TW202412821A - 用於抑制TMPRSS6基因表達之siRNA或其鹽、藥物及其應用 - Google Patents
用於抑制TMPRSS6基因表達之siRNA或其鹽、藥物及其應用 Download PDFInfo
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Abstract
本發明提供了用於抑制人體細胞中TMPRSS6表達之siRNA 或者藥學上可接受之鹽，以及對所述siRNA進行了合適之修飾，以提高靶標之沉默能力並降低非靶點活性。本發明還提供了含有上述siRNA之用於抑制TMPRSS6表達之生物製劑或者藥物製劑。本發明藉由一系列體外和體內試驗中，獲得了具有很好之抑制TMPRSS6表達生物活性之siRNA序列，且比臨床上進展最快之TMPRSS6-HCM-9活性更高，有望應用於臨床上進行與TMPRSS6表達異常相關之疾病或鐵超負荷或鐵超載相關聯疾病，例如紅細胞增多症、地中海貧血等之治療，具有很好之應用前景。
Description
本發明要求2022年9月29日提交之名稱為《用於抑制TMPRSS6基因表達之siRNA、藥物及其應用》之中國專利申請CN2022112033373之優先權，該發明公開之內容藉由援引合併於此；本發明還要求2023年01月19日提交之名稱為《用於抑制TMPRSS6基因表達之siRNA、藥物及其應用》之2023100881658之中國專利申請之優先權，該發明之部分內容藉由援引合併於此。
本發明屬於生物醫藥領域，具體系涉及用於抑制TMPRSS6基因表達之siRNA或其鹽、藥物及其應用。
RNA 干擾 (RNAi) 系信使 RNA (mRNA) 被小干擾RNA (siRNA) 切割之過程，廣泛用於研究各種細胞中基因表達之調節。在體外將siRNA遞送到細胞中可明顯抑制基因表達(Jackson et al. 2006, RNA 12:1179-1187.)。在過去之幾年裡，RNA 干擾(RNAi)已成為最廣泛使用之基因敲除技術(Hu et al. 2020, Signal Transduct Target Ther 5:101.)。小干擾RNA(siRNA)或短單鏈RNA如 microRNA (miRNA)與RNA誘導之沉默複合物(RISC)形成複合物，並進一步與互補之mRNA結合，mRNA在RNA酶之幫助下被切割以抑制特定基因之表達，RNAi之機制可以保護細胞免受病毒和轉座子之侵害(Weingartner et al. 2020, Mol Ther Nucleic Acids 21:242-250.)。
β-地中海貧血系最常見之遺傳性疾病之一，通常系由β珠蛋白基因或其啟動子之突變引起β-珠蛋白合成減少或缺失而導致(Cao and Kan 2013, Cold Spring Harb Perspect Med 3:a011775. Rivella 2015 , Haematologica 100:418-430.)。在β-地中海貧血患者中發現了180多種不同之β-珠蛋白基因突變，它們可能在轉錄、初級信使核糖核酸轉錄物之加工、翻譯或基因產物之翻譯後穩定性之間之任何水準上影響基因功能。β-地中海貧血通常表現為無效之紅細胞生成和隨之而來之髓外造血、脾腫大和系統性鐵負載。受影響之個體表現出極端之臨床異質性，從幾乎無症狀到表現出危及生命之嚴重貧血，β-地中海貧血可以分為重型地中海貧血，相對不太嚴重之貧血（非輸血依賴性地中海貧血或中間型地中海貧血）(Musallam et al. 2021, Am J Hematol 96:1518-1531.)。
在地中海貧血患者中，鐵穩態之主要調節劑鐵調素長期受到抑制。導致膳食鐵之吸收失控和網狀內皮系統之鐵保留不足，患者吸收異常高水準之鐵，加劇了無效之紅細胞生成和貧血，需要鐵螯合以防止與鐵超載相關之臨床後遺症(Richard et al. 2020, Am J Hematol 95:68-77)。需要調節患者體內之鐵穩態，以避免由於其過量而產生毒性(Nualkaew et al. 2021, Mol Ther 29:2841-2853.)。如果不加以治療，鐵超載會導致器官衰竭和死亡。由此，在β-地中海貧血和其他與鐵相關之疾病中，鐵超載之管理已成為主要焦點(Beliveau et al. 2019, Cell Chem Biol 26:1559-1572 e1559.)。鐵調素之產生主要受一種由 TMPRSS6基因編碼之跨膜絲氨酸蛋白酶調節。TMPRSS6（跨膜蛋白酶，絲氨酸6）也稱為蛋白裂解酶-2，這系一種II型絲氨酸蛋白酶。它主要在肝臟中表達，但在腎臟中也發現了高水準之TMPRSS6 mRNA，TMPRSS6藉由切割膜血紅素抑制鐵調素活化，從而在鐵穩態中起作用，抑制TMPRSS6之表達可以調節鐵穩態，降低鐵超載，從而緩解地中海貧血之病症 (Ginzburg and Rivella 2011, Blood 118:4321-4330.)。
TMPRSS6-HCM-9系一種靶向TMRPSS6之siRNA，正在開展用於治療地中海貧血之一期臨床試驗。之前開展之SLN124在健康志願者中之一期臨床試驗資料顯示SLN124在降低血漿鐵水平，安全有效之，並且作用持續時間長。SLN124序列公佈於專利US_11174483_B2中。目前市場上治療地中海貧血藥物少，siRNA療法有很好之市場前景。
紅細胞生成失敗和慢性貧血導致鐵代謝異常和全身鐵超載，進而導致心臟、肝臟和內分泌功能障礙。地中海貧血症之治療選擇有限，主要之治療手段系紅細胞輸血(Longo et al. 2021, Int J Mol Sci 22.)。當前急需新之用於有效治療與鐵負載相關聯之病症之方法。
紅細胞增多症分為繼發性紅細胞增多症和真性紅細胞增多症兩種情況。繼發性紅細胞增多症改善誘發原因即可得到緩解。真性紅細胞增多症(PV)系一種骨髓增生性疾病(MPDS)，常常導致異常高之紅細胞壓積(Hct)和血紅蛋白(Hb)濃度，進而導致血栓栓塞併發症。TMPRSS6基因之功能喪失導致鐵調素之過度產生，進而改善紅細胞生成異常，防止或限制鐵超載。靶向TMPRSS6之寡核苷酸為預防或治療紅細胞增多症提供了一種方案。目前一項靶向TMPRSS6之反義寡核苷酸藥物IONIS-TMPRSS6-LRx (ISIS 702843)治療真性紅細胞增多症二期臨床。開發更多之siRNA，更有助於上述疾病之治療，也為臨床治療提供更多之選擇。
基於此，本發明之目之系提供一種用於抑制TMPRSS6表達之雙鏈siRNA或其藥學上可接受之鹽及其應用，其具有體外、體內試驗效果好、非靶點活性弱之優勢。
本發明之第一個方面，提供了一種用於抑制TMPRSS6表達之siRNA或其藥學上可接受之鹽，由正義鏈及反義鏈組成，正義鏈和反義鏈藉由鹼基配對形成雙鏈，其鹼基序列選自a-e所示中之任意一組或與其相差一個、兩個或三個核苷酸之序列：
a) 雙鏈siRNA SEQ ID NO:BBD-2051，其中正義鏈序列如UGGGAACUUACUACAACUCCA（SEQ ID NO:103）所示，所述反義鏈序列如UGGAGUUGUAGUAAGUUCCCAGG（SEQ ID NO:104）所示。
b) 雙鏈siRNA SEQ ID NO:BBD-2083，其中正義鏈序列如UGCUACUCUGGUAUUUCCUAA所示（SEQ ID NO:167），所述反義鏈序列如UUAGGAAAUACCAGAGUAGCACC所示（SEQ ID NO:168）；
c) 雙鏈siRNA SEQ ID NO:BBD-2047，其中正義鏈序列如CGCCUGGGAACUUACUACAAU所示（SEQ ID NO:95），所述反義鏈序列如AUUGUAGUAAGUUCCCAGGCGGG所示（SEQ ID NO:96）；
d) 雙鏈siRNA SEQ ID NO:BBD-2086，其中正義鏈序列如GGGUGCUACUCUGGUAUUUCA所示（SEQ ID NO:173），所述反義鏈序列如UGAAAUACCAGAGUAGCACCCCC所示（SEQ ID NO:174）；
e) 雙鏈siRNA SEQ ID NO:BBD-2087，其中正義鏈序列如GGGGUGCUACUCUGGUAUUUA所示（SEQ ID NO:175），所述反義鏈序列如UAAAUACCAGAGUAGCACCCCCG所示（SEQ ID NO:176）。
在其中之一些實施例中，所述正義鏈包括不多於3、2、1 或 0 個未修飾之核苷酸，正義鏈中修飾之核苷酸，包含選自 2'-O-甲基修飾之核苷酸、2'-去氧核苷酸、2'-氟修飾之核苷酸，所述正義鏈在 5'-末端含有 1-3 個硫代磷酸酯鍵。
在其中之一些實施例中，所述反義鏈包括不多於 3、2、1 或 0 個未修飾之核苷酸，所述反義鏈中修飾之核苷酸包含選自 2'-O-甲基修飾之核苷酸、2'-去氧核苷酸、2'-氟修飾之核苷酸、5’端-(E)-乙烯基膦酸核苷酸和異甘油核苷酸(isoGNA)，所述反義鏈在 5'-末端和 3'-末端各含有 1-3 個硫代磷酸酯鍵；雙鏈 RNAi 試劑，其中至少一條鏈包含具有至少1-3個核苷酸之3'突出端。
在其中之一些優選之實施例中，所述異甘油核苷酸(isoGNA)位於反義鏈，優選位於5'端起之4-8位中之至少1位，以使所述siRNA或其藥學上可接受之鹽非靶點活性低，具有良好之安全性。
在其中之一些實施例中，互補配對之siRNA反義鏈 5'端第11-13位或正義鏈5'端第9-11位共含有1-6個2'-去氧核苷酸。
其中，所述異甘油核苷酸(isoGNA)為如下結構中之至少一種：
。
其中，所述2'-去氧核苷酸為如下結構中之至少一種：
。
其中，所述尿苷-5’端-(E)-乙烯基膦酸核苷酸之結構如下：
。
在一些優選之實施例中，所述修飾雙鏈體之名稱為：BBD-2051.210、BBD-2051.211、BBD-2051.28、BBD-2083.313、BBD-2083.413、BBD-2051.25、BBD-2051.26、BBD-2051.27、BBD-2051.28、BBD-2051.29、BBD-2051.213、BBD-2051.214、BBD-2051.215、BBD-2051.217、BBD-2051.218、BBD-2051.219；BBD-2083.41、BBD-2083.410、BBD-2083.411、BBD-2083.412、BBD-2083.414、BBD-2083.415、BBD-2083.416、BBD-2083.418、BBD-2083.419、BBD-2083.420、BBD-2083.422、BBD-2083.423、BBD-2083.424。
在一些更優選之實施例中，所述修飾之siRNA為：
BBD-2051.210：
正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
反義鏈：VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
BBD-2051.211：
正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
反義鏈：VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
BBD-2051.28：
正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
反義鏈：VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
BBD-2083.313：
正義鏈：mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA，
反義鏈：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
BBD-2083.413：
正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
反義鏈：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC。
本發明之第二個方面，提供了用於抑制TMPRSS6表達之藥物製劑
，包括上述任一所述siRNA或其藥學上可接受之鹽，以及與所述siRNA連接或包裹之靶向性遞送配體或遞送載體。
在其中一些實施例中，所述配體為N-乙醯基半乳糖胺(Galnac)衍生物。
在其中一些實施例中，所述配體之結構如下：
或
或
或
。
在其中一些實施例中，所述配體與siRNA之正義鏈之3’端連接。
在其中一些實施例中，所述配體與siRNA之正義鏈之3’端連接方式如下：
或
或
或
。
本發明之第三個方面，提供了上述siRNA或其藥學上可接受之鹽在製備抑制TMPRSS6表達之生物製劑或者藥物製劑中之應用。
本發明之第四個方面，系提供所述生物製劑或者藥物製劑在製備預防和治療與TMPRSS6相關之疾病或鐵超負荷或鐵超載相關聯之疾病藥物中之應用。
本發明之第五個方面，系提供一種抑制TMPRSS6在細胞內表達之方法,該方法包括:
(a) 將細胞與上述任一種之siRNA或其藥學上可接受之鹽或上述任一種之生物製劑或者藥物製劑接觸; 和
(b)將步驟(a)中產生之細胞維持足以獲得TMPRSS6基因之mRNA轉錄本之降解之時間,從而抑制TMPRSS6基因在細胞中之表達。
本發明之第六個方面，系提供一種治療患有TMPRSS6相關病症或鐵超負荷或鐵超載相關聯之受驗者之方法，包含對該受驗者給藥治療有效量之上述任一種siRNA或其藥學上可接受之鹽或上述任一種所述之生物製劑或者藥物製劑，從而治療該受驗者。
在其中一些實施例中，其中所述細胞在受試者內。
在其中一些實施例中，其中物件系人。
在其中一些實施例中，其中TMPRSS6 表達被抑制至少約 30%、約 40%、約 50%、約 60%、約 70%、約 80%、約 90%、約 95%、約 98% 或約 100%。
在其中一些實施例中，所述受試者之血清鐵調素濃度增加至少約10%; 及/或其中所述受試者之血清鐵濃度降低至少約20%; 及/或其中所述受試者中之轉鐵蛋白飽和百分比降低至少約20%。
在其中一些實施例中，所述與TMPRSS6相關之疾病或鐵超負荷或鐵超載相關聯之疾病為紅細胞增多症、地中海貧血、血色素沉著症、骨髓增生異常綜合征、遲發性皮膚卟啉症、再生障礙性貧血、鐵粒幼細胞性貧血、鐵難治性缺鐵性貧血、遺傳性貧血、嚴重慢性溶血、帕金森病、阿爾茲海默症、弗裡德賴希氏共濟失調或小細胞貧血、長期輸血導致鐵超載及非酒精性脂肪性肝病（NAFLD）相關之鐵超載。
在其中一些實施例中，所述地中海貧血選自α-地中海貧血、β-地中海貧血和δ-地中海貧血。
在其中一些實施例中，所述紅細胞增多症系真性紅細胞增多症。
在其中一些實施例中，所述遺傳性貧血選自鐮狀細胞性貧血、地中海貧血、範可尼貧血、戴-布二氏貧血、舒-戴二氏綜合征、紅細胞膜障礙、葡萄糖-6-磷酸脫氫酶缺乏和遺傳性出血性毛細血管擴張症。
本申請之發明人經過對siRNA序列研究，並藉由大量之實驗，篩選到多條能夠用於抑制TMPRSS6表達之siRNA序列，並在此基礎上，進一步進行了合適之修飾以提高靶標之沉默能力並降低非靶點活性。其中，藉由一系列體外和體內試驗中，獲得了具有很好之抑制TMPRSS6表達生物活性之siRNA序列，且比臨床上進展最快之TMPRSS6-HCM-9活性更高，有望應用於臨床上進行與TMPRSS6表達相關之疾病或鐵超負荷或鐵超載相關聯疾病之治療。
為了便於理解本發明，下面將對本發明進行更全面之描述。本發明可以以許多不同之形式來實現，並不限於本文所描述之實施例。相反地，提供這些實施例之目之系使對本發明公開內容之理解更加透徹全面。
下列實施例中未注明具體條件之實驗方法，通常按照常規條件，例如Sambrook等人，分子克隆：實驗室手冊(New York: Cold Spring Harbor Laboratory Press, 1989)中所述之條件，或按照製造廠商所建議之條件。實施例中所用到之各種常用化學試劑，均為市售產品。
除非另有定義，本發明所使用之所有之技術和科學術語與屬於本發明之技術領域之技術人員通常理解之含義相同。本發明之說明書中所使用之術語只系為了描述具體之實施例之目之，不用於限制本發明。本發明所使用之術語“及/或”包括一個或多個相關之所列項目之任意之和所有之組合。
定義 為了便於理解本技術，下面定義了一些術語和短語。
鐵超載系鐵在體內過度沉積，並導致重要臟器（尤其系心臟、肝臟、垂體、胰腺和關節）之結構損害和功能障礙。
紅細胞增多症系指單位容積血液中紅細胞數量及血紅蛋白量高於參考值高限。
血色素沉著症系由於高鐵飲食、大量輸血或全身疾病造成體內鐵質儲積過多，而發生之鐵質代謝障礙所致之疾病。
地中海貧血(alphathalassaemia)又稱海洋性貧血(thalassemia)，據全國醫學名詞審定委員會規定應稱為“珠蛋白生成障礙貧血”。系由於一種或多種珠蛋白肽鏈合成受阻或完全抑制，導致Hb成分組成異常，引起慢性溶血性貧血。
TMPRSS6基因之表達可基於與TMPRSS6基因表達相關之任何變數之水準而評價，如組織或血清中之TMPRSS6 mRNA水準、TMPRSS6蛋白質水準、鐵調素mRNA水準、鐵調素蛋白質水準、運鐵蛋白飽和度水準、或鐵水平。抑制可藉由此等變數之一者或多者之絕對水準或相對水準相較于對照水準之下降而評價。該對照水準可為該技術領域中採用之任何類型之對照水準，如給藥前基線水準、或自未治療或經對照治療之類似受驗者、細胞或樣本測得之水準、以及已知族群水準。
在其中一些實施例中，與TMPRSS6表達相關之疾病或鐵超負荷或鐵超載相關聯之疾病為紅細胞增多症、地中海貧血、血色素沉著症、骨髓增生異常綜合征、遲發性皮膚卟啉症、再生障礙性貧血、鐵粒幼細胞性貧血、鐵難治性缺鐵性貧血、遺傳性貧血、嚴重慢性溶血、帕金森病、阿爾茲海默症、弗裡德賴希氏共濟失調或小細胞貧血、長期輸血導致鐵超載及非酒精性脂肪性肝病（NAFLD）相關之鐵超載。
將本發明之siRNA劑遞送至細胞，如受驗者(如人受驗者，如患有TMPRSS6相關病症如血色素沉著症之受驗者)內之細胞，可藉由多種不同路徑達成。舉例而言，可藉由令細胞與本發明之siRNA或於活體外或於活體內接觸而施行遞送。活體內遞送亦可藉由將包含siRNA或其鹽之組成物給藥至受驗者。
通常，任一遞送核酸分子之方法(活體外或活體內可參考已有之遞送技術。對於活體內遞送，為了遞送siRNA分子考慮之因素系包括，舉例而言，所遞送之分子之生物學安定性、非專一性效果之預防、以及所遞送之分子到達目標群組織內之蓄積。可藉由局部給藥(舉例而言，藉由直接注射或植入組織或外用給藥)製劑。局部給藥至治療位元點系最大化該藥物製劑之局部濃度，限制該劑曝露至可被該劑傷害或可令該劑降解之全身性組織，以及容許待給藥之iRNA分子之較低總劑量。
對於全身性給藥iRNA而治療疾病，該RNA可經修飾或使用藥物遞送系統遞送；兩種方法皆作用以防止藉由活體內之核酸內切酶及核酸外切酶造成之dsRNA之快速降解。對RNA或藥學載劑之修飾亦可容許該siRNA組成物靶向至目標群組織，並避免脫靶效果。siRNA分子可藉由化學接合至親脂性基如膽固醇而修飾，例如脂質顆粒，以提升細胞攝取並防止降解。
本發明也包括藥物組成物及製劑，其包括本發明之任一所述siRNA或其鹽。在其中一些實施例中，所述藥物製劑含有本文中描述之siRNA或其鹽以及藥學可接受之載體之藥物組成物。含有該iRNA之藥物組成物有用於治療與TMPRSS6相關之疾病或病症，如血色素沉著症。此等藥物組成物基於遞送模式而配製。
在其中一些實施例中，配製為用於經由不經腸道遞送如靜脈(IV)遞送或皮下(SC)遞送而全身給藥之組成物。于本發明之方法中，該siRNA或其鹽之藥物製劑可於溶液中給藥，優選在無菌溶液中給藥，例如藉由注射給藥。
本發明中，“治療有效量”包括，當對患者給藥用于治療TMPRSS6相關疾病時，siRNA或其鹽之量系足以造成該疾病之治療(如，透過削弱、減輕或維持慣有疾病、或疾病之一種或多種症候)。該“治療有效量”可依據該siRNA或其鹽之藥物製劑、該製劑如何給藥、疾病及其嚴重性、及待治療之患者之病史、年齡、體重、家族病史、基因組成、藉由TMPRSS6表現介導之病理過程之階段、先前治療或聯合治療之類型(若有)、及其他獨立特徵而改變。
本發明中，“預防有效量”包括，當對尚未經歷或顯現TMPRSS6相關疾病之症候但可能易患該疾病之受驗者給藥時，包含siRNA或其鹽之藥物製劑之量足以預防或減輕該疾病或該疾病之一種或多種症候。減輕該疾病包括延緩該疾病之進程或降低後續發展之疾病之嚴重性。該“預防有效量”可依據該siRNA或其鹽之藥物制之如何給藥、疾病之風險程度、及待治療之患者之病史、年齡、體重、家族病史、基因組成、藉由TMPRSS6表現介導之病理過程之階段、先前治療或聯合治療之類型(若有)、及其他獨立特徵而改變。
“治療有效量”或“預防有效量”也包括一種siRNA之量，以任何治療可接受之合理之效益、風險比。
本發明所述靶向性遞送配體或其他類型遞送載體，所述配體優選為N-乙醯基半乳糖胺衍生物（Galnac載體），其他類型遞送載體如特異性遞送到肝臟細胞之脂質體只要能實現siRNA之遞送都可用于本發明 。
近年來，Galnac載體目前已經有比較深入研究，GalNAc-核酸系糖類化合物與核酸形成之單綴合物，將N-乙醯化之半乳糖胺以三價態之方式共價綴合到不同序列之RNA之正義鏈3’及/或5’末端，形成GalNAc-siRNA藥物，從而實現向肝細胞之特異性遞送，並藉由細胞內吞作用使藥物進入細胞和發揮功能。
以下實施例中，Galnac載體及連接siRNA後之結構如下：
核酸序列表示中使用之核苷酸單體之縮寫及結構如下：
| A | 腺苷-3’-磷酸酯 |
| T | 5’-甲基尿苷-3’-磷酸酯 |
| C | 胞苷-3’-磷酸酯 |
| G | 鳥苷-3’-磷酸酯 |
| U | 尿苷-3’-磷酸酯 |
| dA | 2’-去氧腺苷-3’-磷酸酯 |
| dT | 2’-去氧胸苷-3’-磷酸酯 |
| dC | 2’-去氧胞苷-3’-磷酸酯 |
| dG | 2’-去氧鳥苷-3’-磷酸酯 |
| dU | 2’-去氧尿苷-3’-磷酸酯 |
| mA | 2’-O-甲基腺苷-3’-磷酸酯 |
| mC | 2’-O-甲基胞苷-3’-磷酸酯 |
| mG | 2’-O-甲基鳥苷-3’-磷酸酯 |
| mU | 2’-O-甲基尿苷-3’-磷酸酯 |
| fA | 2’-氟代腺苷-3’-磷酸酯 |
| fC | 2’-氟代胞苷-3’-磷酸酯 |
| fG | 2’-氟代鳥苷-3’-磷酸酯 |
| fU | 2’-氟代尿苷-3’-磷酸酯 |
| VPU | 尿苷-5’端-(E)-乙烯基膦酸核苷酸 |
| isoGNA -A | 腺苷-異甘油核苷酸 |
| isoGNA -T | 胸苷-異甘油核苷酸 |
| isoGNA -C | 胞苷-異甘油核苷酸 |
| isoGNA -G | 鳥苷-異甘油核苷酸 |
| isoGNA -U | 尿苷-異甘油核苷酸 |
| * | 硫代磷酸酯鍵聯 |
具體結構如下：
本領域技術人員根據已有之技術，所述siRNA之藥學上可接受之鹽可為鈉鹽或鉀鹽，例如，在純化之時候，產生siRNA之鈉鹽。
以下結合具體實施例對本發明作進一步詳細之說明。
實施例
1 siRNA
之合成
