TW202039843A - 用於抑制pnpla3表現之rnai構建體及其使用方法 - Google Patents
用於抑制pnpla3表現之rnai構建體及其使用方法 Download PDFInfo
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Abstract
本發明係關於用於降低PNPLA3基因表現之RNAi構建體。亦描述使用此等RNAi構建體治療或預防肝病非酒精性脂肪性肝病(NAFLD)之方法。
Description
本發明係關於調節含有patatin樣磷脂酶結構域3(PNPLA3)之肝臟表現的組成物及方法,特別是,本發明係關於用於經由RNA干擾來降低PNPLA3表現之基於核酸的治療劑及使用此等基於核酸的治療劑來治療或預防肝病,例如非酒精性脂肪性肝病(NAFLD)之方法。
非酒精性脂肪性肝病(NAFLD)構成一系列肝臟病變,為世界上最常見的慢性肝病,其患病率在過去20年中翻倍,且如今據估計影響了約20%世界人口(Sattar等人 (2014) BMJ 349:g4596;Loomba及Sanyal (2013) Nature Reviews Gastroenterology & hepatology 10(11):686-690;Kim及Kim (2017) Clin Gastroenterol Hepatol 15(4):474-485;Petta等人 (2016) Dig Liver Dis 48(3):333-342)。NAFLD開始於肝臟中之甘油三酯積累,且定義為個體中超過5%肝細胞中存在細胞質脂滴,該個體1) 無明顯飲酒史且2) 其中其他類型肝病之診斷已被排除在外(Zhu 等人 (2016) World J Gastroenterol 22(36):8226-33;Rinella (2015) JAMA 313(22):2263-73;Yki-Jarvinen (2016) Diabetologia 59(6):1104-11)。在一些個體中,肝臟中異位脂肪之積累(稱為脂肪變性)引發炎症及肝細胞損傷,導致更晚期疾病,稱為非酒精性脂肪性肝炎(NASH)(Rinella,同上)。截至2015年,預計有7500萬至1億美國人患有NAFLD;NASH約佔NAFLD診斷之10%-30%(Rinella, 同上;Younossi等人 (2016) Hepatology 64(5):1577-1586)。
含有patatin樣磷脂酶結構域3(PNPLA3),以前稱為脂聯素(ADPN)及鈣非依賴性磷脂酶A2-ε(iPLA(2)ε),為一種II型跨膜蛋白(Wilson等人 (2006) J Lipid Res 47(9):1940-9;Jenkins等人 (2004) J Biol Chem 279(47):48968-75)。最初在脂肪細胞中鑒定為在小鼠脂肪形成期間誘導的膜相關性脂肪富集蛋白,其現在已充分表徵為在其他組織,包括肝臟中表現(Wilson等人, 同上;Baulande 等人 (2001) J Biol Chem 276(36):33336-44;Moldes等人 (2006) Eur J Endocrinol 155(3):461-8;Faraj等人 (2006) J Endocrinol 191(2):427-35;Liu等人 (2004) J Clin Endocrinol Metab 89(6):2684-9;Lake等人 (2005) J Lipid Res 46(11):2477-87)。在無細胞生化系統中,重組PNPLA3蛋白可以表現出三醯基甘油脂肪酶或轉醯基化活性(Jenkins等人, 同上;Kumari等人(2012) Cell Metab 15(5):691-702;He等人(2010) J Biol Chem 285(9):6706-15)。在肝細胞中,PNPLA3在內質網及脂質膜上表現,且主要表現出三醯基甘油水解酶活性(He等人, 同上;Huang等人 (2010) Proc Natl Acad Sci USA 107(17):7892-7;Ruhanen等人 (2014) J Lipid Res 55(4):739-46;Pingitore等人 (2014) Biochim Biophys Acta 1841(4):574-80)。儘管缺乏分泌信號,但數據表明PNPLA3得以分泌且可在人血漿中作為二硫鍵依賴性多聚體出現(Winberg等人 (2014) Biochem Biophys Res Commun 446(4):1114-9)。因此,靶向PNPLA3功能之新型治療劑代表一種降低PNPLA3水平及治療肝病,例如非酒精性脂肪性肝病的新方法。
本發明部分基於靶向PNPLA3基因且降低肝細胞中PNPLA3表現之RNAi構建體的設計及產生。PNPLA3表現之序列特異性抑制適用於治療或預防與PNPLA3表現相關之病狀,例如肝臟相關疾病,例如單純性脂肪肝(脂肪變性)、非酒精性脂肪性肝炎(NASH)、肝硬化(肝臟不可逆晚期瘢痕形成)、或PNPLA3相關性肥胖。因此,在一個實施方式中,本發明提供一種包含有義鏈及反義鏈之RNAi構建體,其中該反義鏈包含具有與PNPLA3 mRNA序列互補之序列的區域。在某些實施方式中,反義鏈包含具有來自表1或表2中列出之反義序列的至少15個連續核苷酸的區域。在一些實施方式中,相對於不含該等變化之參照等位基因,本發明之RNAi選擇性抑制PNPLA3-rs738409、PNPLA3-rs738408及/或PNPLA3-rs738409-rs738408次要等位基因。
在一些實施方式中,本文所述之RNAi構建體之有義鏈包含與反義鏈之序列充分互補以形成長度為約15至約30個鹼基對的雙鏈體區之序列。在該等及其他實施方式中,有義鏈及反義鏈之長度各為約15至約30個核苷酸。在一些實施方式中,RNAi構建體包含至少一個鈍端。在其他實施方式中,RNAi構建體包含至少一個核苷酸突出端。此類核苷酸突出端可包含至少1至6個未配對核苷酸,且可位於有義鏈3'端、反義鏈3'端或有義鏈及反義鏈3'端。在某些實施方式中,RNAi構建體在有義鏈3'端及反義鏈3'端處包含兩個未配對核苷酸的突出端。在其他實施方式中,RNAi構建體包含在反義鏈3'端處之兩個未配對核苷酸突出端及有義鏈3'端/反義鏈5'端之鈍端。
本發明之RNAi構建體可包含一或多種經修飾之核苷酸,包括對核糖環、核鹼基或磷酸二酯主鏈具有經修飾之核苷酸。在一些實施方式中,RNAi構建體包含一或多種經2'-修飾之核苷酸。此經2'-修飾之核苷酸可包括經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸、經2'-O-甲氧基乙基修飾之核苷酸、經2'-O-烯丙基修飾之核苷酸、雙環核酸(BNA)、乙二醇核酸(GNA)、反向鹼基(例如經倒置之腺苷)或其組合。在一特定實施方式中,RNAi構建體包含一或多個經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸或其組合。在一些實施方式中,RNAi構建體之有義鏈及反義鏈中的所有核苷酸均為經修飾之核苷酸。
在一些實施方式中,RNAi構建體包含至少一個主鏈修飾,例如經修飾之核苷酸間或核苷間鍵。在某些實施方式中,本文所述之RNAi構建體包含至少一個硫代磷酸酯核苷酸間鍵。在特定實施方式中,硫代磷酸酯核苷酸間鍵可位於有義鏈及/或反義鏈之3'端或5'端。
在一些實施方式中,本發明之RNAi構建體之反義鏈及/或有義鏈可包含來自表1或2中所列出之反義及有義序列的序列或由其組成。在某些實施方式中,RNAi構建體可為表1至2中任一者中所列出之任一種雙鏈體化合物。
本發明係關於用於調節含有Patatin樣磷脂酶結構域3(PNPLA3)基因之表現的組成物及方法。在一些實施方式中,基因可處於細胞或受試者內,例如哺乳動物(例如人)。在一些實施方式中,本發明之組成物包含靶向PNPLA3 mRNA且降低細胞或哺乳動物中之PNPLA3表現的RNAi構建體。此類RNAi構建體適用於治療或預防各種形式的肝臟相關疾病,例如單純性脂肪肝(脂肪變性)、非酒精性脂肪性肝炎(NASH)、肝硬化(肝臟不可逆晚期瘢痕形成)或PNPLA3相關性肥胖。
