Genethon, 91000 Evry, France.
INTEGRARE Research Unit UMR_S951 (INSERM, Université Paris-Saclay, Univ Evry), 91000 Evry, France.
Int J Mol Sci. 2024 Sep 27;25(19):10444. doi: 10.3390/ijms251910444.
Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein. Therefore, expressing larger dystrophin proteins may be necessary for a more complete correction of the disease phenotype. In this study, we developed three novel midi-dystrophin constructs using a dual-AAV approach, leveraging split-intein-based protein trans-splicing. The midi-dystrophins include additional domains compared to µDys, such as the central cytoskeleton-binding domain, nNOS and Par1b interacting domains, and a complete C-terminal region. Given the limited capacity of each AAV vector, we strategically partially reduced hinge regions while ensuring that the structural stability of the protein remains intact. We predicted the interactions between the two halves of the split midi-Dys proteins thanks to the deep learning algorithm AphaFold3. We observed strong associations between the N- and C-termini in midi-Dys 1 and 2, while a weaker interaction in midi-Dys 3 was revealed. Our subsequent experiments confirmed the efficient protein trans-splicing both in vitro and in vivo in DBA2/mdx mice of the midi-Dys 1 and 2 and not in midi-Dys 3 as expected from the structural prediction. Additionally, we demonstrated that midi-Dys 1 and 2 exhibit significant therapeutic efficacy in DBA2/mdx mice, highlighting their potential as therapeutic agents for DMD. Overall, these findings highlight the potential of deep learning-based structural modeling for the generation of intein-based dystrophin versions and pose the basis for further investigation of these new midi-dystrophins versions for clinical studies.
杜氏肌营养不良症（DMD）是一种儿科疾病，其特征为进行性肌肉退化和过早死亡，目前尚无治愈方法。目前最有前途的治疗途径是基于腺相关病毒（AAV）的基因替代，使用缩短但仍具有功能的肌营养不良蛋白的微版本（µDys），以适应 AAV 的容量。临床试验中观察到的有限改善表明，µDys 在人类环境中的性能不佳，这可能是由于该蛋白缺乏关键结构域。因此，表达更大的肌营养不良蛋白可能对于更完全地纠正疾病表型是必要的。在这项研究中，我们使用双 AAV 方法，利用基于分裂内含肽的蛋白质转剪接，开发了三种新型的 midi-Dys 构建体。与 µDys 相比，midi-Dys 包含更多的结构域，如中央细胞骨架结合结构域、nNOS 和 Par1b 相互作用结构域以及完整的 C 末端区域。鉴于每个 AAV 载体的容量有限，我们在确保蛋白质结构稳定性完整的情况下，战略性地部分减少了铰链区域。我们借助深度学习算法 AlphaFold3 预测了分裂 midi-Dys 蛋白的两半之间的相互作用。我们观察到 midi-Dys 1 和 2 的 N 端和 C 端之间存在强烈的关联，而 midi-Dys 3 则显示出较弱的相互作用。我们随后的实验证实了 midi-Dys 1 和 2 在 DBA2/mdx 小鼠中的体外和体内高效的蛋白质转剪接，但 midi-Dys 3 并不如此，这与结构预测一致。此外，我们证明 midi-Dys 1 和 2 在 DBA2/mdx 小鼠中具有显著的治疗效果，突出了它们作为 DMD 治疗剂的潜力。总的来说，这些发现强调了基于深度学习的结构建模在产生内含肽肌营养不良蛋白版本方面的潜力，并为进一步研究这些新型 midi-Dys 版本用于临床研究奠定了基础。