在北京擎科生物科技有限公司12通道核酸合成儀上，使用固相寡核苷酸合成方案進行0.2-1μmol之寡核苷酸合成，含Galnac之siRNA序列需要在偶聯Galnac之CPG預填充柱上進行合成。在合成之寡核苷酸中加入氨解試劑，並在45-80℃下孵育，將寡核苷酸與固相載體分離使寡核苷酸游離下來。然後用乙醇沉澱粗制寡核苷酸，高速離心棄去上清液，重複兩遍獲得粗制寡核苷酸，並將沉澱重懸于DEPC水中。藉由離子配對HPLC方法對粗制寡核苷酸進行純化，將收集到之產物在真空離心乾燥器中乾燥至粉末狀。用DEPC水溶解純化後得到之產物，並藉由TOF LC-MS進行分析。測定寡核苷酸濃度，計算等莫耳量之正義鏈、反義鏈所需體積，將等莫耳量之正義鏈、反義鏈混合均勻，藉由95℃加熱5分鐘，然後自然冷卻至室溫之退火方法製備雙鏈。
表1：未修飾siRNA之正義鏈和反義鏈序列。TMPRSS6-hcm-9為Silence Therapeutics公司進入臨床階段之序列（公開號：CN113164768A），用於陽性比對序列。
| 雙鏈體 名稱 | 正義鏈 名稱(SEQID NO.) | 正義鏈序列 5’-3’ | NM_153609.4中之範圍 | 反義鏈 名稱(SEQID NO.) | 反義鏈序列 5’-3’ | NM_153609.4中之範圍 |
| TMPRSS6-hcm-9 | S0（1） | UCACCUGCUUCUUCUGGUU | 560_578 | AS0（2） | AACCAGAAGAAGCAGGUGA | 560_578 |
| BBD-2001 | S1（3） | GCCAAAGCCCAGAAGAUGCUA | 463_483 | AS01（4） | UAGCAUCUUCUGGGCUUUGGCGG | 461_483 |
| BBD-2002 | S2(5) | UCCUUUGGGGAGGGACCCCUA | 541_561 | AS02(6) | UAGGGGUCCCUCCCCAAAGGAAU | 539_561 |
| BBD-2003 | S3(7) | GUGAUCCUGGAAGCCAGUGUA | 724_744 | AS03(8) | UACACUGGCUUCCAGGAUCACUA | 722_744 |
| BBD-2004 | S4(9) | UGAUCCUGGAAGCCAGUGUGA | 725_745 | AS04(10) | UCACACUGGCUUCCAGGAUCACC | 723_745 |
| BBD-2005 | S5(11) | GAUCCUGGAAGCCAGUGUGAA | 726_746 | AS05(12) | UUCACACUGGCUUCCAGGAUCAC | 724_746 |
| BBD-2006 | S6(13) | CCCUCUCUGGACUACGGCUUA | 1234_1244 | AS06(14) | UAAGCCGUAGUCCAGAGAGGGCA | 1232_1244 |
| BBD-2007 | S7(15) | GAUCCAGAACAGGAGGCUGUA | 1326_1346 | AS07(16) | UACAGCCUCCUGUUCUGGAUCGU | 1324_1346 |
| BBD-2008 | S8(17) | AUCCAGAACAGGAGGCUGUGU | 1327_1347 | AS08(18) | ACACAGCCUCCUGUUCUGGAUCG | 1325_1347 |
| BBD-2009 | S9(19) | UCCAGAACAGGAGGCUGUGUA | 1328_1348 | AS09(20) | UACACAGCCUCCUGUUCUGGAUC | 1326_1348 |
| BBD-2010 | S10(21) | CCAGAACAGGAGGCUGUGUGA | 1329_1349 | AS10(22) | UCACACAGCCUCCUGUUCUGGAU | 1327_1349 |
| BBD-2011 | S11(23) | CAGAACAGGAGGCUGUGUGGA | 1330_1350 | AS11(24) | UCCACACAGCCUCCUGUUCUGGA | 1328_1350 |
| BBD-2012 | S12(25) | GAACAGGAGGCUGUGUGGCUU | 1332_1352 | AS12(26) | AAGCCACACAGCCUCCUGUUCUG | 1330_1352 |
| BBD-2013 | S13(27) | UCACCAUCAACUUCACCUCCA | 1406_1426 | AS13(28) | UGGAGGUGAAGUUGAUGGUGAUC | 1404_1426 |
| BBD-2014 | S14(29) | CACCAUCAACUUCACCUCCCA | 1407_1427 | AS14(30) | UGGGAGGUGAAGUUGAUGGUGAU | 1405_1427 |
| BBD-2015 | S15(31) | ACCAUCAACUUCACCUCCCAA | 1408_1428 | AS15(32) | UUGGGAGGUGAAGUUGAUGGUGA | 1406_1428 |
| BBD-2016 | S16(33) | CCAUCAACUUCACCUCCCAGA | 1409_1429 | AS16(34) | UCUGGGAGGUGAAGUUGAUGGUG | 1407_1429 |
| BBD-2017 | S17(35) | CAUCAACUUCACCUCCCAGAU | 1410_1430 | AS17(36) | AUCUGGGAGGUGAAGUUGAUGGU | 1408_1430 |
| BBD-2018 | S18(37) | AUCAACUUCACCUCCCAGAUA | 1411_1431 | AS18(38) | UAUCUGGGAGGUGAAGUUGAUGG | 1409_1431 |
| BBD-2019 | S19(39) | UCAACUUCACCUCCCAGAUCU | 1412_1432 | AS19(40) | AGAUCUGGGAGGUGAAGUUGAUG | 1410_1432 |
| BBD-2020 | S20(41) | CAACUUCACCUCCCAGAUCUA | 1413_1433 | AS20(42) | UAGAUCUGGGAGGUGAAGUUGAU | 1411_1433 |
| BBD-2021 | S21(43) | AACUUCACCUCCCAGAUCUCA | 1414_1434 | AS21(44) | UGAGAUCUGGGAGGUGAAGUUGA | 1412_1434 |
| BBD-2022 | S22(45) | ACUUCACCUCCCAGAUCUCCA | 1415_1435 | AS22(46) | UGGAGAUCUGGGAGGUGAAGUUG | 1413_1435 |
| BBD-2023 | S23(47) | CUUCACCUCCCAGAUCUCCCU | 1416_1436 | AS23(48) | AGGGAGAUCUGGGAGGUGAAGUU | 1414_1436 |
| BBD-2024 | S24(49) | UUCACCUCCCAGAUCUCCCUA | 1417_1437 | AS24(50) | UAGGGAGAUCUGGGAGGUGAAGU | 1415_1437 |
| BBD-2025 | S25(51) | UCACCUCCCAGAUCUCCCUCA | 1418_1438 | AS25(52) | UGAGGGAGAUCUGGGAGGUGAAG | 1416_1438 |
| BBD-2026 | S26(53) | CACCUCCCAGAUCUCCCUCAA | 1419_1439 | AS26(54) | UUGAGGGAGAUCUGGGAGGUGAA | 1417_1439 |
| BBD-2027 | S27(55) | CCAGUGUGAGGACCGGAGCUA | 1725_1745 | AS27(56) | UAGCUCCGGUCCUCACACUGGAA | 1723_1745 |
| BBD-2028 | S28(57) | CACUGUGACUGUGGCCUCCAA | 1810_1830 | AS28(58) | UUGGAGGCCACAGUCACAGUGCU | 1808_1830 |
| BBD-2029 | S29(59) | UUCGGGGUCGACACAUCUGUA | 1904_1924 | AS29(60) | UACAGAUGUGUCGACCCCGAACC | 1902_1924 |
| BBD-2030 | S30(61) | UCGGGGUCGACACAUCUGUGA | 1905_1925 | AS30(62) | UCACAGAUGUGUCGACCCCGAAC | 1903_1925 |
| BBD-2031 | S31(63) | CGGGGUCGACACAUCUGUGGA | 1906_1926 | AS31(64) | UCCACAGAUGUGUCGACCCCGAA | 1904_1926 |
| BBD-2032 | S32(65) | CCAGGAGGACAGCAUGGCCUA | 1974-1994 | AS32(66) | UAGGCCAUGCUGUCCUCCUGGAA | 1972-1994 |
| BBD-2033 | S33(67) | CUUCCAGGAGGACAGCAUGGA | 1971-1991 | AS33(68) | UCCAUGCUGUCCUCCUGGAAGCA | 1969-1991 |
| BBD-2034 | S34(69) | GCUUCCAGGAGGACAGCAUGA | 1970-1990 | AS34(70) | UCAUGCUGUCCUCCUGGAAGCAG | 1968-1990 |
| BBD-2035 | S35(71) | UGCUUCCAGGAGGACAGCAUA | 1969-1989 | AS35(72) | UAUGCUGUCCUCCUGGAAGCAGU | 1967-1989 |
| BBD-2036 | S36(73) | CUGCUUCCAGGAGGACAGCAU | 1968-1988 | AS36(74) | AUGCUGUCCUCCUGGAAGCAGUG | 1966-1988 |
| BBD-2037 | S37(75) | GUGUCCUUCAAGGUGAGCCGA | 2056-2076 | AS37(76) | UCGGCUCACCUUGAAGGACACCU | 2054-2076 |
| BBD-2038 | S38(77) | GGUGUCCUUCAAGGUGAGCCA | 2055-2075 | AS38(78) | UGGCUCACCUUGAAGGACACCUC | 2053-2075 |
| BBD-2039 | S39(79) | AGGUGUCCUUCAAGGUGAGCA | 2054-2074 | AS39(80) | UGCUCACCUUGAAGGACACCUCU | 2052-2074 |
| BBD-2040 | S40(81) | AGCCAUGACUACGACGUGGCA | 2107-2127 | AS40(82) | UGCCACGUCGUAGUCAUGGCUGU | 2105-2127 |
| BBD-2041 | S41(83) | CAGCCAUGACUACGACGUGGA | 2106-2126 | AS41(84) | UCCACGUCGUAGUCAUGGCUGUC | 2104-2126 |
| BBD-2042 | S42(85) | ACAGCCAUGACUACGACGUGA | 2105-2125 | AS42(86) | UCACGUCGUAGUCAUGGCUGUCC | 2103-2125 |
| BBD-2043 | S43(87) | GACAGCCAUGACUACGACGUA | 2104-2124 | AS43(88) | UACGUCGUAGUCAUGGCUGUCCU | 2102-2124 |
| BBD-2044 | S44(89) | GGACAGCCAUGACUACGACGU | 2103-2123 | AS44(90) | ACGUCGUAGUCAUGGCUGUCCUC | 2101-2123 |
| BBD-2045 | S45(91) | AGGACAGCCAUGACUACGACA | 2102-2122 | AS45(92) | UGUCGUAGUCAUGGCUGUCCUCU | 2100-2122 |
| BBD-2046 | S46(93) | GAGGACAGCCAUGACUACGAA | 2101-2121 | AS46(94) | UUCGUAGUCAUGGCUGUCCUCUU | 2099-2121 |
| BBD-2047 | S47(95) | CGCCUGGGAACUUACUACAAU | 505_525 | AS47(96) | AUUGUAGUAAGUUCCCAGGCGGG | 503_525 |
| BBD-2048 | S48(97) | GCCUGGGAACUUACUACAACU | 506_526 | AS48(98) | AGUUGUAGUAAGUUCCCAGGCGG | 504_526 |
| BBD-2049 | S49(99) | CCUGGGAACUUACUACAACUA | 507_527 | AS49(100) | UAGUUGUAGUAAGUUCCCAGGCG | 505_527 |
| BBD-2050 | S50(101) | CUGGGAACUUACUACAACUCA | 508_528 | AS50(102) | UGAGUUGUAGUAAGUUCCCAGGC | 506_528 |
| BBD-2051 | S51(103) | UGGGAACUUACUACAACUCCA | 509_529 | AS51(104) | UGGAGUUGUAGUAAGUUCCCAGG | 507_529 |
| BBD-2052 | S52(105) | GGGAACUUACUACAACUCCAA | 510_530 | AS52(106) | UUGGAGUUGUAGUAAGUUCCCAG | 508_530 |
| BBD-2053 | S53(107) | GGAACUUACUACAACUCCAGA | 511_531 | AS53(108) | UCUGGAGUUGUAGUAAGUUCCCA | 509_531 |
| BBD-2054 | S54(109) | GAACUUACUACAACUCCAGCU | 512_532 | AS54(110) | AGCUGGAGUUGUAGUAAGUUCCC | 510_532 |
| BBD-2055 | S55(111) | AACUUACUACAACUCCAGCUA | 513_533 | AS55(112) | UAGCUGGAGUUGUAGUAAGUUCC | 511_533 |
| BBD-2056 | S56(113) | ACUUACUACAACUCCAGCUCA | 514_534 | AS56(114) | UGAGCUGGAGUUGUAGUAAGUUC | 512_534 |
| BBD-2057 | S57(115) | CUUACUACAACUCCAGCUCCA | 515_535 | AS57(116) | UGGAGCUGGAGUUGUAGUAAGUU | 513_535 |
| BBD-2058 | S58(117) | UUACUACAACUCCAGCUCCGU | 516_536 | AS58(118) | ACGGAGCUGGAGUUGUAGUAAGU | 514_536 |
| BBD-2059 | S59(119) | UACUACAACUCCAGCUCCGUA | 517_537 | AS59(120) | UACGGAGCUGGAGUUGUAGUAAG | 515_537 |
| BBD-2060 | S60(121) | ACUACAACUCCAGCUCCGUCU | 518_538 | AS60(122) | AGACGGAGCUGGAGUUGUAGUAA | 516_538 |
| BBD-2061 | S61(123) | CUACAACUCCAGCUCCGUCUA | 519_539 | AS61(124) | UAGACGGAGCUGGAGUUGUAGUA | 517_539 |
| BBD-2062 | S62(125) | UACAACUCCAGCUCCGUCUAU | 520_540 | AS62(126) | AUAGACGGAGCUGGAGUUGUAGU | 518_540 |
| BBD-2063 | S63(127) | ACAACUCCAGCUCCGUCUAUU | 521_541 | AS63(128) | AAUAGACGGAGCUGGAGUUGUAG | 519_541 |
| BBD-2064 | S64(129) | CAACUCCAGCUCCGUCUAUUA | 522_542 | AS64(130) | UAAUAGACGGAGCUGGAGUUGUA | 520_542 |
| BBD-2065 | S65(131) | AACUCCAGCUCCGUCUAUUCA | 523_543 | AS65(132) | UGAAUAGACGGAGCUGGAGUUGU | 521_543 |
| BBD-2066 | S66(133) | ACUCCAGCUCCGUCUAUUCCU | 524_544 | AS66(134) | AGGAAUAGACGGAGCUGGAGUUG | 522_544 |
| BBD-2067 | S67(135) | CUCCAGCUCCGUCUAUUCCUU | 525_545 | AS67(136) | AAGGAAUAGACGGAGCUGGAGUU | 523_545 |
| BBD-2068 | S68(137) | UCCAGGCCUGUGAAGUGAACA | 1112_1132 | AS68(138) | UGUUCACUUCACAGGCCUGGAAG | 1110_1132 |
| BBD-2069 | S69(139) | CCAGGCCUGUGAAGUGAACCU | 1113_1133 | AS69(140) | AGGUUCACUUCACAGGCCUGGAA | 1111_1133 |
| BBD-2070 | S70(141) | CAGGCCUGUGAAGUGAACCUA | 1114_1134 | AS70(142) | UAGGUUCACUUCACAGGCCUGGA | 1112_1134 |
| BBD-2071 | S71(143) | AGGCCUGUGAAGUGAACCUGA | 1115_1135 | AS71(144) | UCAGGUUCACUUCACAGGCCUGG | 1113_1135 |
| BBD-2072 | S72(145) | GGCCUGUGAAGUGAACCUGAA | 1116_1136 | AS72(146) | UUCAGGUUCACUUCACAGGCCUG | 1114_1136 |
| BBD-2073 | S73(147) | GCCUGUGAAGUGAACCUGACA | 1117_1137 | AS73(148) | UGUCAGGUUCACUUCACAGGCCU | 1115_1137 |
| BBD-2074 | S74(149) | CUGUGAAGUGAACCUGACGCU | 1119_1139 | AS74(150) | AGCGUCAGGUUCACUUCACAGGC | 1117_1139 |
| BBD-2075 | S75(151) | UGUGAAGUGAACCUGACGCUA | 1120_1140 | AS75(152) | UAGCGUCAGGUUCACUUCACAGG | 1118_1140 |
| BBD-2076 | S76(153) | GUGAAGUGAACCUGACGCUGA | 1121_1141 | AS76(154) | UCAGCGUCAGGUUCACUUCACAG | 1119_1141 |
| BBD-2077 | S77(155) | AUCGCUGACCGCUGGGUGAUA | 1936_1956 | AS77(156) | UAUCACCCAGCGGUCAGCGAUGA | 1934_1956 |
| BBD-2078 | S78(157) | UCGCUGACCGCUGGGUGAUAA | 1937_1957 | AS78(158) | UUAUCACCCAGCGGUCAGCGAUG | 1935_1957 |
| BBD-2079 | S79(159) | CGCUGACCGCUGGGUGAUAAU | 1938_1958 | AS79(160) | AUUAUCACCCAGCGGUCAGCGAU | 1936_1958 |
| BBD-2080 | S80(161) | GCUGACCGCUGGGUGAUAACA | 1939_1959 | AS80(162) | UGUUAUCACCCAGCGGUCAGCGA | 1937_1959 |
| BBD-2081 | S81(163) | GACCGCUGGGUGAUAACAGCU | 1942_1962 | AS81(164) | AGCUGUUAUCACCCAGCGGUCAG | 1940_1962 |
| BBD-2082 | S82(165) | ACCGCUGGGUGAUAACAGCUA | 1943_1963 | AS82(166) | UAGCUGUUAUCACCCAGCGGUCA | 1941_1963 |
| BBD-2083 | S83(167) | UGCUACUCUGGUAUUUCCUAA | 326-345 | AS83(168) | UUAGGAAAUACCAGAGUAGCACC | 324-345 |
| BBD-2084 | S84(169) | GUGCUACUCUGGUAUUUCCUA | 325-345 | AS84(170) | UAGGAAAUACCAGAGUAGCACCC | 323-345 |
| BBD-2085 | S85(171) | GGUGCUACUCUGGUAUUUCCU | 324-344 | AS85(172) | AGGAAAUACCAGAGUAGCACCCC | 322-344 |
| BBD-2086 | S86(173) | GGGUGCUACUCUGGUAUUUCA | 323-343 | AS86(174) | UGAAAUACCAGAGUAGCACCCCC | 321-343 |
| BBD-2087 | S87(175) | GGGGUGCUACUCUGGUAUUUA | 322-342 | AS87(176) | UAAAUACCAGAGUAGCACCCCCG | 320-342 |
| BBD-2088 | S88(177) | GGGGGUGCUACUCUGGUAUUU | 321-341 | AS88(178) | AAAUACCAGAGUAGCACCCCCGC | 319-341 |
