Alves Christiano R R, Das Sabyasachi, Krishnan Vijai, Ha Leillani L, Fox Lauren R, Stutzman Hannah E, Shamber Claire E, Kalailingam Pazhanichamy, McCarthy Siobhan, Lino Cardenas Christian L, Fong Claire E, Imai Takahiko, Mitra Sunayana, Yun Shuqi, Wood Rachael K, Benning Friederike M C, Roh Kangsan, Lawton Joseph, Kim Nahye, Silverstein Rachel A, Ferreira da Silva Joana, de la Cruz Demitri, Richa Rashmi, Xie Jun, Gray-Edwards Heather L, Malhotra Rajeev, Chung David Y, Chao Luke H, Tsai Shengdar Q, Maguire Casey A, Lindsay Mark E, Kleinstiver Benjamin P, Musolino Patricia L
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
Nat Biomed Eng. 2025 Sep 11. doi: 10.1038/s41551-025-01499-1.
Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.
α-肌动蛋白同种型2（ACTA2）基因中的致病性错义突变会导致多系统平滑肌功能障碍综合征（MSMDS），这是一种遗传性血管病，与儿童期的中风、主动脉夹层和死亡有关。在这里，我们进行了突变特异性蛋白质工程，以开发一种定制的CRISPR-Cas9酶，该酶对最常见的导致MSMDS的突变ACTA2 R179H具有增强的靶向活性。为了直接纠正R179H突变，我们筛选了数十种碱基编辑器配置，以开发一种高度精确的纠正性A到G编辑，这种编辑产生的有害旁观者编辑最少，而使用野生型SpCas9碱基编辑器时，有害旁观者编辑很常见。我们创建了一个MSMDS小鼠模型，该模型表现出与人类患者一致的表型，包括血管病和过早死亡，以探索这种策略的体内治疗潜力。通过工程化的平滑肌嗜性腺相关病毒（AAV-PR）载体递送定制的碱基编辑器，可显著延长MSMDS小鼠的生存期，并在其整个生命周期内挽救包括血管、主动脉和大脑在内的全身表型。我们的研究结果突出了定制的突变特异性CRISPR-Cas9酶如何能够通过碱基编辑器改善突变校正。
