2015-03-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658494摘要：一般的概念認為血友病的臨床治療已是相當成功，病人多能擁有正常餘命，然而約有 20-30%的治療後血友病人會因產生抗血漿製劑如第八因子(factor VIII, FVIII)的抗體而成 為所謂的難治型病人。臨床上這類病人無法再輸注FVIII而需使用其他”bypass”觀念的血 漿製劑如活化第七因子(Factor VIIa, FVIIa)等作症狀治療(on-demand)才能緩解病灶。但 bypass治療成本高，每人每年約花費健保兩仟八佰萬元(台幣），約為輸注FVIII製劑的7 倍，且止血效果不佳，嚴重影響患者生活品質，因此抗體陽性的難治型血友病可說 是”un-met medical need”。以血友病發生率約1萬分之1估算，對國家財政是一大支出， 尤其與癌症病人不同的是血友病人使用終生的醫療資源，因此難治型病人的醫療策略具開 發的急迫性。治療抗體陽性的血友病人依成人與兒童雖有不同的考量，但輸注血漿製劑以誘導免疫 而于受(immune tolerance induction, ITI)的療法，對病人的身心(如關節磨損導致行動困難或無 法正常生活)及疾病後續照護較有利。在醫療支出層面的統計顯示，ITI成功的患者終生花 費較bypass治療節省五千萬元(台幣）。然而，ITI療效深受個體免疫系統及抗體調控機轉 影響，其中牽涉多種細胞且病患個體異質性大，至今仍無體外診斷試劑(IVD)或體外診斷 方法可確知哪些病人可成功誘導ITI，導致目前臨床醫生實行ITI成功率約只有40-60%。 特別是ITI療程費用平均每人約千萬元(台幣），且療程耗時，若能發展一套診斷試劑或方 法先確認病患是否適用ITI、再予以治療，將能有效提升臨床處置的正確性及減少醫療支 出。本計晝提出開發個人化的體外診斷方法以鑑定出適用ITI治療的難治型血友病人。本 計畫得以執行的因素在於結合兩項新創的尖端科技，包括基因剔除技術的”CRISPR/Cas9” 及免疫缺陷NSG (Nod/Scid-Il2y-/-)小鼠。前者可在任何物種完成基因改造，後者比 NOD/SCID更易接受並擴增自病人移植入的血球細胞。本團隊已利用CRISPR/Cas9技術 開發出全球首例的血友病NSG小鼠系統，目前已著手申請專利。利用此小鼠移植入病人 周邊血球細胞後，可作為一體外診斷平台，診斷病人個體是否為ITI可誘導性(ITI successful, 可誘導成功)或替患者篩選最適合的治療製劑及療程。本計晝第一年將測試診斷10例病人 檢體，預計兩年内完成35例，同時本計晝將應用此體外診斷方法所得結果與病人治療成 效進行臨床驗證，以評估此體外診斷方法的準確性。個人化醫療已成為現今全球醫衛領域 的熱門趨勢，且血友病患為健保平均每人醫療支出點數的第一名，遠高於第二名腎衰竭病 患6倍。本計晝所產製的個人化體外診斷方法及後續製程將為全球第一個運用於難治型血 友病的相關產品(ITI診斷平台及專利），不僅能有效提升臨床ITI治療成功率且具體節省醫 療資源及提升病患生活品質，極具商品化潛能及市場競爭力。<br> Abstract: Current clinical treatment of hemophilia can be considered to be quite successful, with most hemophilia patients achieving a normal average life span. However, in 20-30% patients anti-factor VIII IgG (a-FVIII) is induced after receiving therapeutic FVIII preparation and patients are diagnosed as “intractable hemophiliacs”. Clinical therapy for these patients may use bypass agents, such as factor VIIa as on-demand therapy instead of FVIII replacement. However, the medical expense covered by National Health Insurance averages NT$28 million per year for each patient receiving bypass-on-demand therapy, which is more than 7 times greater than patients who receive FVIII replacement. Furthermore, bypass-on-demand therapy results in less hemostasis and a poorer quality of life for patients. Since the incidence of hemophilia is 1/10,000 in males and patients all need the follow-up health care for their entire lifespan, the national economic burden is large.An alternative strategy for intractable hemophilia is immune tolerance induction (ITI), which typically involves the daily infusion of a large dose of FVIII over many months or 1~3 years to decrease the titer of a-FVIII. ITI can improve the clinical outcome and decrease the complications associated with intractable hemophilia. The total estimated lifetime cost for the ITI strategy is at least NT$50 million less per patient than for non-ITI patients. Nevertheless, the ITI successful rate depends on many complicated characteristics of the patients undergoing treatment, and so far no vitro diagnostic reagent or method has been developed to predict the efficiency rate of ITI resulting in a success rate for the therapy of only 40-60%. Additionally, ITI is usually costly (approaching NT$10 million on average) and it is time consuming. If clinical MDs are able to use a personalized in vitro diagnostic platform to confirm whether patients are suitable for ITI therapy before therapy, it would improve the accuracy of clinical management and decrease medical expenses.Here, we propose the development of an in vitro diagnostic method to identify intractable hemophilia suitable for ITI. We propose the use of two new developed and advanced techniques, the CRISPR/Cas9 system and immunodeficient NSG mice, (Nod/Scid-Il2y_/_). The CRISPR/Cas9 system can be used for genetic modification in any living species. The NSG mice are more acceptable for than NOD/SCID mice for transplanting with human peripheral blood mononucleated cell (PBMC). We have used the CRISPR/Cas9 system to establish hemophilia mice in the NSG background (NSG-Hemo A mice) and we plan to establish a humanized immune system in NSG-Hemo A mice through transplanting the patient’s PBMCs into these mice to establish an in vitro diagnostic platform for identifying intractable hemophilics who are suitable to receive ITI. We will test 10 patients using our in vitro diagnosis platform during the first year of our project and finish diagnosis of 35 cases. After comparing the diagnostic results with the clinical ITI efficiency of these patients, we will evaluate the accuracy of this in vitro diagnostic platform. Personalized medicine is a popular trend in medical fields globally, and hemophilia patients represent the greatest health insurance spending per person on average, much higher than the second highest cost, patients with renal failure. This diagnostic method will not only increase the efficiency of ITI in intractable hemophilia but also save healthcare resources; it has commercialization potential and market competitiveness.