Research Group on Neuromuscular and Mitochondrial Diseases, Vall d'Hebron Research Institute, Universitat Autònoma de Barcelona, and Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Barcelona, Catalonia, Spain.
Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Catalonia, Spain; Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Cerdanyola del Vallès, Catalonia, Spain.
EBioMedicine. 2020 Dec;62:103133. doi: 10.1016/j.ebiom.2020.103133. Epub 2020 Nov 21.
Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches.
dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals.
Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy.
Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype.
This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
临床前研究表明，基因治疗是治疗线粒体神经胃肠脑肌病（MNGIE）的一种可行方法。然而，该疾病的遗传小鼠模型（Tymp/Upp1 双敲除，dKO）除了代谢失衡之外，其功能表型有限，因此证明基因治疗有效性的研究几乎完全依赖于证明生化表型的纠正。慢性口服胸苷（dThd）和脱氧尿苷（dUrd）可使 dKO 小鼠的表型恶化，为测试治疗方法提供了更好的模型。
从断奶到研究结束，dKO 小鼠通过饮用水摄入 dThd 和 dUrd。在 8-11 周龄时，用携带人类 TYMP 编码序列的几种腺相关病毒（AAV）血清型 8 载体（受不同肝特异性启动子（TBG、AAT 或 HLP）控制）治疗小鼠。研究了动物的整个生命周期中的生化特征和功能表型。
核苷暴露导致 dKO 小鼠的血浆核苷水平比未暴露的野生型小鼠高 30 倍。AAV 治疗可提高暴露的 dKO 小鼠肝脏中的 TP 活性并降低全身核苷水平。暴露的 dKO 小鼠的脑室扩大（通过磁共振成像评估）和运动障碍（旋转棒测试）；AAV 治疗均可预防这两种情况。在所测试的所有启动子中，AAT 显示出最佳疗效。
我们的研究结果表明，AAV 介导的基因治疗可恢复 MNGIE 小鼠模型中的生化平衡，并且首次证明该治疗可改善功能表型。
这项工作部分由西班牙卡洛斯三世健康研究所和加泰罗尼亚政府资助。披露的资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。
