a) C46 that interferes fusion between HIV envelope and cellular membranes, thus blocking HIV infections and b) shRNA against HIV LTR (sh516) that destructs all HIV transcripts including the sequence derived from HIV LTR. Altogether, our approach will ensure CNS delivery of monocytes following gene modification to express multiple anti-HIV CARs or multidirectional bNAbs whilst protecting from HIV infections and/or productions. The monocytes will then differentiate into macrophages in the CNS and attack the infected cells via anti-HIV CAR (CAR-M) or secreting multidirectional bNAbs (bNAb-M) inducing antibody-dependent cytotoxicity or phagocytosis (ADCC/ADCP) against the infected cells. In case of the bNAb-M approach, secreted bNAbs will also work on adjacent myeloid cells which support both ADCC/ADCP. In addition, the bNAbs will neutralize new HIV infection. As such, our approach is highly unique, and, if successful, will open a new avenue to defeat HIV reservoirs in the CNS.