Project 4: A permanent off-switch for AAV

项目 4：AAV 的永久开关

• 批准号: 10625294
• 负责人: Michael R. Farzan
• 金额: $ 35.43万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625294
• 关键词: Adverse event; Antibodies; Base Pairing; Binding; Capsid; Catalytic RNA; Cells; Cellular Immunity; Clinical; Collaborations; DNA; Data; Dependovirus; Disease remission; Dose; Duchenne muscular dystrophy; Enterobacteria phage P1 Cre recombinase; FDA approved; HIV-1; Human; Individual; Infection; Innate Immune Response; Intramuscular; Luciferases; Macaca; Macaca mulatta; Measurement; Measures; Mediating; Modeling; Mus; Pathway interactions; Pharmaceutical Preparations; Prophylactic treatment; Proteins; Reporter; Safety; Site; System; Testing; Therapeutic; Time; Trans-Activators; Transgenes; Vaccines; Virus; Work; adaptive immune response; adeno-associated viral vector; antibody engineering; antibody mimetics; antiretroviral therapy; cellular transduction; immune clearance; improved; in vivo; inhibitor; neutralizing antibody; protein expression; simian human immunodeficiency virus; transgene expression; vector; vector genome; viral rebound

PROJECT SUMMARY (Project 4 – A permanent off-switch for AAV transgenes) Adeno-associated virus (AAV)-expressed antibody-like entry inhibitors provide very robust protection from HIV-1 model viruses in rhesus macaques. AAV-mediated expression of broadly neutralizing antibodies or antibody-like molecules can also largely or wholly suppress an established infection in macaques. We thus have an effective vaccine and a pathway to drug-free virologic remission if this same approach could be safely applied to humans. However, there is currently no effective way to inactivate an AAV transgene in case of adverse events, and thus this approach is still considered too risky for most individuals. An effective ‘off-switch’ would have two important uses. First, it would increase the safety of AAV-based vaccines and therapies. Second, it would facilitate eradication studies by allowing sustained expression of a potent antibody or entry inhibitor, and then allowing it to be inactivated so that the rate of viral rebound can be measured. In preliminary data, we show that a morpholino can be used together with a highly efficient ribozyme to induce expression of an AAV transgene. We also show that the Cre recombinase can permanently inactivate an AAV transgene flanked by LoxP sites. Here we proposed to combine these observations to generate a permanent off-switch for an AAV-expressed transgene. We will then demonstrate in mice that this off-switch can halt otherwise efficient expression of two potent HIV-1 entry inhibitors. Finally, we will use the same principles to develop a morpholino-regulated on-switch. We will then use this switch to test whether a 4-month delay in transgene expression from the time of AAV inoculation can limit immune clearance of the transgene. These studies will therefore make AAV-based therapeutics safer, and facilitate study of the impact of long-term antibody expression on the reservoir of latently infected cells.

项目总结（项目4 -AAV转基因的永久关闭开关） 腺相关病毒（AAV）表达的抗体样进入抑制剂提供了非常强大的保护， 恒河猴中的HIV-1模型病毒。AAV介导的广泛中和抗体的表达或 抗体样分子也可以在很大程度上或完全抑制猕猴中已建立的感染。我们因此 如果同样的方法可以， 安全地应用于人类。然而，目前还没有有效的方法将AAV转基因转染到哺乳动物细胞中。 然而，这种方法并不适用于不良事件，因此这种方法对大多数人来说仍然被认为风险太大。有效 “关闭开关”有两个重要的用途。首先，它将提高基于AAV的疫苗的安全性， 治疗第二，它将促进根除研究，允许持续表达一种有效的 抗体或进入抑制剂，然后使其失活，以便可以降低病毒反弹的速率。 测定了在初步的数据中，我们表明，吗啉代可以与高效的 在一些实施方案中，所述方法包括使用核酶来诱导AAV转基因的表达。我们还表明，Cre重组酶可以 永久性地插入侧翼为LoxP位点的AAV转基因。在这里，我们建议将这些联合收割机 在一些实施方案中，本发明提供了一种观察结果，以产生AAV表达的转基因的永久关闭开关。然后我们将 在小鼠中证明，这种关闭开关可以阻止两种有效的HIV-1进入的有效表达 抑制剂的最后，我们将使用相同的原理来开发吗啉代调节的开关。我们将 然后使用这个开关来测试从AAV的时间到转基因表达的4个月延迟是否 接种可以限制转基因的免疫清除。因此，这些研究将使基于AAV的 更安全的治疗方法，并促进研究长期抗体表达对宿主的影响。 潜伏感染细胞