| BBD-2089 | S89(179) | CGGGGGUGCUACUCUGGUAUU | 320-340 | AS89(180) | AAUACCAGAGUAGCACCCCCGCC | 318-340 |
| BBD-2090 | S90(181) | GCUACUCUGGUAUUUCCUAGG | 327-347 | AS90(182) | CCUAGGAAAUACCAGAGUAGCAC | 325-347 |
| BBD-2091 | S91(183) | CUACUCUGGUAUUUCCUAGGG | 328-348 | AS91(184) | CCCUAGGAAAUACCAGAGUAGCA | 326-348 |
| BBD-2092 | S92(185) | UACUCUGGUAUUUCCUAGGGU | 329-349 | AS92（186） | ACCCUAGGAAAUACCAGAGUAGC | 327-349 |
| AD-1554911 | （187） | UGCUACUCUGGUAUUUCCUAU | 326-345 | （188） | AUAGGAAAUACCAGAGUAGCACC | 324-345 |
實施例
2
在
Hep3B
細胞中採用脂質體轉染方法進行
siRNA
體外篩選
細胞培養和96孔板轉染：在Hep3B細胞中進行體外實驗，採用MEM+10%FBS+1X青黴素鏈黴素+1X非必需胺基酸培養基，細胞平鋪面積達80%時用胰蛋白酶消化，並用Scepter自動細胞計數儀（Millipore，#PHCC00000）測定細胞密度，同時在96孔板中將siRNA、Opti-MEM和INTERFERin (Polyplus transfection)混合並置於室溫孵育10分鐘，然後每孔加入含有Hep3B細胞之完全培養基，將96孔板放於37℃，5% CO
2之培養箱中孵育24小時。
未修飾之siRNA以1nM最終濃度進行篩選試驗。
96孔板RNA提取、反轉錄：使用Dynabeads mRNA DIRECT試劑盒（Ambion）提取96孔板細胞mRNA，將96孔板中培養基吸走，用DPBS清洗一次，每孔加入50-300 μl細胞裂解液，再加入20-100 μl beads,在振盪器上震盪，將96孔板置於磁分離架上，吸走孔內裂解液，每孔加入50-300 μl之清洗緩衝液A，吹打後置於磁分離架上，吸走清洗緩衝液A，然後用清洗緩衝液B將beads吹起，轉移到新之96孔板，置於磁分離架，吸走清洗緩衝液B，再用緩衝液B將beads吹起轉移到96孔PCR板中，同時配製反轉試劑，將96孔PCR板置於磁分離架上，吸走清洗緩衝液B，每孔加入20 μl之反轉試劑，用封板膜封板後在PCR儀上進行25℃孵育10分鐘，然後37℃孵育兩小時後85℃，5分鐘，降溫至4°C，反轉錄結束。
即時螢光定量PCR：反轉錄結束後將96孔板置於磁分離架上直到beads都吸附在底部，吸走反轉試劑，將配好之QPCR體系加入到96孔PCR板中，封板膜封板，在StepOnePlus之即時PCR系統(applied biosystems)上進行PCR。
採用ΔΔCt方法分析資料，並採用1nM陰性對照序列轉染之細胞進行測試標準化。
陰性對照AD-1955序列為：CUUACGCUGAGUACUUCGAdTdT（SEQ ID NO.187）
UCGAAGUACUCAGCGUAAGdTdT（SEQ ID NO.188）。
表2為Hep3B細胞中未修飾siRNA在轉染1nM濃度下篩選之實驗結果
| siRNA 雙鏈體 | Hep3B細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 1nM平均值 | SD | |
| TMPRSS6-hcm-9 | 25.85 | 4.68 |
| BBD-2001 | 44.51 | 0.39 |
| BBD-2002 | 90.44 | 4.29 |
| BBD-2003 | 33.34 | 3.75 |
| BBD-2004 | 68.18 | 5.57 |
| BBD-2005 | 26.47 | 1.40 |
| BBD-2006 | 53.87 | 3.90 |
| BBD-2007 | 69.38 | 8.36 |
| BBD-2008 | 61.20 | 8.62 |
| BBD-2009 | 53.58 | 8.57 |
| BBD-2010 | 62.46 | 13.61 |
| BBD-2011 | 74.77 | 5.71 |
| BBD-2012 | 51.13 | 4.14 |
| BBD-2013 | 32.25 | 3.20 |
| BBD-2014 | 29.14 | 4.84 |
| BBD-2015 | 50.11 | 9.25 |
| BBD-2016 | 25.86 | 2.37 |
| BBD-2017 | 40.37 | 2.03 |
| BBD-2018 | 30.26 | 5.31 |
| BBD-2019 | 33.95 | 4.77 |
| BBD-2020 | 36.15 | 3.19 |
| BBD-2021 | 65.42 | 6.82 |
| BBD-2022 | 56.01 | 5.34 |
| BBD-2023 | 45.64 | 7.76 |
| BBD-2024 | 83.71 | 7.26 |
| BBD-2025 | 87.45 | 12.37 |
| BBD-2026 | 67.00 | 6.13 |
| BBD-2027 | 80.47 | 3.76 |
| BBD-2028 | 63.95 | 10.03 |
| BBD-2029 | 99.49 | 17.48 |
| BBD-2030 | 89.02 | 7.22 |
| BBD-2031 | 84.44 | 10.02 |
| BBD-2032 | 88.48 | 5.46 |
| BBD-2033 | 100.49 | 12.70 |
| BBD-2034 | 99.74 | 23.78 |
| BBD-2035 | 67.76 | 9.77 |
| BBD-2036 | 87.30 | 8.49 |
| BBD-2037 | 59.57 | 2.84 |
| BBD-2038 | 63.93 | 2.83 |
| BBD-2039 | 63.40 | 3.87 |
| BBD-2040 | 88.84 | 4.33 |
| BBD-2041 | 74.42 | 7.48 |
| BBD-2042 | 44.17 | 7.48 |
| BBD-2043 | 41.89 | 2.87 |
| BBD-2044 | 37.04 | 4.81 |
| BBD-2045 | 53.40 | 6.91 |
| BBD-2046 | 47.74 | 9.28 |
| BBD-2047 | 18.88 | 2.71 |
| BBD-2048 | 15.07 | 3.47 |
| BBD-2049 | 21.18 | 3.87 |
| BBD-2050 | 18.90 | 2.95 |
| BBD-2051 | 11.19 | 3.86 |
| BBD-2052 | 54.69 | 4.51 |
| BBD-2053 | 71.27 | 5.90 |
| BBD-2054 | 61.06 | 15.96 |
| BBD-2055 | 55.83 | 7.12 |
| BBD-2056 | 35.62 | 7.07 |
| BBD-2057 | 42.90 | 7.61 |
| BBD-2058 | 49.78 | 11.87 |
| BBD-2059 | 49.10 | 11.03 |
| BBD-2060 | 65.92 | 13.24 |
| BBD-2061 | 51.45 | 12.52 |
| BBD-2062 | 14.49 | 0.83 |
| BBD-2063 | 51.63 | 14.27 |
| BBD-2064 | 17.71 | 3.05 |
| BBD-2065 | 60.30 | 7.25 |
| BBD-2066 | 46.68 | 7.23 |
| BBD-2067 | 45.39 | 7.07 |
| BBD-2068 | 56.96 | 4.30 |
| BBD-2069 | 60.30 | 14.22 |
| BBD-2070 | 67.24 | 10.25 |
| BBD-2071 | 58.32 | 8.63 |
| BBD-2072 | 57.56 | 9.09 |
| BBD-2073 | 43.46 | 6.72 |
| BBD-2074 | 36.34 | 7.23 |
| BBD-2075 | 57.95 | 11.28 |
| BBD-2076 | 46.99 | 10.47 |
| BBD-2077 | 63.98 | 12.57 |
| BBD-2078 | 20.54 | 5.66 |
| BBD-2079 | 44.91 | 17.92 |
| BBD-2080 | 57.08 | 13.55 |
| BBD-2081 | 73.76 | 8.10 |
| BBD-2082 | 77.17 | 6.88 |
| BBD-2083 | 20.94 | 2.64 |
所得實驗結果顯示，表1中之siRNA在1nM濃度下對Hep3B細胞中TMPRSS6之表達產生了不同程度之抑制。經過比較和綜合考慮後，我們對其中部分活性好之序列進行修飾，以提高其體內之穩定性及活性。
表3描述了多條siRNA修飾正義鏈序列。
| 修飾正義鏈名稱 | 修飾正義鏈序列5'-3' |
| TMPRSS6-hcm-9B | fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG*mU*fU |
| BBD-2003SM1 | mG*mU*mGmAmUmCfCmUdGfGfAmAfGmCmCmAmGmUmGmUmA |
| BBD-2005SM1 | mG*mA*mUmCmCmUfGmGdAfAfGmCfCmAmGmUmGmUmGmAmA |
| BBD-2013SM1 | mU*mC*mAmCmCmAfUmCdAfAfCmUfUmCmAmCmCmUmCmCmA |
| BBD-2014SM1 | mC*mA*mCmCmAmUfCmAdAfCfUmUfCmAmCmCmUmCmCmCmA |
| BBD-2015SM1 | mA*mC*mCmAmUmCfAmAdCfUfUmCfAmCmCmUmCmCmCmAmA |
| BBD-2016SM1 | mC*mC*mAmUmCmAfAmCdTfUfCmAfCmCmUmCmCmCmAmGmA |
| BBD-2017SM1 | mC*mA*mUmCmAmAfCmUdTfCfAmCfCmUmCmCmCmAmGmAmU |
| BBD-2018SM1 | mA*mU*mCmAmAmCfUmUdCfAfCmCfUmCmCmCmAmGmAmUmA |
| BBD-2019SM1 | mU*mC*mAmAmCmUfUmCdAfCfCmUfCmCmCmAmGmAmUmCmU |
| BBD-2020SM1 | mC*mA*mAmCmUmUfCmAdCfCfUmCfCmCmAmGmAmUmCmUmA |
| BBD-2023SM1 | mC*mU*mUmCmAmCfCmUdCfCfCmAfGmAmUmCmUmCmCmCmU |
| BBD-2042SM1 | mA*mC*mAmGmCmCfAmUdGfAfCmUfAmCmGmAmCmGmUmGmA |
| BBD-2043SM1 | mG*mA*mCmAmGmCfCmAdTfGfAmCfUmAmCmGmAmCmGmUmA |
| BBD-2044SM1 | mG*mG*mAmCmAmGfCmCdAfUfGmAfCmUmAmCmGmAmCmGmU |
| BBD-2047SM1 | mC*mG*mCmCmUmGfGmGdAfAfCmUfUmAmCmUmAmCmAmAmU |
| BBD-2048SM1 | mG*mC*mCmUmGmGfGmAdAfCfUmUfAmCmUmAmCmAmAmCmU |
| BBD-2049SM1 | mC*mC*mUmGmGmGfAmAdCfUfUmAfCmUmAmCmAmAmCmUmA |
| BBD-2050SM1 | mC*mU*mGmGmGmAfAmCdTfUfAmCfUmAmCmAmAmCmUmCmA |
| BBD-2051SM1 | mU*mG*mGmGmAmAfCmUdTfAfCmUfAmCmAmAmCmUmCmCmA |
| BBD-2056SM1 | mA*mC*mUmUmAmCfUmAdCfAfAmCfUmCmCmAmGmCmUmCmA |
| BBD-2057SM1 | mC*mU*mUmAmCmUfAmCdAfAfCmUfCmCmAmGmCmUmCmCmA |
| BBD-2058SM1 | mU*mU*mAmCmUmAfCmAdAfCfUmCfCmAmGmCmUmCmCmGmU |
| BBD-2059SM1 | mU*mA*mCmUmAmCfAmAdCfUfCmCfAmGmCmUmCmCmGmUmA |
| BBD-2062SM1 | mU*mA*mCmAmAmCfUmCdCfAfGmCfUmCmCmGmUmCmUmAmU |
| BBD-2064SM1 | mC*mA*mAmCmUmCfCmAdGfCfUmCfCmGmUmCmUmAmUmUmA |
| BBD-2074SM1 | mC*mU*mGmUmGmAfAmGdTfGfAmAfCmCmUmGmAmCmGmCmU |
| BBD-2078SM1 | mU*mC*mGmCmUmGfAmCdCfGfCmUfGmGmGmUmGmAmUmAmA |
| BBD-2078SM2 | mU*mC*mGmCmUmGfAmCfCfGdCmUfGmGmGmUmGmAmUmAmA |
| BBD-2078SM3 | mU*mC*mGmCmUmGfAmCfCfGfCmUfGmGmGmUmGmAmUmAmA |
| BBD-2083SM1 | mU*mG*mCmUmAmCfUmCdTfGfGmUfAmUmUmUmCmCmUmAmA |
| BBD-2084SM1 | mG*mU*mGmCmUmAfCmUfCfUfGmGmUmAmUmUmUmCmCmUmA |
| BBD-2085SM1 | mG*mG*mUmGmCmUfAmCfUfCfUmGmGmUmAmUmUmUmCmCmU |
| BBD-2086SM1 | mG*mG*mGmUmGmCfUmAfCfUfCmUmGmGmUmAmUmUmUmCmA |
| BBD-2087SM1 | mG*mG*mGmGmUmGfCmUfAfCfUmCmUmGmGmUmAmUmUmUmA |
| BBD-2088SM1 | mG*mG*mGmGmGmUfGmCfUfAfCmUmCmUmGmGmUmAmUmUmU |
| BBD-2089SM1 | mC*mG*mGmGmGmGfUmGfCfUfAmCmUmCmUmGmGmUmAmUmU |
| BBD-2090SM1 | mG*mC*mUmAmCmUfCmUfGfGfUmAmUmUmUmCmCmUmAmGmG |
| BBD-2091SM1 | mC*mU*mAmCmUmCfUmGfGfUfAmUmUmUmCmCmUmAmGmGmG |
| BBD-2092SM1 | mU*mA*mCmUmCmUfGmGfUfAfUmUmUmCmCmUmAmGmGmGmU |
表4描述了多條 siRNA修飾反義鏈序列。
| 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| TMPRSS6-hcm-9A | mA*fA*mCfCmAfGmAfAmGfArnAfGmCfAmGfGmU*fG*mA |
| BBD-2003AM1 | mU*fA*mCmAmCmUmGmGmCfUmUdCmCfAmGfGmAmUmCmAmC*mU*mA |
| BBD-2005AM1 | mU*fU*mCmAmCmAmCmUmGfGmCdTmUfCmCfAmGmGmAmUmC*mA*mC |
| BBD-2013AM1 | mU*fG*mGmAmGmGmUmGmAfAmGdTmUfGmAfUmGmGmUmGmA*mU*mC |
| BBD-2014AM1 | mU*fG*mGmGmAmGmGmUmGfAmAdGmUfUmGfAmUmGmGmUmG*mA*mU |
| BBD-2015AM1 | mU*fU*mGmGmGmAmGmGmUfGmAdAmGfUmUfGmAmUmGmGmU*mG*mA |
| BBD-2016AM1 | mU*fC*mUmGmGmGmAmGmGfUmGdAmAfGmUfUmGmAmUmGmG*mU*mG |
| BBD-2017AM1 | mA*fU*mCmUmGmGmGmAmGfGmUdGmAfAmGfUmUmGmAmUmG*mG*mU |
| BBD-2018AM1 | mU*fA*mUmCmUmGmGmGmAfGmGdTmGfAmAfGmUmUmGmAmU*mG*mG |
| BBD-2019AM1 | mA*fG*mAmUmCmUmGmGmGfAmGdGmUfGmAfAmGmUmUmGmA*mU*mG |
| BBD-2020AM1 | mU*fA*mGmAmUmCmUmGmGfGmAdGmGfUmGfAmAmGmUmUmG*mA*mU |
| BBD-2023AM1 | mA*fG*mGmGmAmGmAmUmCfUmGdGmGfAmGfGmUmGmAmAmG*mU*mU |
| BBD-2042AM1 | mU*fC*mAmCmGmUmCmGmUfAmGdTmCfAmUfGmGmCmUmGmU*mC*mC |
| BBD-2043AM1 | mU*fA*mCmGmUmCmGmUmAfGmUdCmAfUmGfGmCmUmGmUmC*mC*mU |
| BBD-2044AM1 | mA*fC*mGmUmCmGmUmAmGfUmCdAmUfGmGfCmUmGmUmCmC*mU*mC |
| BBD-2047AM1 | mA*fU*mUmGmUmAmGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2048AM1 | mA*fG*mUmUmGmUmAmGmUfAmAdGmUfUmCfCmCmAmGmGmC*mG*mG |
| BBD-2049AM1 | mU*fA*mGmUmUmGmUmAmGfUmAdAmGfUmUfCmCmCmAmGmG*mC*mG |
| BBD-2050AM1 | mU*fG*mAmGmUmUmGmUmAfGmUdAmAfGmUfUmCmCmCmAmG*mG*mC |
| BBD-2051AM1 | mU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2056AM1 | mU*fG*mAmGmCmUmGmGmAfGmUdTmGfUmAfGmUmAmAmGmU*mU*mC |
| BBD-2057AM1 | mU*fG*mGmAmGmCmUmGmGfAmGdTmUfGmUfAmGmUmAmAmG*mU*mU |
| BBD-2058AM1 | mA*fC*mGmGmAmGmCmUmGfGmAdGmUfUmGfUmAmGmUmAmA*mG*mU |
| BBD-2059AM1 | mU*fA*mCmGmGmAmGmCmUfGmGdAmGfUmUfGmUmAmGmUmA*mA*mG |
| BBD-2062AM1 | mA*fU*mAmGmAmCmGmGmAfGmCdTmGfGmAfGmUmUmGmUmA*mG*mU |
| BBD-2064AM1 | mU*fA*mAmUmAmGmAmCmGfGmAdGmCfUmGfGmAmGmUmUmG*mU*mA |
| BBD-2074AM1 | mA*fG*mCmGmUmCmAmGmGfUmUdCmAfCmUfUmCmAmCmAmG*mG*mC |
| BBD-2078AM1 | mU*fU*mAmUmCmAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078AM2 | VPU*fU*mAmUisoGNA-C mAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078AM3 | VPU*fU*mAmUmCisoGNA-A mCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078AM4 | VPU*fU*mAmUmCmAisoGNA-C mCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2083AM1 | mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2084AM1 | mU*fA*mGmGmAmAmAmUmAfCmCdAmGfAmGfUmAmGmCmAmC*mC*mC |
| BBD-2085AM1 | mA*fG*mGmAmAmAmUmAmCfCmAdGmAfGmUfAmGmCmAmCmC*mC*mC |
| BBD-2086AM1 | mU*fG*mAmAmAmUmAmCmCfAmGdAmGfUmAfGmCmAmCmCmC*mC*mC |
| BBD-2087AM1 | mU*fA*mAmAmUmAmCmCmAfGmAdGmUfAmGfCmAmCmCmCmC*mC*mG |
| BBD-2088AM1 | mA*fA*mAmUmAmCmCmAmGfAmGdTmAfGmCfAmCmCmCmCmC*mG*mC |
| BBD-2089AM1 | mA*fA*mUmAmCmCmAmGmAfGmUdAmGfCmAfCmCmCmCmCmG*mC*mC |
| BBD-2090AM1 | mC*fC*mUmAmGmGmAmAmAfUmAdCmCfAmGfAmGmUmAmGmC*mA*mC |
| BBD-2091AM1 | mC*fC*mCmUmAmGmGmAmAfAmUdAmCfCmAfGmAmGmUmAmG*mC*mA |
| BBD-2092AM1 | mA*fC*mCmCmUmAmGmGmAfAmAdTmAfCmCfAmGmAmGmUmA*mG*mC |
表5描述了修飾siRNA雙鏈序列
| 雙鏈體名稱 | 修飾正義鏈名稱 | 修飾正義鏈序列5'-3' | 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| TMPRSS6-hcm-9 | TMPRSS6-hcm-9B | fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG*mU*fU | TMPRSS6-hcm-9A | mA*fA*mCfCmAfGmAfAmGfArnAfGmCfAmGfGmU*fG*mA |
| BBD-2003.11 | BBD-2003SM1 | mG*mU*mGmAmUmCfCmUdGfGfAmAfGmCmCmAmGmUmGmUmA | BBD-2003AM1 | mU*fA*mCmAmCmUmGmGmCfUmUdCmCfAmGfGmAmUmCmAmC*mU*mA |
