Byrne Susan M, Burleigh Stephen M, Fragoza Robert, Jiang Yue, Savva Yiannis, Pabon Ricky, Kania Evan, Rainaldi Joseph, Portell Andrew, Mali Prashant, Briggs Adrian W
Shape Therapeutics, 700 Dexter Avenue North, Seattle, WA, 98109, USA.
University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA.
Nat Commun. 2025 May 26;16(1):4860. doi: 10.1038/s41467-025-60155-z.
Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR's adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies.
利用人类RNA腺苷脱氨酶（ADAR）进行定制RNA碱基编辑，有望实现无DNA损伤或无需使用外源蛋白的治疗性基因编辑。ADAR的腺苷到肌苷（实际上是A到G）脱氨活性可通过反义引导RNA（gRNA）靶向转录本，但体内递送的局限性对其疗效提出了挑战。将gRNA嵌入U7小核RNA（snRNA）框架可极大地增强内源性ADAR的RNA编辑能力，一项750重单细胞诱变筛选进一步优化了该框架。一种具有更强合成U7启动子的优化支架，能使每个细胞从单个DNA构建体在体外实现76%的RNA编辑，在一次全身性腺相关病毒（AAV）注射后，可使黏多糖贮积症I型（Hurler综合征）小鼠大脑实现75%的编辑，超过了环状gRNA方法。该技术还使已发表的杜氏肌营养不良症（DMD）外显子跳跃设计在分化的成肌细胞中提高了25倍。我们设计的U7框架代表了一种用于基于ADAR的RNA编辑和其他反义RNA疗法的通用支架。
