We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT) transcripts. We developed 5′-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD) mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.

我们评估了剪接体介导的RNA反式剪接作为LMNA相关的先天性肌营养不良的治疗方法的Lmna mRNA修复的潜力。这种基因治疗策略可降低突变的转录本表达，从而使相应的野生型（WT）转录本受益。我们开发5'-RNA前反式包含的第一个五年外显子-splicing分子LMNA和定位内含子的5 LMNA前体mRNA。在内含子5中的靶向序列不同并在C2C12成肌细胞中测试的9个反式剪接前分子中，在内源性Lmna上诱导了3个反式剪接事件mRNA，并在蛋白质水平得到证实。在源自LMNA相关的先天性肌营养不良（L-CMD）小鼠模型的原代肌管中进行的进一步分析导致突变表型的部分挽救。最后，我们在新生小鼠中使用腺相关病毒（AAV）递送在体内测试了该方法，并表明在AAV递送后50天，WT小鼠发生了转拼事件，尽管发生率很低。总而言之，尽管这些结果提供了体外重编程LMNA mRNA的第一个证据，但仍需要制定提高反转录事件发生率的策略，以在体内有效应用这种治疗方法。