| BBD-2005.11 | BBD-2005SM1 | mG*mA*mUmCmCmUfGmGdAfAfGmCfCmAmGmUmGmUmGmAmA | BBD-2005AM1 | mU*fU*mCmAmCmAmCmUmGfGmCdTmUfCmCfAmGmGmAmUmC*mA*mC |
| BBD-2013.11 | BBD-2013SM1 | mU*mC*mAmCmCmAfUmCdAfAfCmUfUmCmAmCmCmUmCmCmA | BBD-2013AM1 | mU*fG*mGmAmGmGmUmGmAfAmGdTmUfGmAfUmGmGmUmGmA*mU*mC |
| BBD-2014.11 | BBD-2014SM1 | mC*mA*mCmCmAmUfCmAdAfCfUmUfCmAmCmCmUmCmCmCmA | BBD-2014AM1 | mU*fG*mGmGmAmGmGmUmGfAmAdGmUfUmGfAmUmGmGmUmG*mA*mU |
| BBD-2015.11 | BBD-2015SM1 | mA*mC*mCmAmUmCfAmAdCfUfUmCfAmCmCmUmCmCmCmAmA | BBD-2015AM1 | mU*fU*mGmGmGmAmGmGmUfGmAdAmGfUmUfGmAmUmGmGmU*mG*mA |
| BBD-2016.11 | BBD-2016SM1 | mC*mC*mAmUmCmAfAmCdTfUfCmAfCmCmUmCmCmCmAmGmA | BBD-2016AM1 | mU*fC*mUmGmGmGmAmGmGfUmGdAmAfGmUfUmGmAmUmGmG*mU*mG |
| BBD-2017.11 | BBD-2017SM1 | mC*mA*mUmCmAmAfCmUdTfCfAmCfCmUmCmCmCmAmGmAmU | BBD-2017AM1 | mA*fU*mCmUmGmGmGmAmGfGmUdGmAfAmGfUmUmGmAmUmG*mG*mU |
| BBD-2018.11 | BBD-2018SM1 | mA*mU*mCmAmAmCfUmUdCfAfCmCfUmCmCmCmAmGmAmUmA | BBD-2018AM1 | mU*fA*mUmCmUmGmGmGmAfGmGdTmGfAmAfGmUmUmGmAmU*mG*mG |
| BBD-2019.11 | BBD-2019SM1 | mU*mC*mAmAmCmUfUmCdAfCfCmUfCmCmCmAmGmAmUmCmU | BBD-2019AM1 | mA*fG*mAmUmCmUmGmGmGfAmGdGmUfGmAfAmGmUmUmGmA*mU*mG |
| BBD-2020.11 | BBD-2020SM1 | mC*mA*mAmCmUmUfCmAdCfCfUmCfCmCmAmGmAmUmCmUmA | BBD-2020AM1 | mU*fA*mGmAmUmCmUmGmGfGmAdGmGfUmGfAmAmGmUmUmG*mA*mU |
| BBD-2023.11 | BBD-2023SM1 | mC*mU*mUmCmAmCfCmUdCfCfCmAfGmAmUmCmUmCmCmCmU | BBD-2023AM1 | mA*fG*mGmGmAmGmAmUmCfUmGdGmGfAmGfGmUmGmAmAmG*mU*mU |
| BBD-2042.11 | BBD-2042SM1 | mA*mC*mAmGmCmCfAmUdGfAfCmUfAmCmGmAmCmGmUmGmA | BBD-2042AM1 | mU*fC*mAmCmGmUmCmGmUfAmGdTmCfAmUfGmGmCmUmGmU*mC*mC |
| BBD-2043.11 | BBD-2043SM1 | mG*mA*mCmAmGmCfCmAdTfGfAmCfUmAmCmGmAmCmGmUmA | BBD-2043AM1 | mU*fA*mCmGmUmCmGmUmAfGmUdCmAfUmGfGmCmUmGmUmC*mC*mU |
| BBD-2044.11 | BBD-2044SM1 | mG*mG*mAmCmAmGfCmCdAfUfGmAfCmUmAmCmGmAmCmGmU | BBD-2044AM1 | mA*fC*mGmUmCmGmUmAmGfUmCdAmUfGmGfCmUmGmUmCmC*mU*mC |
| BBD-2047.11 | BBD-2047SM1 | mC*mG*mCmCmUmGfGmGdAfAfCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM1 | mA*fU*mUmGmUmAmGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2048.11 | BBD-2048SM1 | mG*mC*mCmUmGmGfGmAdAfCfUmUfAmCmUmAmCmAmAmCmU | BBD-2048AM1 | mA*fG*mUmUmGmUmAmGmUfAmAdGmUfUmCfCmCmAmGmGmC*mG*mG |
| BBD-2049.11 | BBD-2049SM1 | mC*mC*mUmGmGmGfAmAdCfUfUmAfCmUmAmCmAmAmCmUmA | BBD-2049AM1 | mU*fA*mGmUmUmGmUmAmGfUmAdAmGfUmUfCmCmCmAmGmG*mC*mG |
| BBD-2050.11 | BBD-2050SM1 | mC*mU*mGmGmGmAfAmCdTfUfAmCfUmAmCmAmAmCmUmCmA | BBD-2050AM1 | mU*fG*mAmGmUmUmGmUmAfGmUdAmAfGmUfUmCmCmCmAmG*mG*mC |
| BBD-2051.11 | BBD-2051SM1 | mU*mG*mGmGmAmAfCmUdTfAfCmUfAmCmAmAmCmUmCmCmA | BBD-2051AM1 | mU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2056.11 | BBD-2056SM1 | mA*mC*mUmUmAmCfUmAdCfAfAmCfUmCmCmAmGmCmUmCmA | BBD-2056AM1 | mU*fG*mAmGmCmUmGmGmAfGmUdTmGfUmAfGmUmAmAmGmU*mU*mC |
| BBD-2057.11 | BBD-2057SM1 | mC*mU*mUmAmCmUfAmCdAfAfCmUfCmCmAmGmCmUmCmCmA | BBD-2057AM1 | mU*fG*mGmAmGmCmUmGmGfAmGdTmUfGmUfAmGmUmAmAmG*mU*mU |
| BBD-2058.11 | BBD-2058SM1 | mU*mU*mAmCmUmAfCmAdAfCfUmCfCmAmGmCmUmCmCmGmU | BBD-2058AM1 | mA*fC*mGmGmAmGmCmUmGfGmAdGmUfUmGfUmAmGmUmAmA*mG*mU |
| BBD-2059.11 | BBD-2059SM1 | mU*mA*mCmUmAmCfAmAdCfUfCmCfAmGmCmUmCmCmGmUmA | BBD-2059AM1 | mU*fA*mCmGmGmAmGmCmUfGmGdAmGfUmUfGmUmAmGmUmA*mA*mG |
| BBD-2062.11 | BBD-2062SM1 | mU*mA*mCmAmAmCfUmCdCfAfGmCfUmCmCmGmUmCmUmAmU | BBD-2062AM1 | mA*fU*mAmGmAmCmGmGmAfGmCdTmGfGmAfGmUmUmGmUmA*mG*mU |
| BBD-2064.11 | BBD-2064SM1 | mC*mA*mAmCmUmCfCmAdGfCfUmCfCmGmUmCmUmAmUmUmA | BBD-2064AM1 | mU*fA*mAmUmAmGmAmCmGfGmAdGmCfUmGfGmAmGmUmUmG*mU*mA |
| BBD-2074.11 | BBD-2074SM1 | mC*mU*mGmUmGmAfAmGdTfGfAmAfCmCmUmGmAmCmGmCmU | BBD-2074AM1 | mA*fG*mCmGmUmCmAmGmGfUmUdCmAfCmUfUmCmAmCmAmG*mG*mC |
| BBD-2078.11 | BBD-2078SM1 | mU*mC*mGmCmUmGfAmCdCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM1 | mU*fU*mAmUmCmAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.12 | BBD-2078SM1 | mU*mC*mGmCmUmGfAmCdCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM2 | VPU*fU*mAmUisoGNA-C mAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.13 | BBD-2078SM1 | mU*mC*mGmCmUmGfAmCdCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM3 | VPU*fU*mAmUmCisoGNA-A mCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.14 | BBD-2078SM1 | mU*mC*mGmCmUmGfAmCdCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM4 | VPU*fU*mAmUmCmAisoGNA-C mCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.21 | BBD-2078SM2 | mU*mC*mGmCmUmGfAmCfCfGdCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM1 | mU*fU*mAmUmCmAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.22 | BBD-2078SM2 | mU*mC*mGmCmUmGfAmCfCfGdCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM2 | VPU*fU*mAmUisoGNA-C mAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.23 | BBD-2078SM2 | mU*mC*mGmCmUmGfAmCfCfGdCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM3 | VPU*fU*mAmUmCisoGNA-A mCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.24 | BBD-2078SM2 | mU*mC*mGmCmUmGfAmCfCfGdCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM4 | VPU*fU*mAmUmCmAisoGNA-C mCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.31 | BBD-2078SM3 | mU*mC*mGmCmUmGfAmCfCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM1 | mU*fU*mAmUmCmAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.32 | BBD-2078SM3 | mU*mC*mGmCmUmGfAmCfCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM2 | VPU*fU*mAmUisoGNA-C mAmCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.33 | BBD-2078SM3 | mU*mC*mGmCmUmGfAmCfCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM3 | VPU*fU*mAmUmCisoGNA-A mCmCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2078.34 | BBD-2078SM3 | mU*mC*mGmCmUmGfAmCfCfGfCmUfGmGmGmUmGmAmUmAmA | BBD-2078AM4 | VPU*fU*mAmUmCmAisoGNA-C mCmCfAmGdCmGfGmUfCmAmGmCmGmA*mU*mG |
| BBD-2083.11 | BBD-2083SM1 | mU*mG*mCmUmAmCfUmCdTfGfGmUfAmUmUmUmCmCmUmAmA | BBD-2083AM1 | mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2084.11 | BBD-2084SM1 | mG*mU*mGmCmUmAfCmUfCfUfGmGmUmAmUmUmUmCmCmUmA | BBD-2084AM1 | mU*fA*mGmGmAmAmAmUmAfCmCdAmGfAmGfUmAmGmCmAmC*mC*mC |
| BBD-2085.11 | BBD-2085SM1 | mG*mG*mUmGmCmUfAmCfUfCfUmGmGmUmAmUmUmUmCmCmU | BBD-2085AM1 | mA*fG*mGmAmAmAmUmAmCfCmAdGmAfGmUfAmGmCmAmCmC*mC*mC |
| BBD-2086.11 | BBD-2086SM1 | mG*mG*mGmUmGmCfUmAfCfUfCmUmGmGmUmAmUmUmUmCmA | BBD-2086AM1 | mU*fG*mAmAmAmUmAmCmCfAmGdAmGfUmAfGmCmAmCmCmC*mC*mC |
| BBD-2087.11 | BBD-2087SM1 | mG*mG*mGmGmUmGfCmUfAfCfUmCmUmGmGmUmAmUmUmUmA | BBD-2087AM1 | mU*fA*mAmAmUmAmCmCmAfGmAdGmUfAmGfCmAmCmCmCmC*mC*mG |
| BBD-2088.11 | BBD-2088SM1 | mG*mG*mGmGmGmUfGmCfUfAfCmUmCmUmGmGmUmAmUmUmU | BBD-2088AM1 | mA*fA*mAmUmAmCmCmAmGfAmGdTmAfGmCfAmCmCmCmCmC*mG*mC |
| BBD-2089.11 | BBD-2089SM1 | mC*mG*mGmGmGmGfUmGfCfUfAmCmUmCmUmGmGmUmAmUmU | BBD-2089AM1 | mA*fA*mUmAmCmCmAmGmAfGmUdAmGfCmAfCmCmCmCmCmG*mC*mC |
| BBD-2090.11 | BBD-2090SM1 | mG*mC*mUmAmCmUfCmUfGfGfUmAmUmUmUmCmCmUmAmGmG | BBD-2090AM1 | mC*fC*mUmAmGmGmAmAmAfUmAdCmCfAmGfAmGmUmAmGmC*mA*mC |
| BBD-2091.11 | BBD-2091SM1 | mC*mU*mAmCmUmCfUmGfGfUfAmUmUmUmCmCmUmAmGmGmG | BBD-2091AM1 | mC*fC*mCmUmAmGmGmAmAfAmUdAmCfCmAfGmAmGmUmAmG*mC*mA |
| BBD-2092.11 | BBD-2092SM1 | mU*mA*mCmUmCmUfGmGfUfAfUmUmUmCmCmUmAmGmGmGmU | BBD-2092AM1 | mA*fC*mCmCmUmAmGmGmAfAmAdTmAfCmCfAmGmAmGmUmA*mG*mC |
表6為Hep3B細胞中修飾siRNA雙鏈在轉染1nM濃度下篩選之實驗結果
| siRNA雙鏈體 | Hep3B細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 1nM平均值 | SD | |
| TMPRSS6-hcm-9 | 47.00 | 16.38 |
| BBD-2003.11 | 81.59 | 1.17 |
| BBD-2005.11 | 58.15 | 1.80 |
| BBD-2013.11 | 64.28 | 12.64 |
| BBD-2014.11 | 72.25 | 10.82 |
| BBD-2015.11 | 57.40 | 2.93 |
| BBD-2016.11 | 94.41 | 4.46 |
| BBD-2017.11 | 54.37 | 7.90 |
| BBD-2018.11 | 62.84 | 6.74 |
| BBD-2019.11 | 60.63 | 8.20 |
| BBD-2020.11 | 62.93 | 2.90 |
| BBD-2023.11 | 66.26 | 15.88 |
| BBD-2042.11 | 80.91 | 1.56 |
| BBD-2043.11 | 53.02 | 4.03 |
| BBD-2044.11 | 81.53 | 17.27 |
| BBD-2047.11 | 35.02 | 9.98 |
| BBD-2048.11 | 42.86 | 6.25 |
| BBD-2049.11 | 56.69 | 23.22 |
| BBD-2050.11 | 41.65 | 11.26 |
表7為Hep3B細胞中修飾siRNA雙鏈在轉染10nM濃度下篩選之實驗結果。
| siRNA雙鏈體 | Hep3B細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 10nM平均值 | SD | |
| TMPRSS6-hcm-9 | 24.30 | 3.93 |
| BBD-2005.11 | 36.69 | 4.05 |
| BBD-2015.11 | 32.09 | 8.36 |
| BBD-2017.11 | 32.70 | 2.87 |
| BBD-2019.11 | 33.95 | 5.49 |
| BBD-2043.11 | 43.49 | 2.69 |
| BBD-2047.11 | 20.24 | 1.84 |
| BBD-2048.11 | 23.29 | 2.37 |
| BBD-2049.11 | 24.35 | 4.23 |
| BBD-2050.11 | 22.60 | 2.49 |
| BBD-2051.11 | 19.69 | 2.09 |
| BBD-2056.11 | 34.01 | 8.13 |
| BBD-2057.11 | 35.74 | 4.25 |
| BBD-2058.11 | 31.67 | 6.16 |
| BBD-2059.11 | 26.23 | 1.98 |
| BBD-2062.11 | 20.22 | 2.50 |
| BBD-2064.11 | 15.02 | 1.88 |
| BBD-2074.11 | 35.48 | 1.93 |
| BBD-2078.11 | 18.56 | 1.59 |
| BBD-2083.11 | 25.09 | 3.81 |
| BBD-2084.11 | 61.88 | 9.44 |
| BBD-2085.11 | 67.91 | 3.21 |
| BBD-2086.11 | 22.26 | 3.33 |
| BBD-2087.11 | 26.69 | 1.43 |
| BBD-2088.11 | 49.22 | 3.00 |
| BBD-2089.11 | 45.02 | 4.75 |
| BBD-2090.11 | 104.19 | 16.66 |
| BBD-2091.11 | 155.06 | 15.06 |
| BBD-2092.11 | 75.44 | 2.58 |
接下來對上述活性比較好之BBD-2051.11、BBD-2083.11、BBD-2047.11三條序列分別進行進一步之修飾，以找到最適合之修飾方法。
表8描述了多條BBD-2051修飾正義鏈序列。
| 修飾正義鏈名稱 | 修飾正義鏈序列5'-3' |
| BBD-2051SM1 | mU*mG*mGmGmAmAfCmUdTfAfCmUfAmCmAmAmCmUmCmCmA |
| BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA |
| BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA |
| BBD-2051SM4 | mU*mG*mGmGmAmAfCmUfUdAfCmUmAmCmAmAmCmUmCmCmA |
| BBD-2051SM5 | mU*mG*mGmGmAmAfCmUdTfAfCmUmAmCmAmAmCmUmCmCmA |
表9描述了多條BBD-2051修飾反義鏈序列。
