新型生物纳米结构在杜兴肌肉萎缩症靶向治疗中的基础研究

Duchenne muscular dystrophy (DMD) is a lethal muscle degenerative disorder,there is no treatment available in clinic currently. Antisense oligonucleotide (AO) - mediated exon skipping therapy shows huge potential for DMD as demonstrated by recent clinical trials. However, there remain limitations to current AOs applied in clinical trials e.g. low systemic delivery efficiency, particularly to heart as anticipated from pre-clinical studies. Thus, it becomes imperative to explore new,effective targeted delivery vehicles. Exosomes are small (40-100 nm) membrane vesicles of endocytic origin that are released into the extracellular environment on fusion of multivesicular bodies (MVB) with the plasma membrane. In our previous studies, we demonstrated that exosomes could be harnessed as a targeted delivery vehicle by modifying exosomes with a brain-targeting peptide. It was shown that loaded cargoes were sucessfully delivered cross the blood-brain barrier to targeted neurons. Our recent evidence indicates that exosomes derived from muscle cells bear muscle tissue tropism, suggesting that it can be utilized as a delivery vehicle for the treatment of muscle diseases e.g. DMD. Based on our previous data, in this proposal we plan to fully exploit the potential of muscle cell-derived exosomes as a new delivery vector for DMD. A series of studies will be undertaken including detailed characterization of muscle cell-derived exosomes, evaluation of its systemic delivery efficiency in DMD mouse models, optimization of loaded AOs and related mechanistic studies. We wish to provide a new targeted delivery vehicle and therapeutic method for the treatment of DMD.

杜兴肌肉萎缩症是一种致死性的神经肌肉退行性疾病，目前临床尚无有效的治疗方法。最近反义寡核苷酸介导的外显子跳读的临床试验结果显示了该方法的巨大应用前景，但临床上测试的反义寡核苷酸药物存在着系统运输效率低，特别是在心肌中运输效率有限等问题，因此亟需研发新型的高效的肌肉靶向运输载体。本课题组在前期工作中，利用靶向肽修饰exosome（一种由细胞分泌的纳米级生物囊泡结构），并将其作为外源药物的运输载体，成功地跨越血脑屏障，将药物靶向运输到脑神经，显示其作为运输载体的潜力。另外，在前期工作中，我们发现肌肉细胞来源的exosome具有肌肉组织靶向性，因此本课题拟测试exosome在杜兴肌肉萎缩症上靶向运输反义寡核苷酸药物的潜力。通过对肌肉细胞来源exosome的体内组织分布、运载药物和条件的优化及其系统运输效率的评估和其作用机理的阐释，以期为杜兴肌肉萎缩症的治疗提供一种新的靶向运输载体和治疗途径。

杜兴肌肉萎缩症是一种致死性的神经肌肉退行性疾病，目前临床尚无有效的治疗方法。反义寡核苷酸介导外显子跳读的临床试验结果显示了该方法的巨大应用前景，但临床上测试的反义寡核苷酸药物存在着系统运输效率低，特别是在心肌中运输效率有限等问题。利用exosome（一种由细胞分泌的纳米级生物囊泡结构）作为反义寡核苷酸类药物的运输载体，可有效提高药物系统运输效率。但exosome的药物装载效率极低是制约exosome作为生物纳米药物载体发展的主要因素。本项目系统优化了exosome装载药物条件，包括：电激转化法、Ca(PO4)2转化法、通透方法、超声法、冻干法、pH梯度法等，结果显示通过以上测试方法装载反义寡核苷酸类药物（PMO、PNA等）至exosome的效率较低，无法实现exosome介导的高效药物运输。为实现exosome的药物高效装载，本研究筛选到一种新型exosome锚定肽，通过该锚定肽可以高效负载反义寡核苷酸药物至exosome表面，在杜兴肌肉萎缩症动物模型-mdx小鼠上系统测试了exosome介导药物运输效率，结果显示：EXO-PMO给药组能够显著提高外显子跳读水平、显著提高dystrophin阳性肌纤维的数量，与单独注射等量PMO组相比，诱导dystrophin蛋白表达水平提高至20倍作右，显示出高效的系统运输效率。该新型药物装载方法为exosome介导的药物运输奠定了实验基础。为进一步阐释exosome介导药物系统运输的机理，本研究系统测试了肌肉细胞来源的exosome在小鼠体内的组织分布情况，结果显示C2C12来源的exosome具有一定的肌肉组织倾向性且具有较好的代谢稳定性。该研究为exosome介导的药物系统运输提供了一定的理论基础。综上，本研究针对exosome药物装载效率低的问题，优化并开发出一种新型药物负载方法，该方法能够高效负载药物至exosome表面，显著提高exosome介导的药物运输效率，为杜兴肌肉萎缩症的治疗提供一种新的靶向运输载体和治疗途径。

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