Section on Cellular Differentiation, Division of Translational Medicine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.
Foundation Fighting Blindness, Columbia, Maryland.
J Inherit Metab Dis. 2019 May;42(3):470-479. doi: 10.1002/jimd.12069. Epub 2019 Feb 22.
Glycogen storage disease type-Ia (GSD-Ia), caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), is characterized by impaired glucose homeostasis with a hallmark hypoglycemia, following a short fast. We have shown that G6pc-deficient (G6pc-/-) mice treated with recombinant adeno-associated virus (rAAV) vectors expressing either wild-type (WT) (rAAV-hG6PC-WT) or codon-optimized (co) (rAAV-co-hG6PC) human (h) G6Pase-α maintain glucose homeostasis if they restore ≥3% of normal hepatic G6Pase-α activity. The co vector, which has a higher potency, is currently being used in a phase I/II clinical trial for human GSD-Ia (NCT03517085). While routinely used in clinical therapies, co vectors may not always be optimal. Codon-optimization can impact RNA secondary structure, change RNA/DNA protein-binding sites, affect protein conformation and function, and alter posttranscriptional modifications that may reduce potency or efficacy. We therefore sought to develop alternative approaches to increase the potency of the G6PC gene transfer vectors. Using an evolutionary sequence analysis, we identified a Ser-298 to Cys-298 substitution naturally found in canine, mouse, rat, and several primate G6Pase-α isozymes, that when incorporated into the WT hG6Pase-α sequence, markedly enhanced enzymatic activity. Using G6pc-/- mice, we show that the efficacy of the rAAV-hG6PC-S298C vector was 3-fold higher than that of the rAAV-hG6PC-WT vector. The rAAV-hG6PC-S298C vector with increased efficacy, that minimizes the potential problems associated with codon-optimization, offers a valuable vector for clinical translation in human GSD-Ia.
I 型糖原贮积病（GSD-Ia）是由于葡萄糖-6-磷酸酶-α（G6Pase-α 或 G6PC）缺乏引起的，其特征是葡萄糖稳态受损，禁食后会出现低血糖，这是其标志性特征。我们已经表明，用表达野生型（WT）（rAAV-hG6PC-WT）或密码子优化（co）（rAAV-co-hG6PC）人（h）G6Pase-α的重组腺相关病毒（rAAV）载体治疗的 G6pc 缺陷（G6pc-/-）小鼠，如果恢复≥3%的正常肝 G6Pase-α活性，则可维持葡萄糖稳态。该 co 载体的效力更高，目前正在用于人类 GSD-Ia 的 I/II 期临床试验（NCT03517085）。虽然该 co 载体在临床治疗中经常使用，但它可能并不总是最佳选择。密码子优化可能会影响 RNA 二级结构、改变 RNA/DNA 蛋白结合位点、影响蛋白质构象和功能，并改变可能降低效力或功效的转录后修饰。因此，我们试图开发提高 G6PC 基因转移载体效力的替代方法。通过进化序列分析，我们鉴定出一种丝氨酸 298 到半胱氨酸 298 的取代，这种取代天然存在于犬、鼠、大鼠和几种灵长类 G6Pase-α同工酶中，当它被整合到 WT hG6Pase-α序列中时，会显著增强酶活性。使用 G6pc-/-小鼠，我们表明 rAAV-hG6PC-S298C 载体的疗效比 rAAV-hG6PC-WT 载体高 3 倍。与密码子优化相关的潜在问题最小化的 rAAV-hG6PC-S298C 载体具有更高的疗效，为人类 GSD-Ia 的临床转化提供了有价值的载体。