| 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| BBD-2051AM1 | mU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM2 | mU*fG*mGmAmGmUmUmGmUfAdGmUmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM3 | mU*fG*mGmAmGmUmUmGmUfAmGmUdAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM4 | mU*fG*mGmAmGmUmUmGmUfAmGmUmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM5 | mU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM6 | mU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM7 | mU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM8 | VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM9 | VPU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM10 | VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM11 | VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM12 | mU*dG*mGmAmGmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM13 | mU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM14 | mU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM15 | mU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM16 | VPU*dG*mGmAmGmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM17 | VPU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM18 | VPU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051AM19 | VPU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
表10描述了多條 BBD-2051修飾siRNA雙鏈序列
| 雙鏈體名稱 | 修飾正義鏈名稱 | 修飾正義鏈序列5'-3' | 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| BBD-2051.21 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM1 | mU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.25 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM5 | mU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.26 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM6 | mU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.27 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM7 | mU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.28 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM8 | VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.29 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM9 | VPU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.210 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM10 | VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.211 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM11 | VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.212 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM12 | mU*dG*mGmAmGmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.213 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM13 | mU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.214 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM14 | mU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.215 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM15 | mU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.216 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM16 | VPU*dG*mGmAmGmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.217 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM17 | VPU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.218 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM18 | VPU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.219 | BBD-2051SM2 | mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM19 | VPU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.31 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM1 | mU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.35 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM5 | mU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.36 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM6 | mU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.37 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM7 | mU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.38 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM8 | VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.39 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM9 | VPU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.310 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM10 | VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.311 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM11 | VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.312 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM12 | mU*dG*mGmAmGmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.313 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM13 | mU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.314 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM14 | mU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.315 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM15 | mU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.316 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM16 | VPU*dG*mGmAmGmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.317 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM17 | VPU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.318 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM18 | VPU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
| BBD-2051.319 | BBD-2051SM3 | mU*mG*mGmGmAmAfCmUfUfAdCmUmAmCmAmAmCmUmCmCmA | BBD-2051AM19 | VPU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG |
實施例 3 ：將hTMPRSS6（人源TMPRSS6）基因整合到肝靶向之AVV8表達載體，製備病毒[VectorBuilder，AAV8LP]後感染小鼠，獲得穩定表達hTMPRSS6之轉基因小鼠。
小鼠原代肝細胞提取：藉由下腔靜脈灌注法，用膠原酶消化提取小鼠肝細胞。經組織細胞濾網（BIOLOGIX，15-1070）過濾， 得到活性良好之小鼠原代肝細胞，重懸於DMEM培養基+10%FBS+1X青黴素鏈黴素中，用Scepter自動細胞計數儀（Millipore）測定細胞密度。
96孔板轉染：在小鼠原代肝細胞中進行體外實驗，在96孔板中分別將siRNA、Opti-MEM和INTERFERin (Polyplus transfection)混合並置於室溫孵育10分鐘（自由攝取方式siRNA則無需添加INTERFERin），然後每孔加入含有小鼠原代肝細胞之完全培養基。將96孔板放於37℃，5%CO
2之培養箱中孵育24小時。
96孔板RNA提取、反轉：使用Dynabeads mRNA DIRECT試劑盒（Ambion）提取96孔板細胞mRNA，將96孔板中培養基吸走，用DPBS清洗一次，每孔加入50-300 μl細胞裂解液，再加入20-100 μl beads,在振盪器上震盪後，將96孔板置於磁分離架上，吸走孔內裂解液，每孔加入50-300 μl之清洗緩衝液A，吹打後置於磁分離架上，吸走清洗緩衝液A，然後用清洗緩衝液B將beads吹起，轉移到新之96孔板，置於磁分離架上，吸走清洗緩衝液B，再用緩衝液B將beads吹起轉移到96孔PCR板中，同時配好反轉錄試劑，將96孔PCR板置於磁分離架上，吸走清洗緩衝液B，每孔加入20 μl之反轉試劑，用封板膜封板後在PCR儀上進行25℃孵育10分鐘，然後37℃孵育兩小時後85℃，5分鐘，降溫至4°C，反轉錄結束。
即時螢光定量PCR：反轉結束後將96孔板置於磁分離架上直到beads都吸附在底部，吸走反轉試劑，將配好之QPCR體系加入到96孔PCR板中，封板膜封板，在StepOnePlus之即時PCR系統(applied biosystems)上進行PCR。採用ΔΔCt方法分析資料，並採用1nM AD-1955轉染之細胞進行測試標準化。
表11為在表達hTMPRSS6之小鼠原代肝細胞中多條BBD-2051修飾siRNA雙鏈序列藉由Galnac遞送在1nM濃度下篩選之實驗結果。
| siRNA雙鏈體 | hTMPRSS6 小鼠原代肝細胞中 mRNA水準(%) (相對於陰性對照細胞) | |
| 1nM平均值 | SD | |
| BBD-2051.31-Galnac | 68.10 | 21.32 |
| BBD-2051.35-Galnac | 5.33 | 3.94 |
| BBD-2051.36-Galnac | 24.19 | 5.96 |
| BBD-2051.37-Galnac | 17.61 | 2.60 |
| BBD-2051.21-Galnac | 31.55 | 4.88 |
| BBD-2051.25-Galnac | 0.65 | 0.10 |
| BBD-2051.26-Galnac | 3.03 | 0.85 |
表12為Hep3B細胞中BBD-2051修飾siRNA雙鏈序列在轉染1nM濃度下篩選之實驗結果。
| siRNA雙鏈體 | Hep3B細胞中mRNA水準(%)(相對於陰性對照細胞) | |
| 1nM平均值 | SD | |
| BBD-2051.21-Galnac | 20.05 | 6.29 |
| BBD-2051.25-Galnac | 12.77 | 1.47 |
| BBD-2051.26-Galnac | 13.90 | 2.50 |
| BBD-2051.27-Galnac | 15.63 | 3.54 |
| BBD-2051.29-Galnac | 14.90 | 3.23 |
| BBD-2051.210-Galnac | 15.23 | 1.88 |
| BBD-2051.211-Galnac | 9.40 | 0.51 |
表13為在表達hTMPRSS6之小鼠原代肝細胞中BBD-2051修飾siRNA雙鏈序列藉由Galnac遞送在多個濃度下篩選之實驗結果。
| siRNA雙鏈體 | hTMPRSS6 小鼠原代肝細胞中mRNA水準(%) (相對於陰性對照細胞) | |||||
| 10nM平均值 | SD | 2nM平均值 | SD | 0.4nM平均值 | SD | |
| BBD-2051.29-Galnac | 15.20 | 1.35 | 14.77 | 3.20 | 66.23 | 29.81 |
| BBD-2051.210-Galnac | 1.30 | 0.36 | 15.00 | 3.71 | 51.80 | 6.52 |
| BBD-2051.211-Galnac | 1.77 | 0.49 | 14.53 | 1.46 | 47.47 | 18.07 |
| BBD-2051.28-Galnac | 2.30 | 1.50 | 26.13 | 3.59 | 63.13 | 14.84 |
表14描述了多條BBD-2083修飾正義鏈序列。
| 修飾正義鏈名稱 | 修飾正義鏈序列5'-3' |
| AD-1554911-S | mU*mG*mCmUmAmCmUmCfUfGfGmUmAmUmUmUmCmCmUmAmU |
| BBD-2083SM1 | mU*mG*mCmUmAmCfUmCdTfGfGmUfAmUmUmUmCmCmUmAmA |
| BBD-2083SM2 | mU*mG*mCmUmAmCfUmCfUfGdGmUfAmUmUmUmCmCmUmAmA |
| BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA |
| BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA |
| BBD-2083SM5 | mU*mG*mCmUmAmCfUmCfUdGfGmUmAmUmUmUmCmCmUmAmA |
| BBD-2083SM6 | mU*mG*mCmUmAmCfUmCdTfGfGmUmAmUmUmUmCmCmUmAmA |
表15描述了多條BBD-2083修飾反義鏈序列。
| 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| AD-1554911-A | mA*dT*mAmGdGmAdAmAmUmAmCdCmAfGmAmGmUmAmGmCmA*mC*mC |
| BBD-2083AM1 | mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM2 | mU*fU*mAfGfGfAmAfAmUmAmCmCmAfGmAfGmUfAmGmCmA*mC*mC |
| BBD-2083AM3 | mU*fU*mAmGmGmAmAmAmUfAmCfCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM4 | mU*fU*mAmGmGmAmAmAmUfAmCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM5 | mU*fU*mAmGmGmAmAmAmUmAmCfCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM6 | mU*fU*mAmGmGmAmAmAmUfAfCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM7 | mU*fU*mAmGmGmAmAmAmUmAfCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM8 | mU*fU*mAmGmGmAmAmAmUfAdCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM9 | mU*fU*mAmGmGmAmAmAmUfAmCmCdAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM10 | mU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM11 | mU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM12 | mU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM13 | VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM14 | VPU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM15 | VPU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM16 | VPU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM17 | mU*dT*mAmGmGmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM18 | mU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM19 | mU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM20 | mU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM21 | VPU*dT*mAmGmGmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM22 | VPU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM23 | VPU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083AM24 | VPU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
表16描述了多條BBD-2083修飾siRNA雙鏈序列。
| 雙鏈體名稱 | 修飾正義寡核苷酸名稱 | 修飾正義序列5'-3' | 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| AD-1554911 | AD-1554911-S | mU*mG*mCmUmAmCmUmCfUfGfGmUmAmUmUmUmCmCmUmAmU | AD-1554911-A | mA*dT*mAmGdGmAdAmAmUmAmCdCmAfGmAmGmUmAmGmCmA*mC*mC |
