新型运输辅助剂在杜兴肌肉萎缩症治疗中的基础研究

Duchenne muscular dystrophy (DMD) is a severe muscle degenerative disorder caused by mutations in the DMD gene, which disrupt the synthesis of functional dystrophin protein. Antisense oligonucleotides (AOs) - mediated exon skipping can restore the expression of particially functional dystrophin protein by manipulating the pre-mRNA splicing process. This therapeutic approach is in phase IIb DMD clinical trials. However, there remain limitations to current AOs applied in clinical trials including low systemic exon skipping efficiency and high dosage requried in pre-clinical studies. In order to address these issues, we have recently identified a series of new formulations with high safety profiles in clinic, which can enhance the exon skipping efficiency of various AOs in muscle. Therefore in this proposal, we plan to fully explore the potential of the best candidate formulation in DMD animal models. Different dosing regimens and long-term effect and associated potential toxicity will be investigated in details. To understand the biological mechanism underpinning this novel formulation, detailed in vitro and in vivo studies will be undertaken.Moreover, other nucleic acid drugs will be tested with this novel formulation to see whether it can faciliate the delivery of other nucleic acid drugs. The success of this project will provide a new delivery platform for DMD and other muscle-related diseases.

杜兴肌肉萎缩症（Duchenne Muscular Dystrophy-DMD）是最为严重的一种神经肌肉失调症，由编码dystrophin蛋白的DMD基因突变导致功能性dystrophin蛋白缺失所致。研究表明，利用反义寡核苷酸（AOs）可调控DMD基因前体mRNA的拼接过程，介导功能dystrophin蛋白产生。但目前临床测试的AO药物存在系统运输效率低、用药量大等问题，我们在前期工作中发现一系列临床安全性高的新型运输辅助剂，可有效提高AO在肌肉中的系统运输效率。本项目拟对筛选到的最佳运输辅助剂的使用剂量和周期及长期效果等展开全面、系统的研究，并对其作用机理进行阐释。同时研究这一新型运输辅助剂对其他核酸类药物系统运输的提高作用，为DMD及其他肌肉相关疾病提供一种新的药物运输平台。

杜兴肌肉萎缩症（Duchenne Muscular Dystrophy-DMD）是一种最为严重的神经肌肉失调症，由编码dystrophin蛋白的DMD基因突变导致功能性dystrophin蛋白缺失所致。利用反义寡核苷酸（AOs）可调控Dmd 基因前体mRNA的拼接 过程，介导功能dystrophin蛋白产生，目前临床测试的AO药物存在系统运输效率低、用药量大的问题。在最初的工作发现和PMOs（磷酰二胺吗啉代寡聚物）这种AOs与葡萄糖一起使用的时候可以在杜兴肌肉萎缩症的mdx小鼠模型中，调高外显子跳读的效率。基于这个初级的结果我们从临床上广泛应用的输液注射液作为筛选库，开展最佳的AOs运输辅助剂筛选工作，我们确定了一种葡萄糖 - 果糖（GF）配方，可以增强PMO活性，完全纠正异常的DMD转录物，恢复骨骼肌中的dystrophin蛋白表达水平，实现功能性拯救，无可检测的毒性。而该活性提高归因于肌肉中GF介导的PMO摄取的增强。我们证明PMO被细胞摄取是能量依赖性的，而来自GF代谢的ATP有助于增强肌肉中PMO的细胞摄取。总的来说，结果表明GF通过在mdx小鼠的能量缺乏条件下补充细胞能量储备来增强PMO活性。我们的研究结果为己糖介导的寡核苷酸递送提供了机制洞察，并对DMD外显子跳跃疗法的发展具有重要意义。