在2008年,一項探索與NAFLD相關之非同義序列變異或單核苷酸多態性(SNP)的全基因組關聯研究(GWAS)將PNPLA3中之變異體(rs738409[G],編碼I148M;可稱為PNPLA3- rs738409、PNPLA3-ma或PNPLA3-次要等位基因)鑒定為與肝臟脂肪含量顯著相關。自該初始報告以來,隨後的GWAS確認PNPLA3 rs738409為NAFLD的主要遺傳決定因素,其與以下顯著相關:1) 肝損傷的血清生物標誌物丙胺酸轉胺酶(ALT)之水平升高,2) NAFLD發病率、進展及嚴重程度,3) 肥胖及瘦型個體,以及4) 顯示與所有NAFLD階段:脂肪變性、NASH、肝硬化及肝細胞癌顯著相關之唯一已知SNP。許多GWAS的共識表明PNPLA3 rs738409與NAFLD之關聯性與年齡、性別、種族、代謝症候群、體重指數、胰島素抵抗及血清脂質無關。此外,來自多個來源之統計分析估計大約50% NAFLD患者攜帶PNPLA3 rs738409突變。對於PNPLA3 rs738409突變,患者可為純合的或雜合的。此外,已發現具有PNPLA3 rs738409突變之患者通常亦攜帶3個鹼基對之rs738408突變(Tian 等人 (2010) Nature Genetics 42:21-23)。因此,患者可具有PNPLA3-rs738409次要等位基因、PNPLA3-rs738408次要等位基因或PNPLA3-rs738409-rs738408雙重次要等位基因突變(PNPLA3-dma)。
研究人員已開發用於在活體內探索PNPLA3功能之小鼠模型。迄今為止,尚無可偵測代謝表型被鑒別為Pnpla3
缺陷或Pnpla3
過表現之結果。相比之下,Pnpla3 I148M
在轉基因小鼠及敲入小鼠中之表現導致肝臟甘油三酯水平增加,類似於NAFLD。因此,總而言之,活體內小鼠模型數據表明突變體Pnpla3 I148M
蛋白之表現,而非野生型蛋白質之過表現,為疾病表型的驅動因素。除了受NAFLD影響之個體中的次要等位基因的高頻率及與該疾病之主要關聯之外,該等發現亦強調PNPLA3 rs738409作為NAFLD之主要治療靶標。
RNA干擾(RNAi)為將外源RNA導入細胞,導致編碼靶向蛋白之mRNA特異性降解,從而導致蛋白質表現降低之過程。RNAi技術及肝臟遞送之進步以及藉由其他基於RNAi之療法產生的不斷增加的積極結果表明,RNAi為藉由直接靶向PNPLA3I148M來治療性治療NAFLD之有力手段。許多GWAS表明PNPLA3 rs738409對NAFLD發病率、進展及嚴重程度(GWAS)之劑量依賴性作用;與雜合子攜帶者相比,對於純合子攜帶者,優勢比傾向於為雙倍(若非更多),但與野生型個體相比,對於雜合子仍然多至少兩倍。因此,使用等位基因辨別特異性使PNPLA3緘默既可為降低PNPLA3I148M攜帶者中之肝甘油三酯之潛在手段,亦可提供雜合子可獲得益處而不使野生型等位基因緘默之情形。沿著該等線路,我們鑒別PNPLA3I148M的SNP特異性短干擾RNA(siRNA)且證明活體外概念證據。使用肝細胞瘤細胞株Hep3B(對於參考等位基因PNPLA3I148I而言為純合的)及HEPG2(對於次要等位基因PNPLA3I148M而言為純合的),我們鑒別能夠特異性抑制PNPLA3I148M基因表現之siRNA序列。藉由篩選過表現PNPLA3I148I或PNPLA3I148M之中國倉鼠卵巢(CHO)細胞證實該等序列之抑制作用。使用在活體內過表現人PNPLA3I148M之腺相關病毒(AAV),然後我們證明用次要等位基因特異性SNP處理不僅特異性地降低小鼠中人PNPLA3I148M之表現,而且亦顯著逆轉藉由過表現人PNPLA3I148M而誘導之肝甘油三酯積累。
如本文所用,術語「RNAi構建體」係指包含RNA分子之試劑,當引入細胞時,該RNA分子能夠經由RNA干擾機制下調靶基因(例如PNPLA3)之表現。RNA干擾為核酸分子以序列特異性方式(例如,經由經RNA誘導之緘默複合物(RISC)途徑)誘導靶RNA分子(例如經由信使RNA或mRNA分子)裂解及降解的過程。在一些實施方式中,RNAi構建體包含雙鏈RNA分子,其包含連續核苷酸的兩條反平行鏈,其彼此充分互補以雜交形成雙鏈體區。「雜交(hybridize)」或「雜交(hybridization)」係指互補多核苷酸之配對,通常經由兩個多核苷酸中互補鹼基之間的氫鍵(例如Watson-Crick、Hoogsteen或反向Hoogsteen氫鍵)。包含具有與靶序列(例如靶mRNA)基本上互補之序列之區域的鏈被稱為「反義鏈」。「有義鏈」係指包括與反義鏈之區域基本上互補之區域的鏈。在一些實施方式中,有義鏈可包含具有與靶序列基本上相同之序列的區域。
在一些實施方式中,本發明係針對PNPLA3之RNAi。在一些實施方式中,本發明係結合在PNPLA3 rs738409位點處之RNAi。在一些實施方式中,本發明係結合PNPLA3 rs738408位點處之RNAi。在一些實施方式中,本發明係結合在PNPLA3 rs738409 rs738408位點處之RNAi。在一些實施方式中,本發明係相對於天然PNPLA3序列(PNPLA3-ref)優先結合PNPLA3 rs738409之RNAi。在一些實施方式中,本發明係相對於PNPLA3-ref序列優先結合PNPLA3 rs738408之RNAi。在一些實施方式中,本發明係相對於PNPLA3-ma優先結合PNPLA3-dma之RNAi。在一些實施方式中,本發明係含有表1或2中可見之任何序列的RNAi分子。
雙鏈RNA分子可包括對核糖核苷酸之化學修飾,包括對核糖核苷酸的核糖、鹼基或主鏈組分之修飾,例如本文所述或此項技術已知彼等者。出於本發明之目的,術語「雙鏈RNA」涵蓋用於雙鏈RNA分子(例如siRNA、shRNA或類似者)中之任何此類修飾。
如本文所用,若包含第一序列之多核苷酸可與包含第二序列之多核苷酸雜交以在某些條件(例如生理條件)下形成雙鏈體區,則第一序列與第二序列「互補」。其他此等條件可包括熟習此項技術者已知之中等或嚴格雜交條件。若在一或兩個核苷酸序列的整個長度上包含第一序列之多核苷酸與包含第二序列之多核苷酸鹼基配對而無任何錯配,則認為第一序列與第二序列完全互補(100%互補)。若序列與靶序列至少約80%、85%、90%、95%、96%、97%、98%、99%或100%互補,則序列與靶序列「基本上互補」。互補百分比可以藉由將第一序列中與第二或靶序列中相應位置處的鹼基互補之鹼基數除以第一序列之總長度來計算。若在兩個序列雜交時在30個鹼基對的雙鏈體區上存在不多於5個、4個、3個、2個或1個錯配,則亦可說序列與另一個序列基本上互補。通常,若存在如本文所定義之任何核苷酸突出端,則在判定兩個序列之間的互補程度時不考慮該等突出端之序列。舉例而言,長度為21個核苷酸之有義鏈及長度為21個核苷酸之反義鏈雜交形成在各鏈3'端具有2個核苷酸突出端之19個鹼基對雙鏈體區,這兩條鏈將被認為是完全互補,如該術語在本文中所使用。
在一些實施方式中,反義鏈之區域包含與靶RNA序列(例如PNPLA3 mRNA)之區域完全互補的序列。在此類實施方式中,有義鏈可包含與反義鏈之序列完全互補的序列。在其他此類實施方式中,有義鏈可包含與反義鏈之序列基本上互補的序列,例如,在由有義鏈及反義鏈形成之雙鏈體區中具有1、2、3、4或5個錯配。在某些實施方式中,較佳在末端區域內發生任何錯配(例如在鏈5'端及/或3'端之6、5、4、3、2或1個核苷酸內)。在一個實施方式中,由有義鏈及反義鏈形成之雙鏈體區中的任何錯配發生在反義鏈5'端之6、5、4、3、2或1個核苷酸內。
在某些實施方式中,雙鏈RNA之有義鏈及反義鏈可為雜交形成雙鏈體區但在其他情況下不連接的兩個單獨分子。由兩條單獨鏈形成之此類雙鏈RNA分子稱為「小干擾RNA」或「短干擾RNA」(siRNA)。因此,在一些實施方式中,本發明之RNAi構建體包含siRNA。
當dsRNA之兩條基本上互補的鏈由單獨RNA分子組成時,彼等分子不需要但可以共價連接。當兩條鏈藉由除形成雙鏈體結構之一條鏈的3'端及相應另一條鏈的5'端之間的不間斷核苷酸鏈以外的方式共價連接時,連接結構被稱為「連接子(linker)」。RNA鏈可具有相同或不同數量的核苷酸。雙鏈體中鹼基對之最大數目為dsRNA之最短鏈中的核苷酸數減去雙鏈體中存在的任何突出端。除雙鏈體結構外,RNAi可包含一或多個核苷酸突出端。