| BBD-2083.21 | BBD-2083SM2 | mU*mG*mCmUmAmCfUmCfUfGdGmUfAmUmUmUmCmCmUmAmA | BBD-2083AM1 | mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.31 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM1 | mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.33 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM3 | mU*fU*mAmGmGmAmAmAmUfAmCfCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.43 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM3 | mU*fU*mAmGmGmAmAmAmUfAmCfCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.34 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM4 | mU*fU*mAmGmGmAmAmAmUfAmCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.35 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM5 | mU*fU*mAmGmGmAmAmAmUmAmCfCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.36 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM6 | mU*fU*mAmGmGmAmAmAmUfAfCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.37 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM7 | mU*fU*mAmGmGmAmAmAmUmAfCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.38 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM8 | mU*fU*mAmGmGmAmAmAmUfAdCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.39 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM9 | mU*fU*mAmGmGmAmAmAmUfAmCmCdAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.44 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM4 | mU*fU*mAmGmGmAmAmAmUfAmCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.54 | BBD-2083SM5 | mU*mG*mCmUmAmCfUmCfUdGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM4 | mU*fU*mAmGmGmAmAmAmUfAmCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.64 | BBD-2083SM6 | mU*mG*mCmUmAmCfUmCdTfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM4 | mU*fU*mAmGmGmAmAmAmUfAmCmCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.310 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM10 | mU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.311 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM11 | mU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.312 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM12 | mU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.313 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM13 | VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.314 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM14 | VPU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.315 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM15 | VPU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.316 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM16 | VPU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.317 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM17 | mU*dT*mAmGmGmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.318 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM18 | mU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.319 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM19 | mU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.320 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM20 | mU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.321 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM21 | VPU*dT*mAmGmGmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.322 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM22 | VPU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.323 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM23 | VPU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.324 | BBD-2083SM3 | mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM24 | VPU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.41 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM1 | mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.410 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM10 | mU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.411 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM11 | mU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.412 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM12 | mU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.413 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM13 | VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.414 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM14 | VPU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.415 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM15 | VPU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.416 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM16 | VPU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.417 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM17 | mU*dT*mAmGmGmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.418 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM18 | mU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.419 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM19 | mU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.420 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM20 | mU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.421 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM21 | VPU*dT*mAmGmGmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.422 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM22 | VPU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.423 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM23 | VPU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| BBD-2083.424 | BBD-2083SM4 | mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA | BBD-2083AM24 | VPU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC |
| AD-1554911 | AD-1554911-S | mU*mG*mCmUmAmCmUmCfUfGfGmUmAmUmUmUmCmCmUmAmU | AD-1554911-A | mA*dT*mAmGdGmAdAmAmUmAmCdCmAfGmAmGmUmAmGmCmA*mC*mC |
表17為在表達hTMPRSS6之小鼠原代肝細胞中多條BBD-2083修飾siRNA雙鏈序列和AD-1554911藉由Galnac遞送在1nM濃度下篩選之實驗結果
| siRNA雙鏈體 | hTMPRSS6 小鼠原代肝細胞中mRNA水準(%)(相對於陰性對照細胞) | |
| 1nM平均值 | SD | |
| TMPRSS6-hcm-9-Galnac | 27.69 | 7.00 |
| BBD-2083.21-Galnac | 19.61 | 6.00 |
| BBD-2083.31-Galnac | 8.71 | 1.13 |
| BBD-2083.33-Galnac | 13.36 | 6.68 |
| BBD-2083.43-Galnac | 19.13 | 8.74 |
| BBD-2083.34-Galnac | 13.53 | 7.78 |
| BBD-2083.44-Galnac | 21.65 | 3.11 |
| BBD-2083.35-Galnac | 13.42 | 6.68 |
| BBD-2083.36-Galnac | 8.63 | 2.61 |
| BBD-2083.37-Galnac | 15.09 | 0.41 |
| BBD-2083.38-Galnac | 38.70 | 10.53 |
| BBD-2083.39-Galnac | 12.75 | 2.51 |
| BBD-2083.54-Galnac | 28.72 | 8.42 |
| BBD-2083.64-Galnac | 30.53 | 7.52 |
| BBD-2083.310-Galnac | 5.95 | 2.20 |
| BBD-2083.311-Galnac | 6.37 | 0.99 |
| BBD-2083.312-Galnac | 14.20 | 3.37 |
| BBD-2083.313-Galnac | 0.77 | 0.16 |
| BBD-2083.41-Galnac | 4.48 | 1.46 |
| BBD-2083.410-Galnac | 7.70 | 1.50 |
| BBD-2083.411-Galnac | 13.35 | 11.73 |
| BBD-2083.412-Galnac | 28.42 | 17.90 |
| BBD-2083.413-Galnac | 0.98 | 0.53 |
| AD-1554911-Galnac | 14.60 | 5.68 |
表18為Hep3B細胞中BBD-2083修飾siRNA雙鏈序列和AD-1554911在轉染1nM濃度下篩選之實驗結果
| siRNA雙鏈體 | Hep3B細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 1nM平均值 | SD | |
| BBD-2083.31-Galnac | 17.27 | 2.21 |
| BBD-2083.38-Galnac | 46.75 | 7.71 |
| BBD-2083.39-Galnac | 45.10 | 5.65 |
| BBD-2083.34-Galnac | 40.95 | 3.94 |
| BBD-2083.44-Galnac | 47.13 | 5.55 |
| BBD-2083.54-Galnac | 53.17 | 9.15 |
| BBD-2083.64-Galnac | 37.84 | 3.27 |
| BBD-2083.310-Galnac | 8.66 | 0.23 |
| BBD-2083.311-Galnac | 13.47 | 3.50 |
| BBD-2083.312-Galnac | 11.08 | 0.50 |
| BBD-2083.41-Galnac | 14.94 | 7.68 |
| AD-1554911-Galnac | 19.46 | 8.51 |
表19為Hep3B細胞中BBD-2083修飾siRNA雙鏈序列和AD-1554911在轉染0.3nM濃度下篩選之實驗結果
| siRNA雙鏈體 | Hep3B細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 0.3nM平均值 | SD | |
| BBD-2083.313-Galnac | 33.48 | 7.71 |
| BBD-2083.413-Galnac | 42.68 | 10.02 |
| BBD-2083.414-Galnac | 43.21 | 11.53 |
| BBD-2083.415-Galnac | 52.25 | 13.02 |
| AD-1554911-Galnac | 59.07 | 4.07 |
藉由上述實驗我們發現，2083這條序列，藉由加入VP修飾後，體外活性得到了大幅加強。
表20描述了多條BBD-2047修飾正義鏈序列。
| 修飾正義鏈名稱 | 修飾正義鏈序列5'-3' |
| BBD-2047SM1 | mC*mG*mCmCmUmGfGmGdAfAfCmUfUmAmCmUmAmCmAmAmU |
| BBD-2047SM2 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU |
| BBD-2047SM3 | mC*fG*mCfCmUfGmGfGmAfAmCfUmUfAmCfUmAfCmAfAmU |
| BBD-2047SM4 | mC*mG*mCmCmUmGfGmGfAfAfCmUfUmAmCmUmAmCmAmAmU |
| BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU |
| BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU |
| BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU |
| BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU |
| BBD-2047SM9 | mC*mG*mCmCmUmGfGmGfAdAfCmUmUmAmCmUmAmCmAmAmU |
| BBD-2047SM10 | mC*mG*mCmCmUmGfGmGdAfAfCmUmUmAmCmUmAmCmAmAmU |
表21描述了多條BBD-2047修飾反義鏈序列。
| 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| BBD-2047AM1 | mA*fU*mUmGmUmAmGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM2 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM3 | fA*mU*fUmGfUmAfGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM4 | mA*fU*mUmGfUmAmGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM5 | fA*mU*fUmGfUmAfGmUmAfAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM6 | fA*mU*fUmGfUmAfGmUfAmAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM7 | fA*mU*fUmGfUmAfGmUfAmAdGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM8 | fA*mU*fUmGfUmAfGmUfAmAdGmUmUfCmCfCmAfGmGfCmG*fG*mG |
| BBD-2047AM9 | fA*mU*fUmGfUmAfGmUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM10 | fA*mU*fUmGfUmAfGmUmAmAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM11 | mA*fU*mUfGfUfAmGfUmAmAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM12 | mA*fU*mUfGfUfAmGfUmAmAdGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM13 | mA*fU*mUfGfUfAmGfUmAfAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM14 | mA*fU*mUfGfUfAmGfUmAmAmGfUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM15 | mA*fU*mUfGfUfAmGfUmAmAfGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM16 | mA*fU*mUfGfUfAmGfUmAfAmGfUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM17 | mA*fU*mUfGfUfAmGfUmAfAfGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM18 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM19 | mA*fU*mUfGfUfAmGfUfAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047AM20 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM21 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGfGmCmG*mG*mG |
| BBD-2047AM22 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM23 | mA*fU*mUmGmUmAmGmUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM24 | mA*fU*mUmGmUmAmGmUmAfAmGfUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM25 | mA*fU*mUmGmUmAmGmUmAfAdGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047AM26 | mA*fU*mUmGmUmAmGmUmAfAmGmUdTfCmCfCmAmGmGmCmG*mG*mG |
