Gene Therapy and Regulation of Gene Expression Program, CIMA, FIMA, University of Navarra, Navarra Institute for Health Research (IdisNA), Pamplona, Spain.
Department of Chemistry and Soil Sciences, University of Navarra, IdisNA, Pamplona, Spain.
Hepatology. 2019 Jul;70(1):108-126. doi: 10.1002/hep.30535. Epub 2019 Mar 20.
Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8-ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal-binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80-miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long-term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.
腺相关病毒（AAV）血清型 8 载体介导的人 ATP 酶铜转运β多肽（ATP7B） cDNA（AAV8-ATP7B）基因治疗能够纠正 6 周龄雄性威尔逊病（WD）小鼠的长期铜代谢异常。然而，该基因的大小（5.2 千碱基对[kb]）超过了载体的最佳包装容量，导致产量较低；此外，在 WD 雌性小鼠和疾病进展更为严重的动物中进一步分析表明，与年轻雄性相比，治疗效果降低。为了提高治疗效果，我们生成了一种优化的短 AAV 载体，其中从 ATP7B 编码序列中删除了六个金属结合域（MBDs）中的四个，产生了 miniATP7B 蛋白（Δ57-486-ATP7B）。与 AAV8-ATP7B 不同，AAV8-miniATP7B 可以高滴度生产，能够恢复 6 周龄和 12 周龄雄性和雌性 WD 小鼠的铜稳态。此外，最近开发的携带 miniATP7B 基因的合成 AAV 载体 AAVAnc80 在预防肝损伤、恢复铜稳态和提高治疗 1 年后的生存率方面同样有效。大约 20%的肝细胞转导足以使铜稳态正常化，表明被纠正的肝细胞充当汇流来消除多余的铜。重要的是，AAVAnc80-miniATP7B 在健康小鼠中给药是安全的，不会导致铜缺乏。结论：总之，使用不同 AAV 系统的优化治疗盒进行基因治疗能够为临床相关 WD 小鼠模型提供长期的铜代谢纠正，无论性别或疾病阶段如何。这些结果为 WD 患者实施基因治疗铺平了道路。