在其他實施方式中,雜交形成雙鏈體區之有義鏈及反義鏈可為單個RNA分子之一部分,即有義鏈及反義鏈為單個RNA分子之自身互補區的一部分。在此類情況下,單個RNA分子包含雙鏈體區(亦稱為莖區)及環區。有義鏈之3'端藉由將形成環區之連續未配對核苷酸序列與反義鏈之5'端連接。環區通常具有足夠的長度以允許RNA分子自身向後折疊,使得反義鏈可以與有義鏈鹼基配對以形成雙鏈體或莖區。環區可包含自約3至約25、自約5至約15、或自約8至約12個未配對核苷酸。此類具有至少部分自身互補區之RNA分子稱為「短髮夾RNA」(shRNA)。在一些實施方式中,環區可包含至少1、2、3、4、5、10、20或25個未配對核苷酸。在一些實施方式中,環區可具有10、9、8、7、6、5、4、3、2或更少未配對核苷酸。在某些實施方式中,本發明之RNAi構建體包含shRNA。單個至少部分自身互補RNA分子之長度可為自約35個核苷酸至約100個核苷酸、自約45個核苷酸至約85個核苷酸、或自約50至約60個核苷酸,且包含各具有本文所述之長度的雙鏈體區及環區。
在一些實施方式中,本發明之RNAi構建體包含有義鏈及反義鏈,其中反義鏈包含具有與PNPLA3信使RNA(mRNA)序列基本上或完全互補之序列的區域。如本文所用,「PNPLA3 mRNA序列」係指任何信使RNA序列,包括編碼PNPLA3蛋白之剪接變異體,包括來自任何物種(例如小鼠、大鼠、非人靈長類動物、人)之PNPLA3蛋白變異體或同功型。PNPLA3蛋白亦稱為脂聯素(ADPN)及鈣非依賴性磷脂酶A2-ε(iPLA(2)ε)。
PNPLA3 mRNA序列亦包括表現為其互補DNA(cDNA)序列之轉錄物序列。cDNA序列係指表現為DNA鹼基(例如鳥嘌呤、腺嘌呤、胸腺嘧啶及胞嘧啶)而非RNA鹼基(例如鳥嘌呤、腺嘌呤、尿嘧啶及胞嘧啶)之mRNA轉錄物的序列。因此,本發明之RNAi構建體之反義鏈可以包含具有與靶PNPLA3 mRNA序列或PNPLA3 cDNA序列基本上或完全互補之序列的區域。PNPLA3 mRNA或cDNA序列可包括但不限於任何PNPLA3 mRNA或cDNA序列,例如可衍生自NCBI參考序列NM_025225.2。
反義鏈之區域可以與PNPLA3 mRNA序列之至少15個連續核苷酸基本上互補或完全互補。在一些實施方式中,反義鏈所包含互補區之PNPLA3 mRNA序列的靶區域範圍可為自約15至約30個連續核苷酸、自約16至約28個連續核苷酸、自約18至約26個連續核苷酸、自約17至約24個連續核苷酸、自約19至約25個連續核苷酸、自約19至約23個連續核苷酸、或自約19至約21個連續核苷酸。在某些實施方式中,包含與PNPLA3 mRNA序列基本上或完全互補之序列的反義鏈區域在一些實施方式中可以包含來自表1或表2中列出之反義序列的至少15個連續核苷酸。在其他實施方式中,反義序列包含來自表1或表2中列出之反義序列的至少16、至少17、至少18或至少19個連續核苷酸。在一些實施方式中,有義及/或反義序列包含來自表1或2中列出之序列且具有不多於1、2或3個核苷酸錯配之至少15個核苷酸。
RNAi構建體之有義鏈通常包含與反義鏈之序列充分互補,使得兩條鏈在生理條件下雜交以形成雙鏈體區之序列。「雙鏈體區」係指兩個互補或基本上互補的多核苷酸中之區域,該等多核苷酸藉由Watson-Crick鹼基配對或其他氫鍵相互作用彼此形成鹼基對,以在兩個多核苷酸之間產生雙鏈體。RNAi構建體之雙鏈體區應具有足夠的長度以允許RNAi構建體進入RNA干擾途徑,例如藉由使用Dicer酶及/或RISC複合物。例如,在一些實施方式中,雙鏈體區之長度為約15至約30個鹼基對。在該範圍內之雙鏈體區的其他長度亦為合適的,例如約15至約28個鹼基對、約15至約26個鹼基對、約15至約24個鹼基對、約15至約22個鹼基對、約17至約28個鹼基對、約17至約26個鹼基對、約17至約24個鹼基對、約17至約23個鹼基對、約17至約21個鹼基對、約19至約25個鹼基對、約19至約23個鹼基對、或約19至約21個鹼基對。在一個實施方式中,雙鏈體區之長度為約17至約24個鹼基對。在另一個實施方式中,雙鏈體區之長度為約19至約21個鹼基對。
在一些實施方式中,本發明之RNAi劑含有約24至約30個核苷酸之雙鏈體區,其與靶RNA序列(例如PNPLA3靶mRNA序列)相互作用,以指導靶RNA裂解。不希望受理論束縛,引入細胞內之長雙鏈RNA可藉由稱為Dicer之III型核酸內切酶分解為siRNA(Sharp 等人 (2001) Genes Dev. 15:485)。Dicer為一種核糖核酸酶-III樣酶,將dsRNA加工成具有特徵性兩個鹼基3'突出端之19-23個鹼基對短干擾RNA(Bernstein等人 (2001) Nature 409:363)。然後將siRNA摻入經RNA誘導之緘默複合物(RISC)中,其中一或多個解旋酶解開siRNA雙鏈體,使得互補反義鏈能夠指導靶識別(Nykanen等人 (2001) Cell107:309)。在與適當靶20 mRNA結合後,RISC內之一或多種核酸內切酶裂解靶標以誘導緘默(Elbashir等人 (2001) Genes Dev. 15: 188)。
對於其中有義鏈及反義鏈為兩個單獨分子(例如RNAi構建體包含siRNA)之實施方式,有義鏈及反義鏈之長度不需要與雙鏈體區之長度相同。例如,一或兩條鏈可能長於雙鏈體區,且具有側接雙鏈體區之一或多個未配對核苷酸或錯配。因此,在一些實施方式中,RNAi構建體包含至少一個核苷酸突出端。如本文所用,「核苷酸突出端」係指延伸超出雙鏈體區之鏈末端的一或多個未配對核苷酸或核苷酸。當一條鏈之3'端延伸超出另一條鏈之5'端或當一條鏈之5'端延伸超出另一條鏈之3'端時,通常產生核苷酸突出端。核苷酸突出端之長度通常為1至6個核苷酸、1至5個核苷酸、1至4個核苷酸、1至3個核苷酸、2至6個核苷酸、2至5個核苷酸、或2至4個核苷酸。在一些實施方式中,核苷酸突出端包含1、2、3、4、5或6個核苷酸。在一特定實施方式中,核苷酸突出端包含1至4個核苷酸。在某些實施方式中,核苷酸突出端包含2個核苷酸。突出端中之核苷酸可為如本文所述之核糖核苷酸、去氧核糖核苷酸或經修飾之核苷酸。在一些實施方式中,突出端包含5'-尿苷尿苷-3'(5'-UU-3')二核苷酸。在此類實施方式中,UU二核苷酸可包含核糖核苷酸或經修飾之核苷酸,例如經2'-修飾之核苷酸。在其他實施方式中,突出端包含5'-去氧胸苷-去氧胸苷-3'(5'-dTdT-3')二核苷酸。
核苷酸突出端可位於一或兩條鏈之5'端或3'端。例如,在一個實施方式中,RNAi構建體包含反義鏈之5'端及3'端處之核苷酸突出端。在另一個實施方式中,RNAi構建體在有義鏈之5'端及3'端處包含核苷酸突出端。在一些實施方式中,RNAi構建體包含有義鏈之5'端及反義鏈之5'端之核苷酸突出端。在其他實施方式中,RNAi構建體包含有義鏈之3'端及反義鏈之3'端處之核苷酸突出端。
RNAi構建體可以在雙鏈RNA分子之一端處包含單個核苷酸突出端且在另一個末端包含鈍端。「鈍端」意指有義鏈及反義鏈在分子末端完全鹼基配對,且無未配對核苷酸延伸超出雙鏈體區。在一些實施方式中,RNAi構建體包含有義鏈3'端之核苷酸突出端及有義鏈5'端及反義鏈3'端之鈍端。在其他實施方式中,RNAi構建體包含反義鏈3'端處之核苷酸突出端及反義鏈5'端及有義鏈3'端之鈍端。在某些實施方式中,RNAi構建體在雙鏈RNA分子兩端處包含鈍端。在此類實施方式中,有義鏈及反義鏈具有相同長度,且雙鏈體區之長度與有義鏈及反義鏈相同(即,該分子在其整個長度上為雙鏈的)。
有義鏈及反義鏈可各自獨立地具有約15至約30個核苷酸長度、約18至約28個核苷酸長度、約19至約27個核苷酸長度、約19至約25個核苷酸長度、約19至約23個核苷酸長度、約21至約25個核苷酸長度、或約21至約23個核苷酸長度。在某些實施方式中,有義鏈及反義鏈之長度各為約18、約19、約20、約21、約22、約23、約24、或約25個核苷酸。在一些實施方式中,有義鏈及反義鏈具有相同的長度,但形成短於該等鏈使得RNAi構建體具有兩個核苷酸突出端之雙鏈體區。例如,在一個實施方式中,RNAi構建體包含 (i) 長度各為21個核苷酸之有義鏈及反義鏈、(ii) 長度為19個鹼基對之雙鏈體區、及 (iii) 在有義鏈3'端及反義鏈3'端有2個未配對核苷酸之核苷酸突出端。在另一個實施方式中,RNAi構建體包含 (i) 長度各為23個核苷酸之有義鏈及反義鏈、(ii) 長度為21個鹼基對之雙鏈體區、及 (iii) 在有義鏈3'端及反義鏈3'端有2個未配對核苷酸之核苷酸突出端。在其他實施方式中,有義鏈及反義鏈具有相同長度且在其整個長度上形成雙鏈體區,使得在雙鏈分子之任一端上不存在核苷酸突出端。在一個此類實施方式中,RNAi構建體為鈍端的且包含 (i) 其長度各為21個核苷酸之有義鏈及反義鏈及 (ii) 長度為21個鹼基對之雙鏈體區。在另一個此類實施方式中,RNAi構建體為鈍端的且包含 (i) 其長度各為23個核苷酸之有義鏈及反義鏈及 (ii) 長度為23個鹼基對之雙鏈體區。