表22描述了多條BBD-2047修飾siRNA雙鏈序列。
| 雙鏈體名稱 | 修飾正義鏈名稱 | 修飾正義鏈序列5'-3' | 修飾反義鏈名稱 | 修飾反義鏈序列5'-3' |
| BBD-2047.14 | BBD-2047SM1 | mC*mG*mCmCmUmGfGmGdAfAfCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM4 | mA*fU*mUmGfUmAmGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.13 | BBD-2047SM1 | mC*mG*mCmCmUmGfGmGdAfAfCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM3 | fA*mU*fUmGfUmAfGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.12 | BBD-2047SM1 | mC*mG*mCmCmUmGfGmGdAfAfCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM2 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.42 | BBD-2047SM4 | mC*mG*mCmCmUmGfGmGfAfAfCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM2 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.43 | BBD-2047SM4 | mC*mG*mCmCmUmGfGmGfAfAfCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM3 | fA*mU*fUmGfUmAfGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.53 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM3 | fA*mU*fUmGfUmAfGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.55 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM5 | fA*mU*fUmGfUmAfGmUmAfAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.56 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM6 | fA*mU*fUmGfUmAfGmUfAmAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.57 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM7 | fA*mU*fUmGfUmAfGmUfAmAdGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.58 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM8 | fA*mU*fUmGfUmAfGmUfAmAdGmUmUfCmCfCmAfGmGfCmG*fG*mG |
| BBD-2047.59 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM9 | fA*mU*fUmGfUmAfGmUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.510 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM10 | fA*mU*fUmGfUmAfGmUmAmAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.511 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM11 | mA*fU*mUfGfUfAmGfUmAmAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.512 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM12 | mA*fU*mUfGfUfAmGfUmAmAdGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.513 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM13 | mA*fU*mUfGfUfAmGfUmAfAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.411 | BBD-2047SM4 | mC*mG*mCmCmUmGfGmGfAfAfCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM11 | mA*fU*mUfGfUfAmGfUmAmAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.52 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM2 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.514 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM14 | mA*fU*mUfGfUfAmGfUmAmAmGfUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.516 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM16 | mA*fU*mUfGfUfAmGfUmAfAmGfUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.517 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM17 | mA*fU*mUfGfUfAmGfUmAfAfGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.518 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM18 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.519 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM19 | mA*fU*mUfGfUfAmGfUfAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.520 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM20 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.521 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM21 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGfGmCmG*mG*mG |
| BBD-2047.611 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM11 | mA*fU*mUfGfUfAmGfUmAmAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.612 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM12 | mA*fU*mUfGfUfAmGfUmAmAdGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.613 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM13 | mA*fU*mUfGfUfAmGfUmAfAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.614 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM14 | mA*fU*mUfGfUfAmGfUmAmAmGfUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.615 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM15 | mA*fU*mUfGfUfAmGfUmAmAfGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.616 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM16 | mA*fU*mUfGfUfAmGfUmAfAmGfUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.617 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM17 | mA*fU*mUfGfUfAmGfUmAfAfGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.618 | BBD-2047SM6 | mC*mG*mCmCmUmGfGmGfAmAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM18 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.713 | BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM13 | mA*fU*mUfGfUfAmGfUmAfAmGdTmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.716 | BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM16 | mA*fU*mUfGfUfAmGfUmAfAmGfUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.72 | BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM2 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.723 | BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM23 | mA*fU*mUmGmUmAmGmUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.823 | BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM23 | mA*fU*mUmGmUmAmGmUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.824 | BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM24 | mA*fU*mUmGmUmAmGmUmAfAmGfUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.81 | BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM1 | mA*fU*mUmGmUmAmGmUmAfAmGdTmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.522 | BBD-2047SM5 | mC*mG*mCmCmUmGfGmGfAfAdCmUfUmAmCmUmAmCmAmAmU | BBD-2047AM22 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.718 | BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM18 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.720 | BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM20 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.722 | BBD-2047SM7 | mC*mG*mCmCmUmGfGmGfAfAdCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM22 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.82 | BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM2 | mA*fU*mUfGfUfAmGfUmAmAmGmUmUfCmCfCmAfGmGmCmG*mG*mG |
| BBD-2047.822 | BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM22 | mA*fU*mUfGfUfAmGfUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.923 | BBD-2047SM9 | mC*mG*mCmCmUmGfGmGfAdAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM23 | mA*fU*mUmGmUmAmGmUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.1023 | BBD-2047SM10 | mC*mG*mCmCmUmGfGmGdAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM23 | mA*fU*mUmGmUmAmGmUmAfAmGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.825 | BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM25 | mA*fU*mUmGmUmAmGmUmAfAdGmUmUfCmCfCmAmGmGmCmG*mG*mG |
| BBD-2047.826 | BBD-2047SM8 | mC*mG*mCmCmUmGfGmGfAfAfCmUmUmAmCmUmAmCmAmAmU | BBD-2047AM26 | mA*fU*mUmGmUmAmGmUmAfAmGmUdTfCmCfCmAmGmGmCmG*mG*mG |
表23為在表達hTMPRSS6之小鼠原代肝細胞中多條BBD-2047修飾siRNA雙鏈序列藉由Galnac遞送在10nM濃度下篩選之實驗結果
| siRNA雙鏈體 | hTMPRSS6 小鼠原代肝細胞中 mRNA水準(%)(相對於陰性對照細胞) | |
| 10nM平均值 | SD | |
| TMPRSS6-hcm-9-Galnac | 13.87 | 1.86 |
| BBD-2047.14-Galnac | 6.03 | 1.13 |
| BBD-2047.13-Galnac | 17.90 | 0.79 |
| BBD-2047.12-Galnac | 4.37 | 0.89 |
| BBD-2047.42-Galnac | 4.09 | 0.76 |
| BBD-2047.43-Galnac | 31.04 | 6.71 |
| BBD-2047.53-Galnac | 34.47 | 3.22 |
| BBD-2047.55-Galnac | 18.63 | 1.62 |
| BBD-2047.56-Galnac | 18.57 | 0.32 |
| BBD-2047.57-Galnac | 43.98 | 14.65 |
| BBD-2047.58-Galnac | 48.34 | 12.99 |
| BBD-2047.59-Galnac | 40.37 | 16.07 |
| BBD-2047.510-Galnac | 33.04 | 1.95 |
| BBD-2047.511-Galnac | 4.22 | 0.49 |
| BBD-2047.512-Galnac | 6.08 | 2.02 |
| BBD-2047.513-Galnac | 4.25 | 1.89 |
表24為在表達hTMPRSS6之小鼠原代肝細胞中多條BBD-2047修飾siRNA雙鏈序列藉由Galnac遞送在5nM濃度下篩選之實驗結果
| siRNA雙鏈體 | hTMPRSS6 小鼠原代肝細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 5nM平均值 | SD | |
| TMPRSS6-hcm-9-Galnac | 20.97 | 1.26 |
| BBD-2047.42-Galnac | 5.78 | 1.91 |
| BBD-2047.411-Galnac | 9.79 | 6.34 |
| BBD-2047.52-Galnac | 2.88 | 1.51 |
| BBD-2047.511-Galnac | 17.68 | 6.29 |
| BBD-2047.513-Galnac | 6.11 | 3.97 |
| BBD-2047.514-Galnac | 15.70 | 6.19 |
| BBD-2047.516-Galnac | 7.50 | 1.83 |
| BBD-2047.517-Galnac | 19.90 | 0.76 |
| BBD-2047.518-Galnac | 0.83 | 0.77 |
| BBD-2047.519-Galnac | 2.08 | 0.60 |
| BBD-2047.520-Galnac | 1.25 | 1.62 |
| BBD-2047.521-Galnac | 3.41 | 1.75 |
| BBD-2047.611-Galnac | 12.77 | 3.84 |
| BBD-2047.612-Galnac | 29.73 | 1.21 |
| BBD-2047.613-Galnac | 32.68 | 6.36 |
| BBD-2047.614-Galnac | 23.82 | 11.44 |
| BBD-2047.615-Galnac | 33.94 | 9.72 |
| BBD-2047.616-Galnac | 22.24 | 12.59 |
| BBD-2047.617-Galnac | 29.30 | 4.28 |
| BBD-2047.618-Galnac | 30.85 | 12.61 |
| BBD-2047.713-Galnac | 2.93 | 1.50 |
| BBD-2047.716-Galnac | 2.72 | 2.84 |
| BBD-2047.72-Galnac | 1.18 | 0.61 |
| BBD-2047.723-Galnac | 0.67 | 0.37 |
| BBD-2047.823-Galnac | 1.50 | 0.62 |
| BBD-2047.824-Galnac | 0.73 | 0.15 |
| BBD-2047.81-Galnac | 3.91 | 1.12 |
表25為在表達hTMPRSS6之小鼠原代肝細胞中多條BBD-2047修飾siRNA雙鏈序列藉由Galnac遞送在1nM濃度下篩選之實驗結果
| siRNA雙鏈體 | hTMPRSS6 小鼠原代肝細胞中 mRNA水準(%)(相對於陰性對照細胞) | |
| 1nM平均值 | SD | |
| TMPRSS6-hcm-9-Galnac | 27.69 | 8.97 |
| BBD-2047.52-Galnac | 9.02 | 7.06 |
| BBD-2047.522-Galnac | 13.78 | 8.71 |
| BBD-2047.72-Galnac | 10.44 | 3.25 |
| BBD-2047.718-Galnac | 16.27 | 3.47 |
| BBD-2047.723-Galnac | 5.28 | 1.79 |
| BBD-2047.720-Galnac | 39.13 | 11.85 |
| BBD-2047.722-Galnac | 7.19 | 3.85 |
| BBD-2047.72-Galnac | 13.88 | 1.70 |
| BBD-2047.82-Galnac | 12.11 | 6.81 |
| BBD-2047.822-Galnac | 11.31 | 2.81 |
| BBD-2047.823-Galnac | 5.27 | 2.68 |
| BBD-2047.824-Galnac | 10.75 | 1.63 |
表26為在表達hTMPRSS6之小鼠原代肝細胞中多條BBD-2047修飾siRNA雙鏈序列藉由Galnac遞送在0.5nM濃度下篩選之實驗結果
| siRNA雙鏈體 | hTMPRSS6 小鼠原代肝細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 0.5nM平均值 | SD | |
| TMPRSS6-hcm-9-Galnac | 57.20 | 16.97 |
| BBD-2047.52-Galnac | 87.16 | 35.19 |
