Disabling the replication-competent HIV provirus in the reservoir by combined CRISPR-Cas action

通过 CRISPR-Cas 联合作用使储存库中具有复制能力的 HIV 原病毒失效

• 批准号: 9908049
• 负责人: Benjamin Berkhout
• 金额: $ 52.47万
• 依托单位: ACADEMICAL MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-05 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908049
• 关键词: Adverse effects; Animal Model; Antiviral Agents; CCR5 gene; CD32 Antigens; CD4 Antigens; CD4 Positive T Lymphocytes; CXCR3 gene; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Conserved Sequence; DNA; DNA Repair; Enzymes; Excision; Funding; Future; Gene Delivery; Genome; Guide RNA; HIV; HIV Genome; HIV Infections; Immune system; Infection; Kinetics; Lentivirus Vector; Ligands; Mediating; Modeling; Modification; Mus; Nonhomologous DNA End Joining; Outcome; Patients; Pharmacotherapy; Pre-Clinical Model; Preclinical Testing; Prevention strategy; Process; Protein Isoforms; Proviruses; Publishing; Reagent; Reporting; Research; Resistance to infection; Rest; Role; Route; Specificity; Sterilization; Surface; System; T-Lymphocyte; Terminology; Testing; Therapeutic; Therapeutic Intervention; Time; Translating; Treatment Efficacy; Tropism; Viral; Viral reservoir; Virus; combinatorial; design; ds-DNA; flexibility; genome editing; humanized mouse; in vivo; in vivo Model; innovation; insertion/deletion mutation; mouse model; novel; novel strategies; nuclease; preference; prevent; programs; receptor; repaired; safety testing; screening; superinfection; therapeutic gene; tool; transcription activator-like effector nucleases; vector; viral rebound; zinc finger nuclease

Summary. DNA can be targeted by the CRISPR- Cas (CC) genome editing tool, which was swiftly implemented in anti-HIV studies1-8. We confirmed potent HIV inhibition, but also demonstrated rapid virus escape from CC attack9. Inspection of the escape viruses indicated a role of the NHEJ enzyme, which repairs dsDNA breaks and introduces small insertions/deletions (indels) in the process. In other words, we described a novel viral escape route that is facilitated by a cellular DNA repair process. These results were confirmed by other labs10-21. A combinatorial CC attack against conserved sequences triggered HIV inactivation through hypermutation, quite different from the proposed excision route (Fig 1). Although provirus excision could be detected in our system, it occurred with a very low efficiency. Instead, CC attack generated hypermutated proviruses that apparently lost all replication potential. In other words, continuous CC action can functionally CURE HIV infected cells, leaving the cells with a graveyard of inactivated HIV proviruses. This surprise finding represents a major hallmark in HIV CURE studies. We now propose to first delineate the underlying mechanisms and variables that determine the outcome of CC attack (HIV excision versus hypermutation). This will allow us to formulate the best CURE strategy. At the same time we focus on the major obstacle towards a CURE: delivery of the gene therapeutics to cells that compose the HIV-reservoir. The CC and delivery tools will then be combined for in vivo CURE studies in the HIS mouse.

摘要DNA可以被CRISPR靶向- Cas（CC）基因组编辑工具， 在抗艾滋病毒研究中实施1 -8。我们证实 有效的艾滋病毒抑制，但也表现出快速 病毒从CC攻击中逃逸9.检查 逃逸病毒表明NHEJ的作用 酶，修复dsDNA断裂， 引入小的插入/缺失（indels）， 过程换句话说，我们描述了一种新的病毒， 细胞DNA促进的逃逸途径 修复过程。证实了上述结果 其他实验室10 -21.一种针对 保守序列触发HIV失活 通过超突变，与 建议的切除路线（图1）。尽管前病毒 在我们的系统中可以检测到切除，它 发生的效率非常低。相反，CC 攻击产生高度变异的前病毒， 显然失去了所有的复制潜力换句 换句话说，连续的CC动作可以在功能上 治愈艾滋病毒感染的细胞，使细胞与 灭活的HIV前病毒的墓地。这 一个令人惊讶的发现代表了艾滋病毒的一个主要特征 CURE研究。我们现在建议首先描述 潜在的机制和变量， 确定CC攻击的结果（HIV切除 相对于超突变）。这将使我们能够 制定最佳的治愈策略。与此同时，我们专注于治愈的主要障碍： 将基因治疗剂递送至构成HIV储库的细胞。CC和交付工具 然后将其组合用于HIS小鼠的体内CURE研究。