摘要:A 型血友病(Hemophilia A)是遺傳性出血疾病中發生率最高的(約一萬分之一),其治療主要是輸注第八因子製劑,臨床資料顯示在輸注治療後約有20-30%的病患會產生抗第八因子抗體,造成後續治療無法再使用第八因子製劑而必須改用單價更高的By-pass 療法產品。根據103 年度全民健保醫療給付統計,血友病在門診及住院皆為重大傷病類每人平均醫療支出的第一名,其中第八因子抗體陽性病患治療成本更高,估計每人每年需花費高達兩仟八佰萬元(台幣),為其他血友病患醫療支出的7 倍,故如何預先得知哪個病患易對哪種第八因子製劑產生抗體,並輸注適當的第八因子製劑是當前最具挑戰性的醫療難題且臨床上至今仍無任何篩檢方法。針對病患產生第八因子抗體的議題目前只能以回溯性研究推估出風險因子,包括第八因子基因突變(基因倒轉及終止密碼突變)、人類白血球抗原及免疫調節分子基因(IL-10)的多型性等。在2015 年的SIPPET 研究(19 國77 個機構)顯示不同的第八因子製劑也是風險因子,上述諸多風險因子只能作為概率的參考,無法作為醫師針對個別病患輸注哪種第八因子製劑後是否產生抗體的診斷依據。為解決此難題,本計畫率先提出創新的檢測平台,即建立血友病病人個人化的擬人化小鼠,以實測各病患接受第八因子製劑治療後是否產生抗體。本團隊最近利用CRISPR/Cas9 基因剔除技術在NSG(NOD/Scid-Il2γ-/-) 免疫缺陷小鼠建立了MHC class I 的β2-microglobulin 基因剔除小鼠,由於該小鼠不易產生GVHD (Graft-versus host disease),故適合移植人類周邊血液單核球,本團隊同時在此小鼠將第八因子基因剔除,作成血友病免疫缺陷鼠(命名為NSG/β2mnull HemoA,發表於2015 年國際血栓會議,且於2015 年9 月分讓至美國費城兒童醫院Dr. Poncz團隊進行國際合作),故可利用此小鼠將各個血友病人的周邊血液移植入後,作為預測血友病患對不同第八因子製劑是否產生抗體的檢測平台。本計畫的宗旨在於達到精準醫療(precision medicine),可行性在於團隊具備計畫所需各項新創的尖端技術、獨有的基因改造小鼠及移植的初步成果(見下述)。本計畫第一、二年目標為測試並證實建立的擬人化小鼠經未接觸過的抗原免疫刺激後確能生成抗體。本團隊先前利用NSG 小鼠的初步結果顯示,相較於靜脈或骨髓移植人類周邊血球,以脾臟注射方式移植只需三週即可在小鼠周邊血中測得15-58%的人類白血球,顯示本團隊已可成功製備人類免疫細胞的擬人化小鼠(見初步成果),本計畫第一年將繁殖足量的NSG/β2mnull HemoA 小鼠,並利用脾內移殖技術殖入人類周邊單核球後,接續測試其免疫活性,實驗策略為利用Pneumovax 23 肺炎鏈球菌疫苗測試是否能誘發擬人化小鼠生成新抗體。計畫第二、三年將使用建立的平台對血友病患進行實測。預計第二年及第三年各完成5 例,收案對象為未接受過輸注治療或第八因子抗體效價低於5 BU/mL的血友病患,各病患抽取20 ml 的周邊血即可建立多隻病患獨特的擬人化小鼠,接續再輸注不同的第八因子製劑,即可評估是否產生抗體及其效價,實驗預計以臨床預防性治療的輸注劑量及頻率施打小鼠,每週分析小鼠周邊血;若在實驗指定的30 個暴露天(exposure days,約10 週)後,小鼠確無第八因子抗體或第八因子抗體效價低於0.6 BU/mL,即判定為陰性。本計畫將追蹤及驗證包括抗體生成與否或病患原有低效價抗體是否如預期下降,以評估此平台的準確性。此外,本計畫的結果也將提供合作的醫師作為後續預防性治療(prophylaxis)或症狀治療(on-demand)的用藥參考。個人化醫療已成為現今全球醫衛領域的趨勢,本計畫所建立的個人化檢測平台,不僅能提供醫師在治療病患時選擇最適當的第八因子製劑,也可避免病患產生抗體造成後續治療困難,具體節省醫療資源。
Abstract: Hemophilia A (HemoA) is the most common (1/10,000 population) hereditary bleeding disorder. HemoA istreated with factor VIII (FVIII) concentrates and the treatment may have a 20-30% chance of inducing anti-FVIIIantibody (inhibitor). Inhibitor patients can no longer use FVIII concentrates and require more expensive by-passproducts. According to the National Health Insurance Administration in 2014, hemophilia ranked number one inhealthcare expenses in patients with catastrophic illnesses. Expense on inhibitor-positive HemoA amounted to NTD$28million per patient-year, 7 times the amount spent on other types of hemophilia. Identification of individual patientsprone to inhibitor generation, and selection for appropriate FVIII concentrates for each patient to avoidinhibitor incidence is thus important but challenging, and yet no strategy has been developed. Prediction ofinhibitor generation by risk factors assessment, including types of FVIII mutation (gene inversion, stop-codonmutations, etc.), HLA haplotypes, and SNPs in IL-10, has been studied. The SIPPET study (2015, 77 organizations, 19nations) showed that different FVIII concentrates vary in their risks of FVIII inhibitors. Risk factors are “probability”and not diagnostic, or not predictive for individual patients. To tackle this issue, we propose to use humanizedHemoA mice carrying the immune cells from individual patients as a strategy to directly test FVIII inhibitorgeneration in the mice by treating them with FVIII concentrates. Using CRISPR/Cas9, we have knocked outβ2-microglobulin (MHC class I) on immunodeficient NSG (NOD/Scid-Il2γ-/-) mice to avoid GVHD (Graft-versus-hostDisease). We have also mutated FVIII on these mice to generate immunodeficient HemoA mice (NSG/β2mnullHemoAmice, revealed on the 2015 ISTH meeting, and transferred in Sept. to Children’s Hospital in Philadelphia forcooperation with Dr. Poncz). The mice, after repopulated with peripheral blood mononuclear cells (PBMC), willprovide a platform for testing directly FVIII inhibitor generation to various FVIII concentrates infused to the mice. Theaim of this project is to develop precision medicine and therapies and its success relies on our pioneer KO micetechnology, long-term research interest in hemophilia, and promising results on transplantation (see below).For the first two years of this proposal, we will test the immune capacity of the NSG/β2mnull HemoA mice.We have succeeded in repopulating NSG mice with human PBMC to reach a ratio of 15-58% human leukocytes inmouse peripheral blood after 3 weeks of intrasplenic implantation, superior than intravenous or intrafemuralimplantations (see “Preliminary results”). With support of this project we will breed NSG/β2mnull HemoA mice andchallenge them with PNEUMOVAX® pneumococcal vaccine to demonstrate antibody formation. For the 2nd and 3rdyears, we will bring our platform to clinical trials. A total of 10 patients who have not received FVIII concentrates orwith low inhibitor titers (<5 BU/mL) will be enrolled. Twenty mL of blood will be collected for PBMC preparation andimplantation into multiple NSG/β2mnull HemoA mice, which will then be treated with different FVIII concentrates,with strict adherence to mimic clinical procedures. Mice that do not produce FVIII inhibitors or have a low titer (<0.6BU/mL) after 30 exposure days (~10 weeks) will be deemed as test negative. To prove the accuracy of our platform,continuous analysis will be implemented to track whether the antibody will ultimately be produced or if initial inhibitorwill decrease. Moreover, we hope our results provide guides to individual prophylaxis or on-demand drug selection.Minimization of hemophilia control failure not only alleviates bleeding disorder or improves quality of care but alsoreduces medical expenses burdens.