| BBD-2047.518-Galnac | 23.75 | 12.87 |
| BBD-2047.520-Galnac | 26.51 | 12.58 |
| BBD-2047.72-Galnac | 18.07 | 4.88 |
| BBD-2047.716-Galnac | 78.41 | 6.72 |
| BBD-2047.722-Galnac | 28.27 | 28.46 |
| BBD-2047.723-Galnac | 19.50 | 2.07 |
| BBD-2047.823-Galnac | 23.49 | 25.06 |
| BBD-2047.824-Galnac | 10.01 | 3.03 |
表27為Hep3B細胞中BBD-2047修飾siRNA雙鏈序列和TMPRSS6-HCM-9在10nM濃度下篩選之實驗結果
| siRNA雙鏈體 | Hep3B細胞中mRNA水準(%) (相對於陰性對照細胞) | |
| 10nM平均值 | SD | |
| TMPRSS6-hcm-9 | 21.53 | 2.60 |
| BBD-2047.723 | 17.90 | 2.38 |
| BBD-2047.923 | 18.76 | 1.39 |
| BBD-2047.1023 | 18.16 | 1.78 |
實施例
4
評估不同序列在
Hep3B
細胞中抑制
hTMPRSS6
表達之
IC50
。
細胞培養和96孔板轉染：在Hep3B細胞中進行體外實驗，採用MEM+10%FBS+1X青黴素鏈黴素（Gibco, Cat.15070-063）+1X非必需胺基酸（cell cook, Cat.CM1008L）培養基，細胞平鋪面積達80%時用胰蛋白酶消化，並用Scepter自動細胞計數儀（Millipore，#PHCC00000）測定細胞密度，同時在96孔板中將siRNA與Opti-MEM和INTERFERin (Polyplus transfection)混合並置於室溫孵育10分鐘，然後每孔加入含有Hep3B細胞之完全培養基，將96孔板放於37℃，5% CO
2之培養箱中孵育24小時。
siRNA以100nM為最高濃度，3倍梯度稀釋8個濃度點進行試驗。
96孔板RNA提取、反轉：使用Dynabeads mRNA DIRECT試劑盒（Ambion, Cat.61021）提取96孔板細胞mRNA，將96孔板中培養基吸走，用DPBS清洗一次，每孔加入50-300 μl細胞裂解液，再加入20-100 μl beads,在振盪器上震盪，將96孔板置於磁分離架上，吸走孔內裂解液，每孔加入50-300 μl之清洗緩衝液A，吹打後置於磁分離架上，吸走清洗緩衝液A， 然後用清洗緩衝液B將beads吹起，轉移到新之96孔板，置於磁分離架，吸走清洗緩衝液B，再用緩衝液B將beads吹起轉移到96孔PCR板中，同時配製反轉試劑，將96孔PCR板置於磁分離架上，吸走清洗緩衝液B，每孔加入20ul之反轉試劑，用封板膜封板後在PCR儀上進行25℃孵育10分鐘，然後37℃孵育兩小時後85℃，5分鐘，降溫至4°C，反轉錄結束。
即時螢光定量PCR：反轉錄結束後將96孔板置於磁分離架上直到beads都吸附在底部，吸走反轉試劑，將配好之QPCR體系加入到96孔PCR板中，封板膜封板，在StepOnePlus之即時PCR系統(applied biosystems)上進行PCR。
採用ΔΔCt方法分析資料，並採用1nM陰性對照序列轉染之細胞進行測試標準化。計算各濃度點對陰性對照之降低百分比，並藉由S形劑量-反應曲線擬合計算IC
50值。
表28為Hep3B細胞中不同修飾雙鏈序列之活性結果
| siRNA雙鏈體 | HEP3B(n=3) | |||
| BOTTOM | Top | SLOPE | IC 50(nM) | |
| TMPRSS6-hcm-9-Galnac | 27.6 | 93.9 | -1.1 | 0.442 |
| BBD-2083.415-Galnac | 24.0 | = 100.0 | -1.1 | 0.07841 |
| BBD-2051.210-Galnac | 18.8 | = 100.0 | -1.4 | 0.03669 |
實施例
5
在表達
hTMPRSS6
之小鼠中評估不同修飾之
BBD-2047
序列、
BBD-2051
序列和
BBD-2083
序列對肝中
hTMPRSS6
表達影響
1. 腺病毒整合hTMPRSS6
藉由1-10x10
11個純化重組AAV8病毒顆粒感染小鼠，獲得穩定表達hTMPRSS6之轉基因小鼠。
2. 給藥
小鼠注射病毒14天后，將小鼠平均分組，每組4只。siRNA用生理鹽水溶解，皮下注射，給藥劑量1mg/kg和3mg/kg。
3. 取肝檢測
siRNA注射後不同時間點取肝，藉由TRI REAGENT（MRC，貨號：TR118）提取肝組織中之RNA，用PrimeScript RT regent Kit（Takara,貨號：RR047A）將提取之RNA反轉錄為cDNA。配好之QPCR體系加入到96孔PCR板中，封板膜封板，在StepOnePlus之即時PCR系統(applied biosystems)上進行QPCR，檢測hTMPRSS6表達量。
siRNA注射後14天取肝，檢測hTMPRSS6表達量，結果參見圖1，給藥劑量1mg/kg時不同修飾之序列在體內之活性表達不同，多條序列顯示出顯著之小鼠體內降解靶基因效果，其活性比TMPRSS6-hcm-9-Galnac更高。
siRNA注射後不同時間點取肝，檢測hTMPRSS6表達量，結果參見圖2，給藥劑量3mg/kg，BBD-2083.31-Galnac和BBD-2083.313-Galnac之藥效時間持續比TMPRSS6-hcm-9-Galnac和AD-1554911-Galnac藥效時間長，且我們發現在BBD-2083這條序列中加入VP修飾後，其體內活性更高（BBD-2083.313-GalnacVS BBD-2083.31-Galnac）。
siRNA注射後不同時間點取肝，檢測hTMPRSS6表達量，結果參見圖3，給藥劑量3mg/kg,不同修飾BBD-2051之活性均很好，且藥效時間持續比TMPRSS6-hcm-9-Galnac藥效時間長。
實施例
6
在野生型小鼠中評估不同修飾
BBD-2051
序列對肝中
mTMPRSS6
和
mHepcidin1
表達以及血清中鐵含量之影響
野生型小鼠皮下劑量10mg/kg，7、21天取肝，藉由TRI REAGENT（MRC，貨號：TR118）提取肝組織中之RNA，用PrimeScript RT regent Kit（Takara,貨號：RR047A）將提取之RNA反轉錄為cDNA。配好之QPCR體系加入到96孔PCR板中，封板膜封板，在StepOnePlus之即時PCR系統(applied biosystems)上進行QPCR，檢測mTMPRSS6表達量，結果如圖4所示。結果表明，BBD-2051.210和BBD-2051.211可以降低野生型小鼠內源TMPRSS6表達。
野生型小鼠皮下劑量10mg/kg，第7天斷尾取少量血，室溫靜置30分鐘，1000xg離心10分鐘，取上清血清，按照Iron Assay Kit -Colorimetric檢測試劑盒，稀釋血清後加入Reducer solution，37℃培養15 min後繼續加入100 μl Probe Solution， 繼續37°C培養1 h。檢測593 nm處吸光度，根據標準品曲線計算得到血清樣本之鐵含量，結果參考圖5。結果表明，BBD-2051.210和BBD-2051.211可以降低野生型小鼠血清中鐵之含量。
實施例
7
評估不同修飾
之
BBD-2051
序列和
BBD-2083
序列非靶點活性
設計siRNA對應之Off-Target序列，將Off-target序列合成並連接到psiCheck2載體上。psiCheck2-Off-target質粒與siRNA共轉染，24孔板中進行實驗，在轉染前6~8小時，每孔0.5 mL細胞生長培養基中接種細胞。藉由轉染試劑轉染50ng psiCheck2-Off-target 質粒和0.0001nM至100nM siRNA，37 ℃培養24h。用Dual-Luciferase Reporter Assay System（Promega, cat#E1980）檢測海腎螢光和螢火蟲螢光素酶之螢光信號值。計算歸一化值。歸一化值=海腎螢光素酶讀數酶/螢火蟲螢光素酶讀數。相對表達倍數=實驗組歸一化值/對照組歸一化值。
藉由實驗可知，參見圖6，藉由在反義鏈之第5、6和7位引入ISOGNA，體外實驗結果顯示BBD-2051修飾序列（特別系BBD-2051.29，BBD-2051.210）非靶點活性很低，具有良好之安全性。
參見圖7，體外實驗結果顯示BBD-2083修飾序列非靶點活性低，具有良好之安全性。
實施例
8
評估
BBD-2051.210
在血清和肝臟
S9
中之體外代謝穩定性
將小鼠、大鼠、犬、猴和人血清或肝S9與BBD-2051.210-Galnac在體外37 ℃下孵育24小時，評估BBD-2051.210-Galnac之代謝穩定性。藉由實驗資料，結果參見表29，BBD-2051.210-Galnac雙鏈在不同種屬之血清中，反義鏈在血清穩定性為小鼠＞猴子＞大鼠＞人＞犬，正義鏈在血清穩定性為猴子＞大鼠＞小鼠＞人＞犬；結果參見表30，BBD-2051.210-Galnac雙鏈在不同種屬之肝S9中，反義鏈在肝S9穩定性為犬＞人＞猴子＞小鼠＞大鼠，正義鏈在肝S9穩定性為犬＞小鼠＞人＞猴子＞大鼠。
表29 BBD-2051.210-Galnac血清穩定性結果
| 24小時 留存率(%) 血清 | 小鼠 | 大鼠 | 狗 | 猴子 | 人 |
| 反義鏈 | 102.99 | 91.03 | 71.30 | 96.87 | 87.35 |
| 正義鏈 | 94.63 | 95.28 | 71.76 | 98.94 | 87.92 |
表30 BBD-2051.210-Galnac肝S9穩定性結果
| 24小時 留存率(%)肝S9 | 小鼠 | 大鼠 | 狗 | 猴子 | 人 |
| 反義鏈 | 83.47 | 74.37 | 104.55 | 95.17 | 95.31 |
| 正義鏈 | 99.47 | 85.95 | 105.21 | 95.40 | 96.02 |
實施例
9
評估
BBD-2051.210
在肝勻漿中
之體外代謝穩定性
將大鼠肝勻漿與BBD-2051.210-Galnac在體外37 ℃下孵育72小時，評估BBD-2051.210-Galnac之代謝穩定性。BBD-2051.210-Galnac在肝勻漿中孵育72小時後，反義鏈剩餘之百分比為94.98，正義鏈剩餘之百分比54.24。由數據可知，BBD-2051.210-Galnac鏈在肝勻漿中系穩定之。
以上所述實施例僅表達了本發明之幾種實施方式，其描述較為具體和詳細，但並不能因此而理解為對發明專利範圍之限制。應當指出之系，對於本領域之普通技術人員來說，在不脫離本發明構思之前提下，還可以做出若干變形和改進，這些都屬於本發明之保護範圍。因此，本發明專利之保護範圍應以申請專利範圍為准。
無
圖1在表達hTMPRSS6之小鼠中評估不同修飾之BBD-2047序列、BBD-2051序列、BBD-2083序列和TMPRSS6-HCM-9序列對肝中hTMPRSS6表達影響（1mg/kg）。
圖2在表達hTMPRSS6之小鼠中評估不同修飾之BBD-2083序列和和TMPRSS6-HCM-9序列對肝中hTMPRSS6表達影響（3mg/kg）。
圖3在表達hTMPRSS6之小鼠中評估不同修飾之BBD-2051序列和TMPRSS6-HCM-9序列對肝中hTMPRSS6表達影響（3mg/kg）。
圖4在野生型小鼠中評估不同修飾之BBD-2051序列對肝中mTMPRSS6表達影響（10mg/kg）。圖5在野生型小鼠中評估不同修飾之BBD-2051序列對血中鐵含量之表達影響（10mg/kg）。
圖6評估不同修飾之BBD-2051序列非靶點活性。
圖7評估不同修飾之BBD-2083序列和AD-1554911序列非靶點活性。
TW202412821A_112130286_SEQL.xml
Claims (19)
- 一種用於抑制細胞中TMPRSS6表達之siRNA或其藥學上可接受之鹽，其由正義鏈及反義鏈組成，其鹼基序列選自a-e所示中之任意一組： a) BBD-2051，其正義鏈如SEQ ID NO.103所示，反義鏈如SEQ ID NO.104所示； b) BBD-2083，其正義鏈如SEQ ID NO.167所示，反義鏈如SEQ ID NO.168所示； c) BBD-2047，其正義鏈如SEQ ID NO.95所示，反義鏈如SEQ ID NO.96所示； d) BBD-2086，其正義鏈如SEQ ID NO.173所示，反義鏈如SEQ ID NO.174所示； e) BBD-2087，其正義鏈如SEQ ID NO.175所示，反義鏈如SEQ ID NO.176所示。
- 如請求項1所述之siRNA或其藥學上可接受之鹽，其中所述正義鏈包括不多於3、2、1或0個未修飾之核苷酸，所述正義鏈中之修飾之核苷酸包含分別選自2'-O-甲基修飾之核苷酸、2'-去氧核苷酸、2'-氟修飾之核苷酸，所述正義鏈在5'-末端含有1，或2，或3個硫代磷酸酯鍵，所述2'-去氧核苷酸為如下結構中之至少一種： ； 所述反義鏈包括不多於 3、2、1 或0個未修飾之核苷酸，所述反義鏈中之修飾之核苷酸包括分別選自2'-O-甲基修飾之核苷酸、2'-去氧核苷酸、2'-氟修飾之核苷酸、5’端-(E)-乙烯基膦酸核苷酸和異甘油核苷酸，所述反義鏈在5'-末端和3'-末端各含有1-3個硫代磷酸酯鍵；所述異甘油核苷酸(isoGNA)選自如下結構一種： ； 所述尿苷-5’端-(E)-乙烯基膦酸核苷酸之結構如下： 。
- 如請求項2所述之siRNA或其藥學上可接受之鹽，其中所述異甘油核苷酸位於反義鏈，且位於5'端起之4-8位中之至少1位。
- 如請求項1-3任一項所述之siRNA或其藥學上可接受之鹽，其中所述siRNA之至少一條鏈包含具有至少1-3個核苷酸之3'突出端；且互補配對之siRNA反義鏈5'端第11-13位或正義鏈5'端第9-11位共含有1-6個2'-去氧核苷酸。
- 如請求項1-3任一項所述之siRNA或其藥學上可接受之鹽，其中，經過修飾之siRNA選自以下任意一種： BBD-2051.210： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.211： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.28： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2083.313： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.413： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2051.25： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：mU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.26： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：mU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.27： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：mU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.29： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：VPU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.213： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：mU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG； BBD-2051.214： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：mU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.215： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：mU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.217： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：VPU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.218： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：VPU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2051.219： 正義鏈：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA， 反義鏈：VPU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、 BBD-2083.41： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.410： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：mU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.411： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：mU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.412： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：mU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.414： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.415： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.416： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.418： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：mU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.419： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：mU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.420： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：mU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.422： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.423： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、 BBD-2083.424： 正義鏈：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA， 反義鏈：VPU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC。
- 如請求項1所述之siRNA或其藥學上可接受之鹽，其中所述藥學上可接受之鹽為鈉鹽或鉀鹽。
- 一種如請求項1-6任一項所述之siRNA或其藥學上可接受之鹽在製備用於抑制TMPRSS6表達之生物製劑或者藥物製劑中之應用。
- 一種用於抑制TMPRSS6表達之生物製劑或者藥物製劑，其中，包括請求項1至6中任一項所定义之siRNA或其藥學上可接受之鹽作为活性成分，以及與所述siRNA連接或包裹之靶向性遞送配體或遞送載體。
- 如請求項求8所述之生物製劑或者藥物製劑，其中所述配體為N-乙醯基半乳糖胺衍生物；或遞送載體為特異性遞送到肝臟細胞之脂質體、多肽或抗體。
- 如請求項9所述之生物製劑或者藥物製劑，其中所述N-乙醯基半乳糖胺衍生物之結構如下： 或 或 或 。
- 如請求項10所述之生物製劑或者藥物製劑，其中所述N-乙醯基半乳糖胺衍生物與siRNA之正義鏈之3'端連接方式如下： 或 或 或 。
- 一種如請求項8-11任一項所述之生物製劑或者藥物製劑在製備用於抑制細胞中TMPRSS6表達之藥物中之應用。
- 一種如請求項8-11任一項所述之生物製劑或者藥物製劑在製備預防或治療與鐵超負荷或鐵超載相關聯之病症之藥物中之應用。
- 如請求項12或13所述應用，其中所述與TMPRSS6表達相關之疾病或鐵超負荷或鐵超載相關聯之疾病為紅細胞增多症、地中海貧血、血色素沉著症、骨髓增生異常綜合征、遲發性皮膚卟啉症、再生障礙性貧血、鐵粒幼細胞性貧血、鐵難治性缺鐵性貧血、遺傳性貧血、嚴重慢性溶血、帕金森病、阿爾茲海默症、弗裡德賴希氏共濟失調或小細胞貧血、長期輸血導致鐵超載及非酒精性脂肪性肝病（NAFLD）相關之鐵超載。
- 如請求項14所述應用，其中所述地中海貧血為α-地中海貧血、β-地中海貧血或δ-地中海貧血。
- 如請求項14所述應用，其中所述紅細胞增多症系真性紅細胞增多症。
- 如請求項14所述應用，其中所述遺傳性貧血選自鐮狀細胞性貧血、地中海貧血、範可尼貧血、戴-布二氏貧血、舒-戴二氏綜合征、紅細胞膜障礙、葡萄糖-6-磷酸脫氫酶缺乏和遺傳性出血性毛細血管擴張症。
- 一種抑制TMPRSS6在細胞內表達之方法，所述方法包括: (a) 將細胞與請求項1 所述之siRNA或其藥學上可接受之鹽或請求項8 所述之生物製劑或者藥物製劑接觸； 和 (b)將步驟(a)中產生之細胞維持足以獲得TMPRSS6基因之mRNA轉錄本之降解之時間,從而抑制TMPRSS6基因在細胞中之表達。
- 一種治療患有TMPRSS6相關病症或鐵超負荷或鐵超載相關聯之受驗者之方法，包含對該受驗者給藥治療有效量之請求項1 所述之siRNA或其藥學上可接受之鹽或請求項8所述之生物製劑或者藥物製劑，從而治療該受驗者。
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