Divsion of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA.
Deparment of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.
Blood Adv. 2021 Mar 9;5(5):1324-1332. doi: 10.1182/bloodadvances.2019000405.
The high-specific-activity factor IX (FIX) variant Padua (R338L) is the most promising transgene for hemophilia B (HB) gene therapy. Although R338 is strongly conserved in mammalian evolution, amino acid substitutions at this position are underrepresented in HB databases. We therefore undertook a complete 20 amino acid scan and determined the specific activity of human (h) and canine (c) FIX variants with every amino acid substituted at position 338. Notably, we observe that hFIX-R338L is the most active variant and cFIX-R338L is sevenfold higher than wild-type (WT) cFIX. This is consistent with the previous identification of hFIX-R338L as a cause of a rare X-linked thrombophilia risk factor. Moreover, WT hFIX and cFIX are some of the least active variants. We confirmed the increased specific activity relative to FIX-WT in vivo of a new variant, cFIX-R338I, after gene therapy in an HB dog. Last, we screened 232 pediatric subjects with thromboembolic disease without identifying F9 R338 variants. Together these observations suggest a surprising evolutionary pressure to limit FIX activity with WT FIX rather than maximize FIX activity.
高比活性因子 IX（FIX）变体帕多瓦（R338L）是血友病 B（HB）基因治疗最有前途的转基因。尽管 R338 在哺乳动物进化中高度保守，但该位置的氨基酸取代在 HB 数据库中代表性不足。因此，我们进行了完整的 20 个氨基酸扫描，并确定了在位置 338 取代每个氨基酸的人（h）和犬（c）FIX 变体的比活性。值得注意的是，我们观察到 hFIX-R338L 是最活跃的变体，而 cFIX-R338L 比野生型（WT）cFIX 高七倍。这与之前确定 hFIX-R338L 是一种罕见的 X 连锁血栓形成倾向风险因子的发现一致。此外，WT hFIX 和 cFIX 是一些活性最低的变体。我们在 HB 犬的基因治疗后，确认了新变体 cFIX-R338I 的比活性相对于 FIX-WT 的增加，在体内。最后，我们筛查了 232 名患有血栓栓塞性疾病的儿科患者，未发现 F9 R338 变体。综上所述，这些观察结果表明，存在一种令人惊讶的进化压力，即用 WT FIX 而不是最大限度地提高 FIX 活性来限制 FIX 活性。
