Dual AAV gene therapy in combination with rapamycin rescues muscle and liver impairment in a mouse model of GSDIII.

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation in liver, and in cardiac and skeletal muscles, responsible for impaired hepatic metabolism, heart function impairment, and muscle weakness. To date, there is no curative treatment for GSDIII. The large 4.6 kb coding sequence of GDE represents a major limitation toward the development of clinically relevant AAV gene transfer strategy for GSDIII. We previously reported that liver and heart/muscle correction of the GSDIII phenotype can be achieved in a mouse model of GSDIII by using two distinct overlapping AAV vectors encoding GDE. Here, results of a long-term stability of an approach based on a dual AAV vector encoding for GDE under the control of a recently developed tandem liver-muscle promoter suggests that stable but partial correction of the muscle phenotype can be achieved at very long-term (12 months). In liver, the dual AAV had an only transient efficacy supporting the need for an optimization of the approach. We then assessed the efficacy of rapamycin, an autophagy inducer used in the clinic as an immunosuppressive drug that has shown efficacy in GSDIII. Combination of rapamycin with the dual vector expressing GDE resulted in better correction of glycogen accumulation and muscle strength impairment than AAV vector alone thus supporting a synergic effect of the two treatments. The combined treatment synergic effect was also demonstrated at the molecular level by transcriptomic analysis that indicated a better rescue of the lysosomal pathway in muscle, possibly due to the induction of autophagy by rapamycin and the clearance of glycogen achieved with the combination therapy. In GSDIII mice liver, rapamycin was also able to counteract an unexpected immune reaction to the AAV vector that seems specific of this model. In conclusion, these results indicate that correction of both liver and muscle can be achieved in symptomatic GSDIII mice by an overlapping vector expressing GDE with a tandem promoter when combined with rapamycin treatment. These data also indicate that the effect of rapamycin on AAV gene transfer although disease- and tissue-specific has a net positive impact on dual AAV gene therapy for GSDIII thus opening the way to the clinical translation of this combination approach. Overall design: Comparative gene expression profiling analysis of RNA-seq data of triceps or liver biopsies from Agl+/+ and Agl-/- mice, treated with the AAV9-LiMP-GDEov in combination or not with rapamycin.

糖原贮积症III型（Glycogen storage disease type III, GSDIII）是一类因糖原脱支酶（glycogen debranching enzyme, GDE）缺陷引发的罕见代谢紊乱。GDE活性降低会导致病理性糖原在肝脏、心肌及骨骼肌中蓄积，进而引发肝脏代谢异常、心功能受损与肌肉无力。迄今，GSDIII尚无根治性治疗手段。GDE长达4.6 kb的编码序列，是开发临床适用的腺相关病毒（adeno-associated virus, AAV）基因转移策略治疗GSDIII的主要障碍。

本课题组此前已报道，通过两种携带GDE编码序列的重叠AAV载体，可在GSDIII小鼠模型中实现肝脏与心肌/肌肉表型的矫正。本研究针对基于双AAV载体的治疗方案开展长期稳定性评估，该载体搭载新近开发的串联肝-肌肉启动子以调控GDE表达，结果显示，在极长期（12个月）观测中，该方案可实现肌肉表型的稳定但部分矫正。在肝脏中，双AAV载体仅表现出短暂疗效，提示需对该方案进行优化。

随后，本课题组评估了雷帕霉素（rapamycin）的治疗效果：该药作为临床常用的免疫抑制药物，同时也是一种自噬（autophagy）诱导剂，既往研究已证实其对GSDIII具有治疗潜力。将雷帕霉素与表达GDE的双AAV载体联合使用，相较于单独使用AAV载体，可更有效地改善糖原蓄积与肌肉力量受损状况，证实两种治疗方式存在协同效应。该协同效应还在分子层面通过转录组分析（transcriptomic analysis）得到验证：分析结果显示，肌肉组织中的溶酶体通路（lysosomal pathway）得到了更充分的挽救，这可能源于雷帕霉素诱导的自噬作用与联合疗法实现的糖原清除。在GSDIII小鼠的肝脏中，雷帕霉素还能够抵消该小鼠模型特有的、针对AAV载体的意外免疫反应。

综上，本研究结果表明，搭载串联启动子的重叠GDE编码载体联合雷帕霉素治疗，可在有症状的GSDIII小鼠中同时实现肝脏与肌肉表型的矫正。本研究数据同时证实，雷帕霉素对AAV基因转移的影响虽存在疾病与组织特异性，但总体上对GSDIII的双AAV基因治疗具有净正向作用，为该联合方案的临床转化开辟了道路。

整体实验设计：对经AAV9-LiMP-GDEov单独处理或联合雷帕霉素处理的Agl野生型（Agl+/+）与Agl敲除型（Agl-/-）小鼠的肱三头肌或肝脏活检组织的RNA测序（RNA-seq）数据开展比较基因表达谱分析。