Brain myeloid cell-targeted multiplexed gene editing for SIV/HIV eradication

项目摘要 中枢神经系统中的长寿命髓系细胞，如血管周围巨噬细胞和小胶质细胞 (CNS)持续携带艾滋病毒。这些被感染的细胞可能是残余病毒血症的来源 长期抗逆转录病毒治疗(ART)或ART停止后病毒反弹。毫无疑问，这是紧迫的。 需要开发新的战略，专门针对中枢神经系统髓系细胞根除艾滋病毒和治愈艾滋病毒。就是- 因为被广泛研究的“休克并杀死”方法的影响可能会加剧神经炎症，基因 治疗成为最佳策略，特别是先进的CRISPR基因组编辑技术。猿猴 免疫缺陷病毒(SIV)感染猕猴是检测新策略的最佳模型 到临床研究。我们已经证明，SIV感染在中枢神经系统中广泛传播，甚至在ART上的动物中也是如此。 因为病毒在感染后几天内进入大脑并建立了潜伏的蓄水池 发生得很早，艺术的发起应该越早越好。此外，大量研究表明， CCR5/CCR2在HIV进入和神经炎症中起主要作用。最重要的是，我们使用了AAV CRISPR/Cas基因组编辑器在人源化小鼠和非人源化小鼠模型中根除HIV/SIV前病毒 人类灵长类动物。然而，缺乏高效和可靠的AAV血清型来转导髓系 中枢神经系统中的细胞仍然是一个关键挑战。因此，我们假设多靶点基因编辑 系统通过血脑屏障(BBB)可以清除感染的髓系细胞中的SIV前病毒，保护细胞 抗新感染，抑制神经炎症。为了验证这一假设，我们将优化一种新型的AAV血清- 具有血脑屏障穿透和髓系特异性转导的类型(即AAV-BM)，以有效地传递较小的 CjCas9和特定于4个目标站点(SIV LTR、GAG和主机CCR5、CCR2)的多路sgRNAs(BMCj4) 用于体内根除艾滋病毒/SIV的整个中枢神经系统(目标1)。我们将评估BMCj4早期治疗的疗效 在急性SIV感染中预防脑SIV感染或/和切除脑髓细胞中的SIV前病毒DNA 早期艺术(目标2a)。我们还将用ART来确定BMCj4的治疗效果，然后用 用于根除持续性脑SIV潜伏感染的替代AAV-BM(目标2b)。我们预计早些时候或 长期重复的BMCj4AAV基因治疗将有效根除急性和潜伏感染的艾滋病毒前病毒 并广泛减少脑部病毒库的大小，以实现灭菌或功能治愈 艾滋病毒/艾滋病，特别是神经艾滋病。

Project Summary The long-lived myeloid cells such as perivascular macrophages and microglia in the central nervous system (CNS) persistently harbor HIV. These infected cells could contribute to the source of residual viremia during long-term antiretroviral therapy (ART) or to rebounding virus upon ART cessation. It is undoubtfully and urgently needed to develop novel strategies to specifically target CNS myeloid cells for HIV eradication and a cure. Be- cause the effects of the widely-studied “Shock and Kill” approach could exacerbate neuroinflammation, gene therapy emerges as the optimal strategy, particularly the advanced CRISPR genome editing technology. Simian immunodeficiency virus (SIV) infection of macaques is the best available model for testing novel strategies prior to clinical studies. We have shown that SIV infection has a broad spread in the CNS even in animals on ART. Because the virus enters the brain within a few days after infection and the establishment of the latent reservoir occurs very early, the initiation of ART should be as early as possible. In addition, numerous studies suggest that CCR5/CCR2 play a major role in HIV entry and neuroinflammation. Most importantly, we have used AAV delivery of a CRISPR/Cas genome editor to eradicate HIV/SIV provirus in models of humanized mice and non- human primates. However, the lack of AAV serotypes that are highly effective and reliable to transduce myeloid cells in the CNS remains a key challenge. Therefore, we hypothesize that the multiple-targeting gene editing system across the blood brain barrier (BBB) can remove SIV provirus in infected myeloid cells, protect cells against new infection, and inhibit neuroinflammation. To test this hypothesis, we will optimize a novel AAV sero- type with BBB penetration and myeloid-specific transduction (namely AAV-BM) to effectively deliver the smaller cjCas9 with multiplex sgRNAs (BMCj4) specific for 4 target sites (SIV LTR, gag, and host CCR5, CCR2) into the entire CNS for in vivo HIV/SIV eradication (Aim 1). We will evaluate the efficacy of BMCj4 early treatment in preventing brain SIV infection or/and excising SIV proviral DNA from brain myeloid cells in acute SIV infection with early ART (Aim 2a). We will also determine the therapeutic effect of BMCj4 with ART and then boost it with an alternative AAV-BM for eradication of persistent brain SIV latent infection (Aim 2b). We expect that early or long-term repeated BMCj4 AAV gene therapy will effectively eradicate acute and latently-infected HIV provirus and extensively minimize the size of the brain viral reservoir to achieve a sterilizing or functional cure of HIV/AIDS, particularly NeuroAIDS.