Site-directed RNA editing with endogenous ADARs

定点RNA编辑是一种在RNA水平上操纵遗传信息的新方法。目前，最大的挑战是实现高编辑产量，同时保持低脱靶编辑。在这个项目中，我们专注于募集内源性ADAR进行定点RNA编辑。该策略具有两个主要优点，正如我们最近所证明的：a）重编程遗传信息仅需要施用/表达短RNA（类似物），因此在技术上非常简单，以及B）：由于不需要异位过表达过度活性的脱氨酶，因此已经解决了脱靶问题。然而，目前的方法还远远不是最佳的，并导致内源性转录物的编辑效率太低。在项目A）中，我们的目标是用概念上新的、多价的和遗传上可编码的向导RNA设计来招募内源性阿达尔，以有效地编辑内源性转录物，这在当时还不可能。我们进一步的目标是以病毒方式递送这样的指导以准备体内应用。在部分B）中，我们用化学稳定的反义寡核苷酸募集内源性ADAR。同样，我们从一个新的设计开始，它比前一个小得多，并且可以允许我们通过招募更合适的阿达尔同种型来实现更高的编辑效率。在C）部分中，我们的目标是应用来自A）和B）部分的向导RNA和ASO用于小鼠中的体内编辑。后者是我们实验室建立的一项关键技术，旨在研究该技术在未来的全部治疗潜力。

Site-directed RNA editing is a novel approach to manipulate genetic information at the RNA level. Currently, the biggest unmet challenge is to achieve high editing yields while keeping off-target editing low. In this project, we focus on the recruitment of endogenous ADARs for site-directed RNA editing. This strategy has two major advantages, as we have recently demonstrated: a) reprogramming genetic information solely requires administration/expression of a short RNA (analog), thus is technically very simple, and b): the off-targted issue has been solved since no hyperactive deaminase needs to be ectopically overexpressed. Nevertheless, the current methods are yet far from being optimal and result in too little editing efficiency at endogenous transcripts. In project A), we aim to recruit endogenous ADAR with a conceptually new, multivalent and genetically encodable guideRNA design to edit endogenous transcripts efficiently, which was not yet possible. We further aim to deliver such guides virally to prepare for the in vivo application. In part B), we recruit endogenous ADARs with chemically stabilised antisense oligonucleotides. Again, we start from a new design, which is much smaller than the preceding one and which may allow us to achieve much higher editing efficiencies by recruiting a more apropriate ADAR isoform. In part C), we aim to apply the guideRNAs and ASOs from part A) and B) for in vivo editing in mice. The latter is a key technology to establish in our lab with regard to study the full therapeutic potential of this technology in the future.