以B型血友病膝損傷小鼠模型探討人類凝血第九因子FIX-Triple的療效

Abstract

B型血友病是一種性聯遺傳疾病，病人主要由於缺乏凝血第九因子而出現關節反覆性出血，進而造成關節炎、軟硬骨破壞和關節腫脹變形，導致關節功能喪失，稱之為血友病性關節病，在病人中為最常見的併發症，嚴重影響病人的生活品質。針對B型血友病患者的治療，一直以來最常被使用的方式為注射第九因子蛋白濃縮液 (Factor IX concentration)。然而，儘管血漿濃縮與合成的第九因子凝血蛋白注射治療已實施多年，但關節部位出血產生的關節病變仍然嚴重影響病人的日常生活。這顯示目前的第九因子蛋白濃縮液 Factor IX (FIX)-WT 無法完全有效地避免血友病性關節病的發生，原因可能是FIX-WT在病人體內於最低濃度劑量時的凝血活性太低而缺乏保護療效所導致。本實驗室先前已研發出具有三個單點突變 (FIX-V86A/E277A/R338A) 的高專一活性第九因子 (FIX–Triple)，具有比 FIX-WT高13倍的專一活性，因此我想探討高專一活性的 FIX-Triple 是否能比 FIX-WT 更為有效的減低血友病性關節病發生的機會。本論文利用膝損傷血友病小鼠模型，將高劑量 (2×1010 vg/mouse）AAV載體注入至血友病小鼠體內以表達不同變異型第九因子，發現各組小鼠中均未發生膝關節病變，且AAV-Triple 治療之小鼠具有比 AAV-WT 高 7.2 倍的專一活性。確認此法在基因治療的可行性後，接著以中劑量病毒載體 (8×108 vg/mouse）進行治療。我發現小鼠血中蛋白凝血活性可達正常人5~10 % ，且以 AAV-Triple 治療的小鼠能以較少的蛋白量表現就達到和 AAV-WT具有相同的療效。為了模擬病人出血後的需求性治療，我先誘導血友病小鼠在關節產生出血性損傷，接著進行第九因子蛋白輸注治療 (5 micro;g/mouse)。結果顯示，分析以 FIX-Triple 治療之膝損傷血友病小鼠的關節病變程度，發現在膝關節外觀表現、膝關節出血評分和關節直徑長度變化皆比 FIX-WT及已在臨床試驗二期的 FIX-338L 具有更好的保護療效，綜合上述結果得知， FIX-Triple 是一個具有治療潛力的第九因子，可以取代現今所採用的 FIX-WT，若未來成功應用於臨床上，將更能幫助病友們避免關節出血產生之病變情況。

Hemophilia B is an X-linked bleeding disorder that results from a deficiency or dysfunction of plasma coagulation factor IX (FIX). Bleeding into joints that consequently develops into haemophilic arthropathy (HA) is the most common morbidity of haemophilia, and impacts negatively on quality of life. Patients are treated with administration of FIX protein concentration. Despite administration of high quality plasma-derived and recombinant FIX protein products readily available for several decades, many patients still suffer from HA. This suggests that the current FIX (FIX-WT) protein concentration products are not protective against HA. We suspect that this may be due to the low coagulation activity of the currently used FIX products on trough level in patients. Our laboratory previously demonstrated that the high specific activity of FIX-Triple (FIX-V86A/E277A/R338A) is superior to FIX-WT for 13 folds specific activity. Therefore, we aim to test whether FIX-Triple will be more effective than FIX-WT in the reduction of the occurrence of HA. To this purpose, I established a hemophilia B mouse model of synovitis which is raised from haemorrhage-induced joint injury. Gene therapy of hemophilia B mice was performed on adeno-associated virus (AAV) expressing human FIX (hFIX) at a dose of 2×1010 vector genome (vg)/mouse. All the Mice treated with AAV-hFIX were no longer observed development of HA and the mice treated with AAV showed 7.2-fold higher specific clotting activity than those treated with AAV-WT. Under the therapy at a medium dose of 8×108 vector genome (vg)/mouse, the coagulation activity of the mice showed 5~10 % level to normal people. It also indicated that the mice treated with AAV-Triple could exhibit the same protective effect as AAV-WT in the less circulating level of FIX Triple. To mimic the on-demand treatment of patients suffering from bleeding, the hemophilia B mice were treated with intravenous administration of FIX following induction of joint hemorrhage. The result suggested that the mice received intravenous FIX-Triple developed minimal histopathological findings of synovitis after blood-induced joint injury, when compared with the mice that received FIX-WT and FIX-338L, a gain-of-function FIX variant which is undergoing evaluation in phase 2 clinical trial for gene therapy of hemophilia B. These studies demonstrate that FIX-Triple is a potential therapeutic substitute for FIX-WT to improve the bleeding phenotype to prevent the development of HA.