在其他實施方式中,有義鏈或反義鏈長於另一條鏈,且兩條鏈形成長度等於較短鏈長度使得RNAi構建體包含至少一個核苷酸突出端之雙鏈體區。例如,在一個實施方式中,RNAi構建體包含 (i) 長度為19個核苷酸之有義鏈、(ii) 長度為21個核苷酸之反義鏈、(iii) 長度為19個鹼基對之雙鏈體區、及 (iv) 在反義鏈3'端處的2個未配對核苷酸之單核苷酸突出端。在另一個實施方式中,RNAi構建體包含(i)長度為21個核苷酸之有義鏈、(ii)長度為23個核苷酸之反義鏈、(iii) 長度為21個鹼基對之雙鏈體區、及 (iv) 在反義鏈3'端處的2個未配對核苷酸之單核苷酸突出端。
本發明之RNAi構建體之反義鏈可包含表1或表2中列出之任一反義序列之序列或任何該等反義序列之核苷酸1-19之序列。表1及6中列出之各反義序列包含19個連續核苷酸的序列(自5'末端開始計數的前19個核苷酸),其與PNPLA3 mRNA序列加上兩個核苷酸突出端序列互補。因此,在一些實施方式中,反義鏈包含SEQ ID NO:1-166或167-332中任一者之核苷酸1-19的序列。經修飾之核苷酸
本發明之RNAi構建體可包含一或多種經修飾之核苷酸。「經修飾之核苷酸」係指對核苷、核鹼基、戊糖環或磷酸基團具有一或多種經化學修飾之核苷酸。如本文所用,經修飾之核苷酸不包括含有腺苷一磷酸、鳥苷一磷酸、尿苷一磷酸及胞苷一磷酸之核糖核苷酸,以及含有去氧腺苷一磷酸、去氧鳥苷一磷酸、去氧胸苷一磷酸及去氧胞苷一磷酸之去氧核糖核苷酸。然而,RNAi構建體可包含經修飾之核苷酸、核糖核苷酸及去氧核糖核苷酸之組合。將經修飾之核苷酸摻入雙鏈RNA分子之一或兩條鏈中可以改善RNA分子之活體內穩定性,例如藉由降低分子對核酸酶及其他降解過程之易感性。亦可藉由摻入經修飾之核苷酸來增強RNAi構建體降低靶基因表現之效力。
在某些實施方式中,經修飾之核苷酸具有核糖的修飾。該等糖修飾可包括戊糖環之2'及/或5'位置處之修飾以及雙環糖修飾。經2'-修飾之核苷酸係指具有戊糖環之核苷酸,該戊糖環在2'位置具有除H或OH以外的取代基。此類2'修飾包括但不限於2'-O-烷基(例如經O-C1-C10或O-C1-C10取代之烷基)、2'-O-烯丙基(O-CH2CH=CH2)、2'-C-烯丙基、2'-氟基、2'-O-甲基(OCH3)、2'-O-甲氧基乙基(O-(CH2)2OCH3)、2'-OCF3、2'-O(CH2)2SCH3、2'-O-胺基烷基、2'-胺基(例如NH2)、2'-O-乙胺、及2'-疊氮基。在戊糖環之5'位置處之修飾包括但不限於5'-甲基(R或S);5'-乙烯基及5'-甲氧基。
「雙環糖修飾」係指戊糖環的修飾,其中橋接基連接環之兩個原子以形成第二環,產生雙環糖結構。在一些實施方式中,雙環糖修飾包含戊糖環之4'與2'碳之間的橋接基。包含具有雙環糖修飾之糖部分的核苷酸在本文中稱為雙環核酸或BNA。例示性雙環糖修飾包括但不限於α-L-次甲氧基(4'-CH2-O-2')雙環核酸(BNA);β-D-次甲氧基(4'-CH2-O-2')BNA(亦稱為鎖核酸或LNA);伸乙氧基(4' - (CH2)2-O-2')BNA;胺氧基(4'-CH2-O-N(R)-2')BNA;氧基胺基(4'-CH2-N(R)-O-2')BNA;甲基(次甲氧基)(4'-CH(CH3)-O-2')BNA(亦稱為約束乙基或cEt);次甲基 - 硫代(4'-CH2-S-2')BNA;次甲基-胺基(4'-CH2-N(R)-2')BNA;甲基碳環(4'-CH2-CH(CH3)-2')BNA;丙烯碳環(4'-(CH2)3-2')BNA;及甲氧基(伸乙氧基)(4'-CH(CH2OMe)-O-2')BNA(亦稱為約束MOE或cMOE)。可以摻入本發明之RNAi構建體中之該等及其他經糖修飾之核苷酸描述於美國專利第9,181,551號、美國專利公佈第2016/0122761號及Deleavey及Damha,Chemistry and Biology,第19卷:937-954,2012,所有該等文獻均全文以引用之方式併入本文。
在一些實施方式中,RNAi構建體包含一或多個經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸、經2'-O-甲氧基乙基修飾之核苷酸、經2'-O-烯丙基修飾之核苷酸、雙環核酸(BNA)、或其組合。在某些實施方式中,RNAi構建體包含一或多個經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸、經2'-O-甲氧基乙基修飾之核苷酸、或其組合。在一特定實施方式中,RNAi構建體包含一或多個經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸、或其組合。
RNAi構建體之有義鏈及反義鏈均可包含一或多個經修飾之核苷酸。例如,在一些實施方式中,有義鏈包含1、2、3、4、5、6、7、8、9、10或更多個經修飾之核苷酸。在某些實施方式中,有義鏈中之所有核苷酸均為經修飾之核苷酸。在一些實施方式中,反義鏈包含1、2、3、4、5、6、7、8、9、10或更多個經修飾之核苷酸。在其他實施方式中,反義鏈中之所有核苷酸均為經修飾之核苷酸。在某些其他實施方式中,有義鏈中之所有核苷酸及反義鏈中之所有核苷酸均為經修飾之核苷酸。在該等及其他實施方式中,經修飾之核苷酸可為經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸、或其組合。
在一些實施方式中,有義鏈、反義鏈或兩條鏈中之腺苷核苷酸之前的所有嘧啶核苷酸均為經修飾之核苷酸。例如,當序列5'-CA-3'或5'-UA-3'出現在任一鏈中時,胞苷及尿苷核苷酸為經修飾之核苷酸,較佳經2'-O-甲基修飾之核苷酸。在某些實施方式中,有義鏈中之所有嘧啶核苷酸均為經修飾之核苷酸(例如經2'-O-甲基修飾之核苷酸),且在反義鏈中之序列5'-CA-3'或5'-UA-3'的所有出現中之5'核苷酸為經修飾之核苷酸(例如經2'-O-甲基修飾之核苷酸)。在其他實施方式中,雙鏈體區中之所有核苷酸均為經修飾之核苷酸。在此類實施方式中,經修飾之核苷酸較佳為經2'-O-甲基修飾之核苷酸、經2'-氟修飾之核苷酸、或其組合。
在RNAi構建體包含核苷酸突出端之實施方式中,突出端中之核苷酸可為核糖核苷酸、去氧核糖核苷酸或經修飾之核苷酸。在一個實施方式中,突出端中之核苷酸為去氧核糖核苷酸,例如去氧胸苷。在另一個實施方式中,突出端中之核苷酸為經修飾之核苷酸。例如,在一些實施方式中,突出端中之核苷酸為經2'-O-甲基修飾之核苷酸、經2'-氟修飾之核苷酸、經2'-甲氧基乙基修飾之核苷酸、或其組合。
本發明之RNAi構建體亦可以包含一或多個經修飾之核苷酸間鍵。如本文所用,術語「經修飾之核苷酸間鍵」係指除天然3'至5'磷酸二酯鍵外的核苷酸間鍵。在一些實施方式中,經修飾之核苷酸間鍵為含磷核苷酸間鍵,例如磷酸三酯、胺基烷基磷酸三酯、膦酸烷基酯(例如膦酸甲酯、膦酸3' - 伸烷酯)、次膦酸酯、胺基磷酸酯(例如,3'-胺基胺基磷酸酯及胺基烷基胺基磷酸酯)、硫代磷酸酯(P = S)、對掌硫代磷酸酯、二硫代磷酸酯、硫羰基胺基磷酸酯、硫羰基膦酸烷基酯、硫羰基烷基磷酸三酯、及硼烷磷酸酯。在一個實施方式中,經修飾之核苷酸間鍵為2'至5'磷酸二酯鍵。在其他實施方式中,經修飾之核苷酸間鍵為不含磷核苷酸間鍵,且因此可稱為經修飾之核苷間鍵。此類不含磷鍵包括但不限於𠰌啉基鍵(部分由核苷之糖部分形成);矽氧烷鍵(-O-Si(H)2-O-);硫化物、亞碸及碸鍵;甲醯基及硫代甲醯基鍵;含有烯烴之主鏈;胺基磺酸主鏈;次甲基甲基亞胺基(-CH2-N(CH3)-O-CH2-)及次甲基肼基鍵;磺酸鹽及磺醯胺鍵;醯胺鍵;及具有混合N、O、S及CH2組分之其他鍵。在一個實施方式中,經修飾之核苷間鍵為產生肽核酸或PNA之基於肽之鍵(例如胺基乙基甘胺酸),例如美國專利第5,539,082;5,714,331;及5,719,262號中所述之彼等者。可以在本發明之RNAi構建體中使用之其他合適的經修飾之核苷酸間及核苷間鍵描述於美國專利第6,693,187號、美國專利第9,181,551號、美國專利公佈第2016/0122761號、及Deleavey及Damha,Chemistry and Biology,第19卷:937-954, 2012,所有該等文獻均全文以引用之方式併入本文。
