Alves Christiano R R, Ha Leillani L, Yaworski Rebecca, Lazzarotto Cicera R, Christie Kathleen A, Reilly Aoife, Beauvais Ariane, Doll Roman M, de la Cruz Demitri, Maguire Casey A, Swoboda Kathryn J, Tsai Shengdar Q, Kothary Rashmi, Kleinstiver Benjamin P
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
bioRxiv. 2023 Jan 21:2023.01.20.524978. doi: 10.1101/2023.01.20.524978.
Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. is a paralogous gene that mainly differs from by a C•G-to-T•A transition in exon 7, resulting in the skipping of exon 7 in most transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient mutation. Here, we optimized a base editing approach to precisely edit , reverting the exon 7 mutation via an A•T-to-G•C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise editing in an SMA mouse model. This base editing approach to correct should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.
脊髓性肌萎缩症（SMA）是一种由该基因的突变引起的毁灭性神经肌肉疾病。尽管已经开发了各种疗法，但SMA婴儿的治疗效果可能仍然不理想，而且这些疗法的持续时间也不确定。是一个旁系同源基因，其与主要的区别在于外显子7中的C•G到T•A转换，导致大多数转录本中外显子7的跳跃，仅产生低水平的存活运动神经元（SMN）蛋白。针对外显子7突变的基因组编辑技术可以提供一种治疗策略，无论患者的突变如何，都能将SMN蛋白表达恢复到正常水平。在这里，我们优化了一种碱基编辑方法来精确编辑，通过A•T到G•C的碱基编辑来逆转外显子7突变。我们测试了一系列不同的腺苷碱基编辑器（ABE）和Cas9酶，在SMA患者来源的成纤维细胞中实现高达99%的预期编辑，同时外显子7转录本表达和SMN蛋白水平增加。我们生成并表征了与高保真Cas9变体融合的ABE，减少了潜在的脱靶编辑。通过双腺相关病毒（AAV）载体递送这些优化的ABE，在SMA小鼠模型中实现了精确的编辑。这种纠正的碱基编辑方法应该为SMA提供一种持久的基因治疗，与目前的核酸、小分子或外源基因替代疗法相比具有优势。更广泛地说，我们的工作突出了无PAM的SpRY碱基编辑器高效安全地进行编辑的潜力。
