Molecular and Systems Pharmacology Graduate Program, Graduate Division of Biological and Biomedical Sciences, Laney Graduate School, Emory University School of Medicine, Atlanta, Georgia, USA.
Expression Therapeutics, Inc, Atlanta, Georgia, USA.
J Thromb Haemost. 2024 Mar;22(3):633-644. doi: 10.1016/j.jtha.2023.11.010. Epub 2023 Nov 26.
Laboratory resurrection of ancient coagulation factor (F) IX variants generated through ancestral sequence reconstruction led to the discovery of a FIX variant, designated An96, which possesses enhanced specific activity independent of and additive to that provided by human p.Arg384Lys, referred to as FIX-Padua.
The goal of the current study was to identify the amino acid substitution(s) responsible for the enhanced activity of An96 and create a humanized An96 FIX transgene for gene therapy application.
Reductionist screening approaches, including domain swapping and scanning residue substitution, were used and guided by one-stage FIX activity assays. In vitro characterization of top candidates included recombinant high-purity preparation, specific activity determination, and enzyme kinetic analysis. Final candidates were packaged into adeno-associated viral (AAV) vectors and delivered to hemophilia B mice.
Five of 42 total amino acid substitutions in An96 appear sufficient to retain the enhanced activity of An96 in an otherwise human FIX variant. Additional substitution of the Padua variant further increased the specific activity 5-fold. This candidate, designated ET9, demonstrated 51-fold greater specific activity than hFIX. AAV2/8-ET9 treated hemophilia B mice produced plasma FIX activities equivalent to those observed previously for AAV2/8-An96-Padua, which were 10-fold higher than AAV2/8-hFIX-Padua.
Starting from computationally inferred ancient FIX sequences, novel amino acid substitutions conferring activity enhancement were identified and translated into an AAV-FIX gene therapy cassette demonstrating high potency. This ancestral sequence reconstruction discovery and sequence mapping refinement approach represents a promising platform for broader protein drug and gene therapy candidate optimization.
通过祖先序列重建生成的古代凝血因子 (F) IX 变体的实验室复活导致发现了一种 FIX 变体，称为 An96，它具有增强的特异性活性，独立于并可累加于人 p.Arg384Lys，称为 FIX-Padua。
本研究的目的是确定导致 An96 活性增强的氨基酸取代，并创建用于基因治疗应用的人源化 An96 FIX 转基因。
采用简化筛选方法，包括结构域交换和扫描残基取代，并通过一步法 FIX 活性测定进行指导。对顶级候选物进行体外表征，包括重组高纯度制备、比活性测定和酶动力学分析。最终候选物被包装到腺相关病毒 (AAV) 载体中，并递送到血友病 B 小鼠中。
在 An96 的 42 个总氨基酸取代中，有 5 个足以保留在其他人类 FIX 变体中保留 An96 的增强活性。进一步取代 Padua 变体可使比活性进一步增加 5 倍。该候选物命名为 ET9，其比活性比 hFIX 高 51 倍。用 AAV2/8-ET9 治疗的血友病 B 小鼠产生的血浆 FIX 活性与之前用 AAV2/8-An96-Padua 观察到的活性相当，比用 AAV2/8-hFIX-Padua 高 10 倍。
从计算推断的古代 FIX 序列开始，确定了赋予活性增强的新氨基酸取代，并将其转化为具有高效力的 AAV-FIX 基因治疗盒。这种祖先序列重建发现和序列映射细化方法代表了更广泛的蛋白质药物和基因治疗候选物优化的有前途的平台。