在某些實施方式中,RNAi構建體包含一或多個硫代磷酸酯核苷酸間鍵。硫代磷酸酯核苷酸間鍵可以存在於RNAi構建體之有義鏈、反義鏈或兩條鏈中。例如,在一些實施方式中,有義鏈包含1、2、3、4、5、6、7、8或更多個硫代磷酸酯核苷酸間鍵。在其他實施方式中,反義鏈包含1、2、3、4、5、6、7、8或更多個硫代磷酸酯核苷酸間鍵。在其他實施方式中,兩條鏈包含1、2、3、4、5、6、7、8或更多個硫代磷酸酯核苷酸間鍵。RNAi構建體可以在有義鏈、反義鏈或兩條鏈之3'-端、5'-端或3'-端及5'-端處包含一或多個硫代磷酸酯核苷酸間鍵。例如,在某些實施方式中,RNAi構建體在有義鏈、反義鏈或兩條鏈之3'-端處包含約1至約6或更多(例如,約1、2、3、4、5、6或更多)個連續硫代磷酸酯核苷酸間鍵。在其他實施方式中,RNAi構建體在有義鏈、反義鏈或兩條鏈之5'-端處包含約1至約6或更多(例如,約1、2、3、4、5、6或更多)個連續硫代磷酸酯核苷酸間鍵。在一個實施方式中,RNAi構建體包含在有義鏈3'端處之單個硫代磷酸酯核苷酸間鍵及在反義鏈3'端之單個硫代磷酸酯核苷酸間鍵。在另一個實施方式中,RNAi構建體在反義鏈3'端處包含兩個連續硫代磷酸酯核苷酸間鍵(即在反義鏈3'端處之第一及第二核苷酸間鍵處的硫代磷酸酯核苷酸間鍵)。在另一個實施方式中,RNAi構建體在反義鏈之3'端及5'端處包含兩個連續硫代磷酸酯核苷酸間鍵。在另一個實施方式中,RNAi構建體包含反義鏈之3'端及5'端處之兩個連續硫代磷酸酯核苷酸間鍵及有義鏈5'端處之兩個連續硫代磷酸酯核苷酸間鍵。在另一個實施方式中,RNAi構建體包含反義鏈3'端及5'端處之兩個連續硫代磷酸酯核苷酸間鍵及有義鏈3'端及5'端處之兩個連續硫代磷酸酯核苷酸間鍵(即,在反義鏈5'端及3'端處之第一及第二核苷酸間鍵處的硫代磷酸酯核苷酸間鍵及在有義鏈5'端及3'端處之第一及第二核苷酸間鍵處的硫代磷酸酯核苷酸間鍵)。在一或兩條鏈包含一或多個硫代磷酸酯核苷酸間鍵之任何實施方式中,鏈內其餘核苷酸間鍵可為天然3'至5'磷酸二酯鍵。例如,在一些實施方式中,有義鏈及反義鏈之各核苷酸間鍵選自磷酸二酯及硫代磷酸酯,其中至少一個核苷酸間鍵為硫代磷酸酯。
在RNAi構建體包含核苷酸突出端之實施方式中,突出端中之兩或更多個未配對核苷酸可藉由硫代磷酸酯核苷酸間鍵來連接。在某些實施方式中,反義鏈及/或有義鏈之3'端處的核苷酸突出端中的所有未配對核苷酸藉由硫代磷酸酯核苷酸間鍵來連接。在其他實施方式中,反義鏈及/或有義鏈之5'端處的核苷酸突出端中的所有未配對核苷酸藉由硫代磷酸酯核苷酸間鍵來連接。在其他實施方式中,任何核苷酸突出端中之所有未配對核苷酸藉由硫代磷酸酯核苷酸間鍵來連接。
在某些實施方式中,摻入本發明之RNAi構建體之一或兩條鏈中的經修飾之核苷酸具有核鹼基(在本文中亦稱為「鹼基」)的修飾。「經修飾之核鹼基」或「經修飾之鹼基」係指除天然存在的嘌呤鹼基腺嘌呤(A)及鳥嘌呤(G)以及嘧啶鹼基胸腺嘧啶(T)、胞嘧啶(C)及尿嘧啶(U)以外的鹼基。經修飾之核鹼基可為合成的或天然存在的修飾,包括但不限於通用鹼基、5-甲基胞嘧啶(5-me-C)、5-羥甲基胞嘧啶、黃嘌呤(X)、次黃嘌呤(I)、2-胺基腺嘌呤、6-甲基腺嘌呤、6-甲基鳥嘌呤、及腺嘌呤及鳥嘌呤之其他烷基衍生物、腺嘌呤及鳥嘌呤之2-丙基及其他烷基衍生物、2-硫尿嘧啶、2-硫代胸腺嘧啶及2-硫胞嘧啶、5-鹵代尿嘧啶及胞嘧啶、5-丙炔基尿嘧啶及胞嘧啶、6-偶氮尿嘧啶、胞嘧啶及胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-鹵素、8-胺基、8-硫醇、8-硫代烷基、8-羥基及其他8-取代腺嘌呤及鳥嘌呤、5-鹵素,特別是5-溴、5-三氟甲基及其他5-取代尿嘧啶及胞嘧啶、7-甲基鳥嘌呤及7-甲基腺嘌呤、8-氮雜鳥嘌呤及8-氮雜腺嘌呤、7-脫氮鳥嘌呤及7-脫氮腺嘌呤、及3-脫氮鳥嘌呤及3-脫氮腺嘌呤。
在一些實施方式中,經修飾之鹼基為通用鹼基。「通用鹼基」係指鹼基類似物,其與RNA及DNA中的所有天然鹼基不加區別地形成鹼基對,而不改變所得雙鏈體區之雙螺旋結構。通用鹼基為熟習此項技術者已知者,且包括但不限於肌苷、C-苯基、C-萘基及其他芳族衍生物、唑類羧醯胺及硝基唑衍生物,諸如3-硝基吡咯、4-硝基吲哚、5-硝基吲哚、及6-硝基吲哚。
可以摻入本發明之RNAi構建體中之其他合適的經修飾鹼基包括在 Herdewijn, Antisense Nucleic Acid Drug Dev., 第10卷: 297-310, 2000及Peacock等人, J. Org. Chern., 第76卷: 7295-7300, 2011中描述之彼等者,該等文獻全文以引用之方式併入本文。熟習此項技術者充分瞭解到鳥嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶及尿嘧啶可被其他核鹼基替代,例如上述經修飾之核鹼基,而基本上不改變包含攜帶此置換核鹼基之核苷酸的多核苷酸之鹼基配對性質。
在本發明之RNAi構建體之一些實施方式中,有義鏈、反義鏈或反義鏈及有義鏈之5'端包含磷酸酯部分。如本文所用,術語「磷酸酯部分」係指包括未經修飾之磷酸酯(-O-P=O)(OH)OH)以及經修飾之磷酸酯的末端磷酸酯基團。經修飾之磷酸酯包括磷酸酯,其中一或多個O及OH基團被H、O、S、N(R)或烷基取代,其中R為H、胺基保護基團或未經取代或經取代之烷基。例示性磷酸酯部分包括但不限於5'-一磷酸;5'-二磷酸;5'-三磷酸;5'-鳥苷帽(7-甲基化或非甲基化);5'-腺苷帽或任何其他經修飾或未經修飾之核苷酸帽結構;5'-單硫代磷酸酯(硫代磷酸酯);5'-單二硫代磷酸酯(二硫代磷酸酯);5'-α-硫代三磷酸酯;5'-γ-硫代三磷酸酯、5'-胺基磷酸酯;5'-乙烯基磷酸酯;5'-烷基膦酸酯(例如烷基 = 甲基、乙基、異丙基、丙基等);及5'-烷基醚膦酸酯(例如,烷基醚 = 甲氧基甲基、乙氧基甲基等)。
可摻入本發明之RNAi構建體中之經修飾之核苷酸可具有本文所述之多於一種化學修飾。例如,經修飾之核苷酸可以具有對核糖之修飾以及對核鹼基之修飾。舉例而言,經修飾之核苷酸可包含2'糖修飾(例如2'-氟基或2'-甲基)且包含經修飾之鹼基(例如5-甲基胞嘧啶或假尿嘧啶)。在其他實施方式中,經修飾之核苷酸可以包含糖修飾以及對5'磷酸之修飾,當經修飾之核苷酸摻入多核苷酸時,該等修飾將產生經修飾之核苷酸間或核苷間鍵。例如,在一些實施方式中,經修飾之核苷酸可包含糖修飾,例如2'-氟修飾、2'-O-甲基修飾、或雙環糖修飾、以及5'硫代磷酸酯基團。因此,在一些實施方式中,本發明之RNAi構建體之一或兩條鏈包含經2'修飾之核苷酸或BNA與硫代磷酸酯核苷酸間鍵之組合。在某些實施方式中,本發明之RNAi構建體之有義鏈及反義鏈均包含經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸及硫代磷酸酯核苷酸間鍵之組合。包含經修飾之核苷酸及核苷酸間鍵的例示性RNAi構建體顯示在表2中。RNAi 構建體之功能
較佳地,本發明之RNAi構建體降低或抑制PNPLA3在細胞,特別是肝細胞中之表現。因此,在一個實施方式中,本發明提供一種藉由使細胞與本文所述之任何RNAi構建體接觸來降低細胞中之PNPLA3表現的方法。細胞可為活體外或活體內的。PNPLA3表現可以藉由量測PNPLA3 mRNA、PNPLA3蛋白或與PNPLA3表現相關之另一種生物標誌物之量或水平來評估。可以相對於未用RNAi構建體處理或用對照RNAi構建體處理之細胞或動物中之PNPLA3表現來判定用本發明之RNAi構建體處理之細胞或動物中之PNPLA3表現之降低。例如,在一些實施方式中,藉由以下步驟來評估PNPLA3表現之降低:(a) 量測用本發明之RNAi構建體處理之肝細胞中之PNPLA3 mRNA之量或水平、(b) 量測用對照RNAi構建體(例如,針對未在肝細胞中表現之RNA分子的RNAi劑或具有無義或亂序序列之RNAi構建體)處理或不用構建體處理之肝細胞中之PNPLA3 mRNA之量或水平、及 (c) 比較來自 (a) 中之經處理細胞的經量測PNPLA3 mRNA水平與來自 (b) 中之對照細胞的經量測PNPLA3 mRNA水平。在比較之前,可以將處理細胞及對照細胞中之PNPLA3 mRNA水平標準化至對照基因(例如18S核糖體RNA)之RNA水平。PNPLA3 mRNA水平可藉由多種方法量測,包括北方墨點分析、核酸酶保護測定、螢光原位雜交(FISH)、逆轉錄酶(RT)-PCR、即時RT-PCR、定量PCR及類似方法。
