背景:贝克尔肌营养不良症(BMD)是一种遗传性神经肌肉疾病,由帧内引起,肌营养不良蛋白(DMD)基因的部分功能丧失突变。与Duchenne型肌营养不良(DMD)相比,BMD的严重程度降低,完全肌营养不良蛋白缺乏的等位基因疾病。在DMD方面取得了显著的治疗进展,包括四种FDA批准的药物。BMD,然而,研究不足,服务不足——没有药物,临床试验也很少。治疗努力中的不一致部分是由于缺乏BMD小鼠模型,该模型可以在首次人体试验之前更好地了解疾病和降低风险的潜在治疗方法。重要的是,BMD小鼠模型变得越来越重要,因为新兴的DMD肌营养不良蛋白恢复疗法旨在将DMD基因型转化为BMD表型。
方法:我们使用CRISPR/Cas9技术产生bmx(Becker肌营养不良,X连接的)小鼠,在鼠Dmd基因中表达外显子45-47的框内〜40000bp缺失,复制最常见的BMD患者突变。这里,我们使用分子和组织学技术表征肌肉发病机制,然后使用肌肉功能测定和超声心动图测试骨骼肌和心脏功能。
结果:总体而言,与野生型(WT)小鼠相比,bmx小鼠表现出明显的肌肉无力和心脏功能障碍,尽管在病理学上显著改善了肌营养不良蛋白缺失的mdx52小鼠。bmx小鼠在握力方面表现出运动功能受损(-39%,P<0.0001),钢丝吊挂(P=0.0025),和体内以及离体力测定。在老化的BMX中,超声心动图显示心脏功能通过缩短分数降低(-25%,P=0.0036)。此外,肌肉特异性血清CK增加>60倍(P<0.0001),表明肌肉损伤增加。组织学上,bmx肌肉显示增加的肌纤维大小变异性(最小Feret直径:P=0.0017)和位于中央的核表明变性/再生(P<0.0001)。bmx肌肉也表现出营养不良的病理;然而,与mdx52相比,以下参数的水平适中:炎症/坏死灶(P<0.0001),胶原蛋白沉积(+1.4倍,P=0.0217),和细胞内IgM测量的肌膜损伤(P=0.0878)。像BMD患者一样,bmx肌肉显示降低的肌营养不良蛋白水平(约20-50%的WT),而Dmd转录水平不变。在分子水平上,bmx肌肉表达炎症基因水平升高,炎性miRNA和纤维化基因。
结论:bmx小鼠用组织学,分子和功能缺陷。重要的是,它可以告知BMD病理学和DMD肌营养不良蛋白恢复治疗。这种新的模型将能够进一步表征BMD疾病的进展,识别生物标志物,确定治疗靶点和新的临床前药物研究,旨在开发BMD患者的治疗方法。
Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype.
We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography.
Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret\'s diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes.
The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.