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

基本信息

批准号：
9753408
负责人：
Benjamin Berkhout
金额：
$ 53.42万
依托单位：
ACADEMICAL MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/9753408
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

项目摘要

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.

概括。 DNA可以由CRISPR- CAS（CC）基因组编辑工具，迅速在抗HIV研究中实施1-8。我们确认有效的艾滋病毒抑制，但也证明了迅速病毒从CC攻击中逃脱。检查逃生病毒表示NHEJ的作用酶，维修dsDNA断裂，并且引入小插入/删除（indels）过程。换句话说，我们描述了一种新颖的病毒由细胞DNA促进的逃生路线维修过程。这些结果通过其他实验室10-21。组合CC攻击保守的序列触发HIV失活通过超名，与提出的切除路线（图1）。虽然遗嘱病毒可以在我们的系统中检测到切除以非常低的效率发生。而是CC 攻击产生的过度解释的病毒显然失去了所有复制潜力。在其他单词，连续的CC动作可以在功能上治愈感染HIV感染的细胞，使细胞与A 灭活的艾滋病毒病毒的墓地。这惊喜发现代表了艾滋病毒的主要标志治疗研究。我们现在建议首先描述基本机制和变量确定CC攻击的结果（HIV切除与超名）。这将使我们能够制定最佳的治疗策略。同时，我们专注于治愈的主要障碍：将基因疗法递送到组成HIV-粘膜的细胞。 CC和交付工具然后将组合在他的老鼠中进行体内治疗研究。