在其他實施方式中,藉由以下步驟來評估PNPLA3表現之降低:(a) 量測用本發明之RNAi構建體處理之肝細胞中之PNPLA3蛋白質之量或水平、(b) 量測用對照RNAi構建體(例如,針對未在肝細胞中表現之RNA分子的RNAi劑或具有無義或亂序序列之RNAi構建體)處理或不用構建體處理之肝細胞中之PNPLA3蛋白質之量或水平、及 (c) 比較來自 (a) 中之經處理細胞的經量測PNPLA3蛋白質水平與來自 (b) 中之對照細胞的經量測PNPLA3蛋白質水平。量測PNPLA3蛋白水平之方法為熟習此項技術者已知,包括西方墨點、免疫測定(例如ELISA)及流式細胞術。用於評估PNPLA3蛋白表現之例示性基於免疫測定之方法描述於實例2中。實例3描述了一種用於使用RNA FISH量測PNPLA3 mRNA之例示性方法。任何能夠量測PNPLA3 mRNA或蛋白質之方法可用於評估本發明之RNAi構建體之功效。
在一些實施方式中,評估PNPLA3表現水平之方法在活體外在天然表現PNPLA3之細胞(例如肝細胞)或已經工程化以表現PNPLA3之細胞中進行。在某些實施方式中,該等方法在活體外在肝細胞中進行。合適的肝細胞包括但不限於原代肝細胞(例如人、非人靈長類動物或齧齒動物肝細胞)、HepAD38細胞、HuH-6細胞、HuH-7細胞、HuH-5-2細胞、BNLCL2細胞、Hep3B細胞或HepG2細胞。在一個實施方式中,肝細胞為Hep3B細胞。在另一個實施方式中,肝細胞為HepG2細胞。
在其他實施方式中,評估PNPLA3表現水平之方法在活體內進行。RNAi構建體及任何對照RNAi構建體可以投與動物(例如齧齒動物或非人靈長類動物)且在處理後,在自動物收穫之肝組織中評估PNPLA3 mRNA或蛋白質水平。或者或另外,可以在經處理之動物中評估與PNPLA3表現相關之生物標誌物或功能表型。
在某些實施方式中,藉由本發明之RNAi構建體,使PNPLA3之表現在肝細胞中降低至少10%、至少15%、至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、或至少50%。在一些實施方式中,藉由本發明之RNAi構建體,使PNPLA3之表現在肝細胞中降低至少60%、至少65%、至少70%、至少75%、至少80%、或至少85%。在其他實施方式中,藉由本發明之RNAi構建體,使PNPLA3之表現在肝細胞中降低約90%或更多,例如91%、92%、93%、94%、95%、96%、97%、98%、99%、或更多。PNPLA3表現之降低百分比可藉由本文所述之任何方法以及此項技術已知之其他方法量測。例如,在某些實施方式中,本發明之RNAi構建體在活體外在Hep3B細胞(含有野生型PNPLA3)中以5 nM將PNPLA3表現抑制至少45%。在相關實施方式中,本發明之RNAi構建體在活體外在Hep3B細胞中以5 nM將PNPLA3表現抑制至少50%、至少55%、至少60%、至少65%、至少70%、或至少75%。在其他實施方式中,本發明之RNAi構建體在活體外在Hep3B細胞中以5 nM將PNPLA3表現抑制至少80%、至少85%、至少90%、至少92%、至少94%、至少96%、或至少98%。在某些實施方式中,本發明之RNAi構建體在活體外在HepG2細胞(含有PNPLA3-rs738409-rs738408雙重次要等位基因)中以5 nM將PNPLA3表現抑制至少45%。在相關實施方式中,本發明之RNAi構建體在活體外在HepG2細胞中以5 nM將PNPLA3表現抑制至少50%、至少55%、至少60%、至少65%、至少70%、或至少75%。在其他實施方式中,本發明之RNAi構建體在活體外HepG2細胞中以5 nM將PNPLA3表現抑制至少80%、至少85%、至少90%、至少92%、至少94%、至少96%、或至少98%。在某些實施方式中,本發明之RNAi構建體在活體外在表現人PNPLA3 I148I或I148M細胞之CHO轉染細胞中以5 nM將PNPLA3表現抑制至少45%。在相關實施方式中,本發明之RNAi構建體在活體外在表現人PNPLA3 I148I或I148M之CHO轉染細胞中以5 nM將PNPLA3表現抑制至少50%、至少55%、至少60%、至少65%、至少70%、或至少75%。在其他實施方式中,本發明之RNAi構建體在活體外在表現人PNPLA3 I148I或I148M之CHO轉染細胞中以5 nM將PNPLA3表現抑制至少80%、至少85%、至少90%、至少92%、至少94%、至少96%、或至少98%。如實例2及3中所述,可以使用包括RNA FISH或微滴式數位PCR在內之多種技術量測PNPLA3之降低。
在一些實施方式中,計算IC50值以評估本發明之RNAi構建體抑制肝細胞中之PNPLA3表現的效力。「IC50值」為實現50%生物或生物化學功能抑制所需之劑量/濃度。任何特定物質或拮抗劑之IC50值可以藉由構建劑量-反應曲線且在任何測定中檢查不同濃度的物質或拮抗劑對表現水平或功能活性之影響來判定。給定拮抗劑或物質之IC50值可以藉由判定抑制一半最大生物反應或天然表現水平所需之濃度來計算。因此,任何RNAi構建體之IC50值可以藉由在任何測定中判定抑制肝細胞中之一半天然PNPLA3表現水平(例如對照肝細胞中之PNPLA3表現水平)所需之RNAi構建體的濃度來計算,該等測定例如實例中描述之免疫測定或RNA FISH測定或微滴式數位PCR測定。本發明之RNAi構建體可以小於約20 nM之IC50抑制肝細胞(例如Hep3B細胞)中之PNPLA3表現。例如,RNAi構建體以以下IC50抑制肝細胞中之PNPLA3表現:約0.001 nM至約20 nM、約0.001 nM至約10 nM、約0.001 nM至約5 nM、約0.001 nM至約1 nM、約0.1 nM至約10 nM、約0.1 nM至約5 nM、或約0.1 nM至約1 nM。在某些實施方式中,RNAi構建體以約1 nM至約10 nM之IC50抑制肝細胞(例如Hep3B細胞)中之PNPLA3表現。本發明之RNAi構建體可以小於約20 nM之IC50抑制肝細胞(例如HepG2細胞)中之PNPLA3表現。例如,RNAi構建體以以下IC50抑制肝細胞中之PNPLA3表現:約0.001 nM至約20 nM、約0.001 nM至約10 nM、約0.001 nM至約5 nM、約0.001 nM至約1 nM、約0.1 nM至約10 nM、約0.1 nM至約5 nM、或約0.1 nM至約1 nM。在某些實施方式中,RNAi構建體以約1 nM至約10 nM之IC50抑制肝細胞(例如HepG2細胞)中之PNPLA3表現。本發明之RNAi構建體可以小於約20 nM之IC50抑制肝細胞(例如表現人PNPLA3 I148I或I148M之CHO轉染細胞)中之PNPLA3表現。例如,RNAi構建體以以下IC50抑制肝細胞中之PNPLA3表現:約0.001 nM至約20 nM、約0.001 nM至約10 nM、約0.001 nM至約5 nM、約0.001 nM至約1 nM、約0.1 nM至約10 nM、約0.1 nM至約5 nM、或約0.1 nM至約1 nM。在某些實施方式中,RNAi構建體以約1 nM至約10 nM之IC50抑制肝細胞(例如表現人PNPLA3 I148I或I148M之CHO轉染細胞)中之PNPLA3表現。
本發明之RNAi構建體可使用此項技術已知之技術,例如使用習知的核酸固相合成來容易地製成。RNAi構建體之多核苷酸可使用標準核苷酸或核苷先質(例如亞磷醯胺)在合適的核酸合成儀上組裝。自動化核酸合成儀由若干供應商商業銷售,包括來自應用生物系統公司(Applied Biosystems)(加利福尼亞州福斯特城)之DNA/RNA合成儀、來自生物自動化公司(BioAutomation)(德克薩斯州歐文市)之MerMade合成儀、及來自通用電氣醫療集團生命科學部(GE Healthcare Life Sciences)(賓夕法尼亞州匹茲堡)之OligoPilot合成儀。
