The hCNS-HIV/ARV assay system, a platform utilizing human iPSC-neurons/-microglia/-astrocytes to test HIV/AIDS therapeutics for neurotoxicity related to HIV-associated cognitive disroder (HAND)

在全球范围内，约有4000万艾滋病毒/艾滋病患者，艾滋病毒抗逆转录病毒药物(ARV)是数十亿 医药板块随着代理商的不断发展而不断发展。多亏了联合抗逆转录病毒 治疗(CART，同时给予2到4个抗逆转录病毒药物)HIV感染者的寿命接近正常。 艾滋病毒携带者孕妇也接受购物车，她们的孩子在出生时预防性给予购物车(在 一般情况下，所有HIV+的婴儿和儿童都可以获得CART)。对于所有HIV+患者，如果停止CART，则HIV 复制重新开始；因此，艾滋病毒携带者必须在其余生中继续使用购物车。对于HIV+成人，抗逆转录病毒药物 在衰老过程中存在，其中中枢神经系统(CNS)神经元的丧失导致 认知/痴呆症。对于HIV阳性的婴儿和儿童，抗逆转录病毒药物存在于神经发育过程中。HIV+ 个人经常患上艾滋病毒相关认知障碍(HAND)，其中包括与艾滋病毒相关的 痴呆症(HAD，在艾滋病中常见)和较轻的形式(无症状神经认知障碍和轻度 神经认知障碍(MND)。虽然CART降低了HAD的发生率，但ANI和MND仍处于较高水平 水平，即使在艾滋病毒负荷非常低的情况下也是如此。事实上，某些抗逆转录病毒药物可能会导致Hand，令人担忧的是， 购物车上的HIV阳性儿童在智力表现上表现出发育迟缓。此外，艾滋病毒感染/复制在 小胶质细胞(MG)是中枢神经系统的一种先天免疫细胞，它们不是ARV所能及的，因为ARV在 跨越血液：脑屏障。更多的脑穿透性抗逆转录病毒药物被开发出来，但出乎意料的是，这些 与更高的手部发病率有关。ARV神经毒性的临床前研究已在初级 啮齿动物神经元和神经胶质细胞。我们的目标是开发一种检测系统(称为hCNS-HIV/ARV平台)，用于 测试抗逆转录病毒药物的神经毒性和抑制HIV、神经元、MG和星形胶质细胞(另一种胶质细胞)的有效性， 来源于人类诱导的多能干细胞(HiPSC-神经元、HiPSC-MG和HiPSC-ACS)。在第一阶段， 我们开发了在384孔培养皿中培养的HiPSC神经元上测试抗逆转录病毒药物的方法，利用高通量 数字显微镜/分析。我们发现了神经毒性(神经突起、突触和钙瞬变减少) 对于elvitegravir(EVG，作用最强，与大鼠神经元匹配的数据)，多洛替格韦(DTG，与出生有关 缺陷)，以及替诺福韦富马酸二异丙酯(TDF)和代表CART的某些ARV组合 比抗逆转录病毒药物单独使用的效果更强。TDF还降低了人的存活率，并引发了表观遗传学变化 神经前体细胞(HNPC)，提示TDF可能影响神经发生，这是一个关键的过程 神经发育和认知。在第二阶段，我们将制定标准的分析方案/分析方法 在HiPSC-神经元/-MG/-ACS和HiPSC-NPC上测试抗逆转录病毒和艾滋病毒感染本身。HCNS- 艾滋病毒/抗逆转录病毒平台将使抗逆转录病毒药物的开发能够以最小的神经毒性抑制艾滋病毒，并将 向开发艾滋病毒/艾滋病新疗法的制药公司销售。

Globally, about 40 million people live with HIV/AIDS, and HIV antiretroviral agents (ARVs) are a multi-billion dollar pharmaceutical sector with continued development of agents. Thanks to combination antiretroviral therapy (cART, in which 2 to 4 ARVs are given, simultaneously) HIV+ people have a near-normal life-span. HIV+ pregnant women also receive cART, and their children are given cART prophylactically at birth (in general, cART is given to all HIV+ infants and children). For all HIV+ patients, if cART is stopped, HIV replication restarts; thus, HIV+ people must remain on cART for the rest of their lives. For HIV+ adults, ARVs are present during aging, in which loss of central nervous system (CNS) neurons contributes to loss of cognition/dementia. For HIV+ infants and children, ARVs are present during neurodevelopment. HIV+ individuals frequently develop HIV-Associated Cognitive Disorders (HAND), which includes HIV-associated dementia (HAD,common in AIDS), and less severe forms (asymptomatic neurocognitive impairment and mild neurocognitive disorder (MND). While cART has reduced the incidence of HAD, ANI and MND remain at high levels, even when HIV load is very low. In fact, certain ARVs may contribute to HAND, and alarmingly, HIV+children on cART exhibit developmental delays in intellectual performance. Also, HIV infects/replicates in microglia (MG), an innate immune cell of the CNS, and MG are out of the reach of ARVs that are poor at crossing the blood:brain barrier. More brain-penetrant ARVs were developed, but, unexpectedly, these are linked to greater incidence of HAND. Preclinical research on ARV neurotoxicity has been conducted on primary rodent neurons and glial. Our goal is to develop an assay system (dubbed the hCNS-HIV/ARV platform) for testing ARVs for neurotoxicity and efficacy at inhibiting HIV, neurons, MG and astrocytes (another glial cells), derived from human induced pluripotent stem cells (hiPSC-neurons, hiPSC-MG, and hiPSC-ACs). In phase I, we developed methods for testing ARVs on hiPSC-neurons plated in 384-well dishes, utilizing high-throughput digital microscopy/analysis. We found neurotoxicity (reductions in neurites, synapses, and calcium transients) for elvitegravir (EVG, strongest effect, matching data with rat neurons), dolutegravir (DTG, linked to birth defects), and tenofovir disoproxil fumerate (TDF) and certain combinations of ARVs representing cART had stronger effects than the ARVs, alone. TDF also reduced the viability and elicited epigenetic changes in human neural precursor cells (hNPCs), suggesting that TDF may affect neurogenesis, a process critical for neurodevelopment and cognition. In phase II we will develop standard assay protocols/analysis methods for testing ARVs and HIV infection, itself, on hiPSC-neurons/-MG/-ACs, and on hiPSC-NPCs. The hCNS- HIV/ARV platform will enable development of ARVs that inhibit HIV with minimal neurotoxicity and will be marketed to pharmaceutical companies developing novel therapeutics for HIV/AIDs.