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

用于根除 SIV/HIV 的脑髓细胞靶向多重基因编辑

• 批准号: 10476684
• 负责人: Wenhui Hu
• 金额: $ 91.54万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-05 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10476684
• 关键词: AIDS/HIV problem; Acute; Animals; Bar Codes; Blood - brain barrier anatomy; Brain; Brain region; CCL2 gene; CCR5 gene; CRISPR/Cas technology; Capsid; Cells; Chinese; Chronic Phase; Clinical; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Data; Early treatment; Epidemic; Excision; Genes; Genome; Goals; Guide RNA; HIV; HIV Infections; HIV-1; In Vitro; Individual; Infection; Injections; Intervention; Intravenous; Latent virus infection phase; Libraries; Macaca; Macaca mulatta; Measurement; Mediating; Microglia; Modeling; Monitor; Monkeys; Mus; Myelogenous; Myeloid Cells; Names; Neuraxis; Outcome; Plasma; Play; Prevention; Prevention therapy; Proviruses; Regimen; Reporter; Residual state; Rhesus; Role; SIV; Seeds; Serotyping; Shock; Site; Source; System; Technology; Testing; Therapeutic Effect; Time; Tissues; Vero Cells; Viral reservoir; Viremia; Virus; Virus Diseases; antiretroviral therapy; base; blood-brain barrier penetration; brain size; cell type; efficacy evaluation; gene therapy; genome editing; high risk; high risk population; humanized mouse; in vivo; macrophage; nervous system disorder; neuroAIDS; neurocognitive disorder; neuroinflammation; nonhuman primate; novel; novel strategies; prevent; promoter; relating to nervous system; simian human immunodeficiency virus; treatment guidelines; viral rebound

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.

项目摘要 中枢神经系统中的长寿命髓样细胞，如血管周围巨噬细胞和小胶质细胞 (CNS)持续携带艾滋病毒。这些感染的细胞可能有助于在治疗过程中残留病毒血症的来源。 长期抗逆转录病毒治疗（ART）或ART停止后病毒反弹。这是明智和紧迫的， 需要开发新的策略，专门针对中枢神经系统骨髓细胞的艾滋病毒根除和治愈。是- 因为被广泛研究的“休克和杀死”方法的影响可能会加剧神经炎症， 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）感染的猕猴是最好的可用模型，用于测试新的策略， 临床研究。我们已经证明SIV感染在CNS中广泛传播，甚至在接受ART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潜伏的储库 如果发生得很早，ART应该尽早开始。此外，许多研究表明， CCR 5/CCR 2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 在人源化小鼠和非人源化小鼠模型中递送CRISPR/Cas基因组编辑器以根除HIV/SIV前病毒 人类灵长类动物然而，缺乏对骨髓造血干细胞高度有效和可靠的AAV血清型， 中枢神经系统中的细胞仍然是一个关键挑战。因此，我们假设多靶向基因编辑 系统通过血脑屏障（BBB）可以清除感染的骨髓细胞中的SIV前病毒，保护细胞 对抗新的感染，抑制神经炎症。为了验证这一假设，我们将优化一种新的AAV血清- 具有BBB穿透和骨髓特异性转导的AAV-BM型（即AAV-BM）， cjCas 9与对4个靶位点（SIV LTR、gag和宿主CCR 5、CCR 2）特异性的多重sgRNA（BMCj 4）一起进入细胞内。 用于体内HIV/SIV根除的整个CNS（目的1）。我们将评估BMCj 4早期治疗在以下患者中的疗效： 预防脑SIV感染或/和在急性SIV感染中从脑髓样细胞切除SIV前病毒DNA 早期抗逆转录病毒疗法（Aim 2a）。我们还将确定BMCj 4与ART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（目的2b）。我们预计， 长期重复的BMCj 4 AAV基因治疗将有效地根除急性和潜伏感染的HIV前病毒 并广泛地最小化脑病毒储存库的大小，以实现消毒或功能性治愈， 艾滋病毒/艾滋病，特别是神经艾滋病。