2'矽基保護基團可與核糖核苷之5'位酸不穩定性二甲氧基三苯甲基(DMT)結合使用,以藉由亞磷醯胺化學合成寡核苷酸。已知最終脫保護條件不會顯著降解RNA產物。所有合成均可在任何自動或手動合成儀中以大、中、小規模進行。合成亦可以在多個孔板、柱或載玻片中進行。
2'-O-矽基可以經由暴露於氟離子來除去,氟離子可以包括任何氟離子源,例如含有與無機相對離子配對之氟離子的彼等鹽,例如氟化銫及氟化鉀,或含有與有機相對離子配對之氟離子的彼等鹽,例如四烷基氟化銨。冠醚催化劑可以與無機氟化物組合用於脫保護反應中。較佳的氟離子源為四丁基氟化銨或胺基氫氟化物(例如,將HF水溶液與三乙胺組合在偶極非質子溶劑如二甲基甲醯胺中)。
選擇用於亞磷酸三酯及磷酸三酯上之保護基團可以改變三酯對氟化物的穩定性。磷酸三酯或亞磷酸三酯之甲基保護可以穩定與氟離子之鍵聯且提高製程產率。
由於核糖核苷具有反應性2'羥基取代基,因此可能需要用與5'-O-二甲氧基三苯甲基保護基正交之保護基團(例如對用酸處理穩定之保護基團)保護RNA中的反應性2'位置。矽基保護基團符合該標準,且可以在最終的氟化物去保護步驟中容易地除去,這可以導致最少RNA降解。
四唑催化劑可用於標準亞磷醯胺偶聯反應。較佳催化劑包括例如四唑、S-乙基 - 四唑、苄基硫代四唑、對硝基苯基四唑。
如熟習此項技術者可以理解的,合成本文所述之RNAi構建體之其他方法對於普通熟習此項技術者而言為顯而易見的。另外,各種合成步驟可以交替序列或順序進行,以得到所要化合物。其他合成化學轉化、保護基團(例如,對於鹼基上存在之羥基、胺基等)及適用於合成本文所述RNAi構建體之保護基團方法(保護及去保護)為此項技術已知的且包括例如以下文獻中描述之彼等者:R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989);T. W. Greene及P. G. M. Wuts, Protective Groups in Organic Synthesis, 第2版, John Wiley and Sons (1991);L. Fieser及M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994);及L. Paquette編 Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995),及其後續版本。RNAi劑之定製合成亦可自若干商業供應商處獲得,包括Dharmacon公司(Dharmacon, Inc.)(科羅拉多州拉斐特)、AXO實驗室股份有限公司(AxoLabs GmbH)(德國庫爾姆巴赫)及Ambion公司(Ambion, Inc.)(加利福尼亞州福斯特城)。
本發明之RNAi構建體可包含配位基。如本文所用,「配位基」係指能夠直接或間接與另一種化合物或分子相互作用的任何化合物或分子。配位基與另一種化合物或分子之相互作用可引發生物反應(例如,啟動信號轉導級聯反應,誘導受體介導之內吞作用)或者可僅為物理締合。配位基可以修改所連接之雙鏈RNA分子的一或多種性質,例如RNA分子之藥效學、藥動學、結合、吸收、細胞分佈、細胞攝取、電荷及/或清除特性。
配位基可包含血清蛋白(例如,人血清白蛋白、低密度脂蛋白、球蛋白)、膽固醇部分、維生素(生物素、維生素E、維生素B12)、葉酸部分、類固醇、膽汁酸(例如膽酸)、脂肪酸(例如棕櫚酸、肉豆蔻酸)、碳水化合物(例如葡聚糖、支鏈澱粉、甲殼質、殼聚糖、菊粉、環糊精或透明質酸)、糖苷、磷脂、或抗體或其結合片段(例如,將RNAi構建體靶向特定細胞類型,例如肝臟之抗體或結合片段)。配位基的其他實例包括染料、嵌入劑(例如吖啶)、交聯劑(例如補骨脂素、絲裂黴素C)、卟啉(TPPC4、泰克薩菲瑞、賽卟啉(Sapphyrin))、多環芳烴(例如吩𠯤、二氫吩𠯤)、人工核酸內切酶(例如EDTA)、親脂性分子,例如金剛烷乙酸、1-芘丁酸、二氫睾酮、1,3-二O(十六烷基)甘油、香葉氧基己基、十六烷基甘油、冰片、薄荷醇、1,3-丙二醇、十七烷基、03-(油醯基)石膽酸、03-(油醯基)膽酸、二甲氧基三苯甲基或㗁吩𠯤)、肽(例如,觸角肽、Tat肽、RGD肽)、烷化劑、聚合物,諸如聚乙二醇(PEG)(例如,PEG-40K)、聚胺基酸及多胺(諸如精胺、亞精胺)。
在某些實施方式中,配位基具有內體溶解特性。內體溶解配位基促進內體裂解及/或本發明之RNAi構建體或其組分自細胞之內體轉運到細胞質。內體溶解配位基可為聚陽離子肽或肽模擬物,其顯示pH依賴性膜活性及融合性。在一個實施方式中,內體溶解配位基在內體pH下呈現其活性構象。「活性」構象為如此構象,其中內體溶解配位基促進內體裂解及/或本發明之RNAi構建體或其組分自細胞之內體轉運到細胞質。例示性內體溶解配位基包括GALA肽(Subbarao等人, Biochemistry, 第26卷: 2964-2972, 1987)、EALA肽(Vogel等人, J. Am. Chern. Soc.,第118卷: 1581-1586, 1996)及其衍生物(Turk等人, Biochem. Biophys. Acta, 第1559卷: 56-68, 2002)。在一個實施方式中,內體溶解組分可含有化學基團(例如胺基酸),其響應於pH之變化將經歷電荷或質子化之變化。內體溶解組分可為直鏈或支鏈的。
在一些實施方式中,配位基包含脂質或其他疏水分子。在一個實施方式中,配位基包含膽固醇部分或其他類固醇。據報道經膽固醇綴合之寡核苷酸較其未經綴合對應物更具活性(Manoharan,Antisense Nucleic Acid Drug Development,第12卷:103-228,2002)。包含用於與核酸分子綴合之膽固醇部分及其他脂質的配位基亦已描述於美國專利第7,851,615號;第7,745,608號;及第7,833,992號;其全文以引用之方式併入本文。在另一個實施方式中,配位基包含葉酸部分。與葉酸部分綴合之多核苷酸可以經由經受體介導之胞吞作用途徑被細胞攝取。此類葉酸-多核苷酸綴合物描述於美國專利第8,188,247號中,其全文以引用之方式併入本文。
鑒於PNPLA3在肝臟細胞(例如肝細胞)中表現,在某些實施方式中,期望將RNAi構建體特異性遞送至彼等肝細胞。在一些實施方式中,RNAi構建體可以藉由使用與肝細胞表面上表現之蛋白質結合或相互作用之配位基而特異性靶向肝臟。例如,在某些實施方式中,配位基可包含特異性結合肝細胞上表現之受體之抗原結合蛋白(例如抗體或其結合片段(例如Fab、scFv))。
在某些實施方式中,配位基包含碳水化合物。「碳水化合物」係指由一或多個具有至少6個碳原子(可為直鏈、支鏈或環狀)及與各碳原子鍵合之氧、氮或硫原子之單醣單元構成的化合物。碳水化合物包括但不限於糖(例如,單醣、二醣、三醣、四醣及含有約4、5、6、7、8或9個單醣單元之寡糖)及多醣,例如澱粉、糖原、纖維素及多醣膠。在一些實施方式中,摻入配位基中之碳水化合物為選自戊糖、己糖或庚糖之單醣及包括此類單醣單元之二醣及三醣。在其他實施方式中,摻入配位基中之碳水化合物為胺基糖,例如半乳糖胺、葡糖胺、N-乙醯半乳糖胺及N-乙醯葡糖胺。
在一些實施方式中,配位基包含己糖或己糖胺。己糖可選自葡萄糖、半乳糖、甘露糖、岩藻糖或果糖。己糖胺可選自果糖胺、半乳糖胺、葡糖胺或甘露糖胺。在某些實施方式中,配位基包含葡萄糖、半乳糖、半乳糖胺或葡糖胺。在一個實施方式中,配位基包含葡萄糖、葡糖胺或N-乙醯葡糖胺。在另一個實施方式中,配位基包含半乳糖、半乳糖胺或N-乙醯基 - 半乳糖胺。在特定實施方式中,配位基包含N-乙醯基 - 半乳糖胺。包含葡萄糖、半乳糖及N-乙醯基 - 半乳糖胺(GalNAc)之配位基特別有效於將化合物靶向肝細胞。參見例如D'Souza及Devarajan, J. Control Release, 第203卷: 126-139, 2015。可以摻入本發明之RNAi構建體中之含GalNAc或半乳糖之配位基的實例描述於美國專利第7,491,805號;第8,106,022號;及第8,877,917號;美國專利公佈第20030130186號;及WIPO公佈第WO 2013166155號,所有該等文獻均全文以引用之方式併入本文。
