通过祖先序列重建识别的增强凝血因子IX变体的人性化和功能性特征
Christopher W Coyle1, Kristopher A Knight1, Harrison C Brown2
1Molecular and Systems Pharmacology Graduate Program, Graduate Division of Biological and Biomedical Sciences, Laney Graduate School, Emory University School of Medicine, Atlanta, Georgia, USA.
Journal of thrombosis and haemostasis : JTH
|November 28, 2023
概括
研究人员复活了古老的凝血因子IX (FIX) 变体,发现了具有增强活性的An96. 他们确定了一个强大的FIX基因治疗候选人的关键替代品 (ET9) 血友病B.
科学领域:
- 生物化学和分子生物学
- 基因治疗 基因治疗
- 蛋白质工程是指蛋白质工程.
背景情况:
- 实验室对古老的凝血因子 (F) IX变体的复苏确定了An96,一种具有增强特异性活性的变体.
- 这种增强的活性独立于FIX-Padua变体 (p.Arg384Lys) 并附加到它.
研究的目的:
- 确定An96中负责其增强活性的特定氨基酸替代物.
- 开发一个人性化的An96 FIX转基因,用于血友病B的潜在基因治疗应用.
主要方法:
- 采用了减少主义查,包括域互换和扫描残留物替代,以一阶段的FIX活动测定为指导.
- 进行了顶级候选物的体外表征,包括重组蛋白制剂,特异性活性和酶动态分析.
- 包装的候选者变成腺相关病毒 (AAV) 载体,用于输送给血友病B小鼠模型.
主要成果:
- 发现An96中的五种氨基酸替代足以在人类FIX背景下保持增强的活性.
- 额外的替代 (ET9) 与帕多瓦变体相结合,使特定活性增加了5倍,比野生类型的人类FIX (hFIX) 增加了51倍.
- 接受AAV2/8-ET9治疗的B型血友病小鼠的血FIX活性比接受AAV2/8-hFIX-Padua治疗的小鼠高10倍.
结论:
- 通过祖先序列重建确定了赋予增强FIX活性的新型氨基酸替代物,并在AAV-FIX基因疗法磁带中验证.
- 开发的基因疗法候选者 (ET9) 在临床前模型中显示出高强度.
- 这种祖先序列重建和改进的方法是优化蛋白质药物和基因治疗候选人的有希望的平台.
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