Project 4: A permanent off-switch for AAV

项目总结(项目4--AAV转基因的永久开关) 腺相关病毒(AAV)表达的抗体样进入抑制剂可提供非常强大的保护作用 恒河猴体内的HIV-1模型病毒。腺相关病毒介导的广谱中和抗体的表达 抗体样分子也可以在很大程度上或完全抑制猕猴的既定感染。因此，我们 有一种有效的疫苗和一条无药物病毒学缓解的途径，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来灭活AAV转基因。 在发生不良事件的情况下，这种方法仍然被认为对大多数人来说风险太大。卓有成效的 “关闭开关”有两个重要的用途。首先，它将增加以AAV为基础的疫苗的安全性和 治疗。其次，它将通过允许有效的持续表达来促进根除研究 抗体或进入抑制物，然后允许其灭活，以便病毒反弹的速度 量过了。在初步数据中，我们表明吗啉可以与高效的 核酶诱导AAV转基因表达。我们还证明了Cre重组酶可以 永久灭活位于loxP位点两侧的AAV转基因。在这里，我们建议将这些组合在一起 对AAV表达的转基因产生永久开关的观察。到时候我们会的 在小鼠中证明这种开关可以阻止两个有效HIV-1进入的高效表达 抑制剂。最后，我们将使用相同的原理来开发一种吗啉调节的开关。我们会 然后使用这个开关来测试从AAV开始的4个月的转基因表达延迟 接种可以限制转基因的免疫清除。因此，这些研究将使AAV成为 治疗更安全，并便于研究长期抗体表达对宿主的影响 潜伏感染的细胞。

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.