在某些實施方式中,配位基包含多價碳水化合物部分。如本文所用,「多價碳水化合物部分」係指包含兩或更多個能夠獨立地與其他分子結合或相互作用之碳水化合物單元之部分。例如,多價碳水化合物部分包含兩或更多個由碳水化合物組成之結合域,其可以結合兩或更多個不同分子或同一分子上之兩或更多個不同位點。碳水化合物部分之化合價表示該碳水化合物部分內之單個結合域的數目。例如,關於碳水化合物部分之術語「單價」、「二價」、「三價」及「四價」分別是指具有一個、兩個、三個及四個結合域的碳水化合物部分。多價碳水化合物部分可包含多價乳糖部分、多價半乳糖部分、多價葡萄糖部分、多價N-乙醯基 - 半乳糖胺部分、多價N-乙醯基 - 葡糖胺部分、多價甘露糖部分、或多價岩藻糖部分。在一些實施方式中,配位基包含多價半乳糖部分。在其他實施方式中,配位基包含多價N-乙醯基-半乳糖胺部分。在該等及其他實施方式中,多價碳水化合物部分為二價、三價或四價的。在此類實施方式中,多價碳水化合物部分可為雙觸角或三觸角的。在一個特定實施方式中,多價N-乙醯基-半乳糖胺部分為三價或四價的。在另一個特定實施方式中,多價半乳糖部分為三價或四價的。用於摻入本發明之RNAi構建體中之例示性含三價及四價GalNAc之配位基在下文詳細描述。
配位基可以直接或間接地連接或綴合到RNAi構建體至RNA分子上。例如,在一些實施方式中,配位基直接共價連接至RNAi構建體之有義鏈或反義鏈。在其他實施方式中,配位基經由連接子共價連接至RNAi構建體之有義鏈或反義鏈。配位基可以連接到本發明之RNAi構建體之多核苷酸(例如有義鏈或反義鏈)的核鹼基、糖部分或核苷酸間鍵。與嘌呤核鹼基或其衍生物之綴合或連接可發生在包括環內及環外原子在內的任何位置。在某些實施方式中,嘌呤核鹼基之2-、6-、7-或8-位連接至配位基。與嘧啶核鹼基或其衍生物之綴合或連接亦可發生在任何位置處。在一些實施方式中,嘧啶核鹼基之2-、5-及6-位可以連接至配位基。與核苷酸之糖部分的綴合或連接可以發生在任何碳原子處。糖部分之可連接至配位基的示例性碳原子包括2'、3'及5'碳原子。1'位置亦可以連接至配位基,例如在鹼性殘基中。核苷酸間鍵亦可以支持配位基連接。對於含磷鍵(例如,磷酸二酯、硫代磷酸酯、二硫代磷酸酯、胺基磷酸酯及類似者),配位基可直接連接至磷原子或與磷原子鍵合之O、N或S原子上。對於含胺或醯胺的核苷間鍵(例如PNA),配位基可連接至胺或醯胺之氮原子或連接至相鄰碳原子。
在某些實施方式中,配位基可以連接有義鏈或反義鏈之3'端或5'端。在某些實施方式中,配位基共價連接至有義鏈之5'端。在其他實施方式中,配位基共價連接至有義鏈之3'端。例如,在一些實施方式中,配位基連接至有義鏈之3'端。在某些這樣的實施方式中,配位基連接在有義鏈之3'-端核苷酸之3'-位置處。在替代實施方式中,配位基連接在有義鏈之3'端附近,但在一或多個末端核苷酸之前(即在1、2、3或4個末端核苷酸之前)。在一些實施方式中,配位基連接在有義鏈之3'-端核苷酸的糖的2'-位置處。
在某些實施方式中,配位基經由連接子連接至有義鏈或反義鏈。「連接子」為將配位基共價連接到RNAi構建體之多核苷酸組分的原子或原子團。連接子可為自約1至約30個原子長度、自約2至約28個原子長度、自約3至約26個原子長度、自約4至約24個原子長度、自約6至約20個原子長度、自約7至約20個原子長度、自約8至約20個原子長度、自約8至約18個原子長度、自約10至約18個原子長度、及自約12至約18個原子長度。在一些實施方式中,連接子可包含雙官能連接部分,其通常包含具有兩個官能基之烷基部分。選擇一個官能基以結合目標化合物(例如RNAi構建體之有義鏈或反義鏈),且選擇另一個官能基以基本上結合任何選定基團,例如本文所述之配位基。在某些實施方式中,連接子包含重複單元,例如乙二醇或胺基酸單元之鏈結構或寡聚物。通常用於雙官能連接部分之官能基的實例包括但不限於用於與親核基團反應之親電子試劑及用於與親電子基團反應之親核試劑。在一些實施方式中,雙官能連接部分包括胺基、羥基、羧酸、硫醇、不飽和度(例如,雙鍵或三鍵)、及類似基團。
可用於將配位基連接至本發明之RNAi構建體中之有義鏈或反義鏈的連接子包括但不限於吡咯啶、8-胺基-3,6-二氧雜辛酸、4-(N-馬來醯亞胺基甲基)環己烷-1-羧酸琥珀醯亞胺酯、6-胺基己酸、經取代之C1-C10烷基、經取代或未經取代之C2-C10烯基或經取代或未經取代之C2-C10炔基。此類連接子之較佳取代基包括但不限於羥基、胺基、烷氧基、羧基、苄基、苯基、硝基、硫醇、硫代烷氧基、鹵素、烷基、芳基、烯基及炔基。
在某些實施方式中,連接子為可裂解的。可裂解連接子為在細胞外足夠穩定,但是在進入靶細胞後經裂解以釋放連接子保持在一起之兩個部分的連接子。在一些實施方式中,可裂解連接子在靶細胞中或在第一參考條件下(其可以例如經選擇以模擬或代表細胞內條件)中比在受試者血液中或在第二參考條件下(其可以例如經選擇以模擬或代表在血液或血清中發現之條件)裂解快至少10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍或更多、或至少100倍。
可裂解連接子易受裂解劑影響,例如pH、氧化還原電位或降解分子之存在。通常,裂解劑在細胞內比在血清或血液中更普遍或以更高水平或活性被發現。此類降解劑之實例包括:經選擇用於特定受質或不具有受質特異性之氧化還原劑,包括例如存在於細胞中之氧化或還原酶或還原劑,諸如硫醇,其可藉由還原來使氧化還原可裂解連接子降解;酯酶;可形成酸性環境之內體或藥劑,例如產生pH 5或更低之彼等者;藉由充當一般酸來使酸可裂解連接子水解或降解之酶、肽酶(可為受質特異性的)、及磷酸酶。
可裂解的連接子可包含對pH敏感之部分。人血清之pH為7.4,而平均細胞內pH略低,範圍為自約7.1-7.3。內體具有更高酸性pH,在5.5-6.0範圍內,且溶酶體具有約5.0之甚至更高酸性pH。一些連接子將具有可裂解基團,其在較佳pH下裂解,從而將RNA分子自配位基釋放到細胞內,或進入細胞之所要區室。
連接子可包括可由特定酶裂解之可裂解基團。摻入連接子中之可裂解基團的類型可取決於待靶向之細胞。例如,肝靶向配位基可以藉由包括酯基之連接子連接至RNA分子。肝細胞富含酯酶,且因此該連接子在肝細胞中將比在不富含酯酶之細胞類型中更有效地裂解。富含酯酶之其他類型的細胞包括肺、腎皮質及睾丸之細胞。當靶向富含肽酶之細胞(例如肝細胞及滑膜細胞)時,可以使用含有肽鍵之連接子。
通常,可以藉由測試降解劑(或條件)裂解候選連接子之能力來評估候選可裂解連接子的適用性。亦期望亦測試候選可裂解連接子在血液中或當與其他非靶組織接觸時抵抗裂解之能力。因此,可以判定第一條件與第二條件之間對裂解之相對易感性,其中第一條件經選擇為指示靶細胞中之裂解,而第二條件經選擇為指示其他組織或生物流體,例如血液或血清中之裂解。評估可以在無細胞系統、細胞、細胞培養物、器官或組織培養物或整個動物中進行。在無細胞或培養條件下進行初步評估且藉由對整個動物進行進一步評估來確認可能是有用的。在一些實施方式中,有用候選連接子在細胞中(或在經選擇以模擬細胞內條件之活體外條件下)與血液或血清(或在經選擇以模擬細胞外條件之活體外條件下)相比,裂解快至少2、4、10、20、50、70或100倍。
在其他實施方式中,使用氧化還原可裂解連接子。氧化還原可裂解連接子在還原或氧化時裂解。還原性可裂解基團之實例為二硫鍵連接基團(-S-S-)。為判定候選可裂解連接子是否為合適的「可還原裂解連接子」,或者例如適合與特定RNAi構建體及特定配位基一起使用,可以使用一或多種本文所述之方法。例如,可以藉由與二硫蘇糖醇(DTT)或此項技術已知之其他還原劑一起溫育來評估候選連接子,其模擬將在細胞(例如靶細胞)中觀察到之裂解速率。候選連接子亦可以在經選擇以模擬血液或血清條件之條件下進行評估。在一特定實施方式中,候選連接子在血液中裂解至多10%。在其他實施方式中,有用候選連接子在細胞中(或在經選擇以模擬細胞內條件之活體外條件下)與血液(或在經選擇以模擬細胞外條件之活體外條件下)相比,降解快至少2倍、4倍、10倍、20倍、50倍、70倍或100倍。
在其他實施方式中,基於磷酸酯之可裂解連接子藉由降解或水解磷酸基團之試劑來裂解。水解細胞中之磷酸酯基團之試劑的實例為酶,例如細胞中之磷酸酶。基於磷酸酯之可裂解基團之實例為-O-P(O)(ORk)-O-、-O-P(S)(ORk)-O-、-O-P(S)(SRk)-O-、-S-P(O)(ORk)-O-、-O-P(O)(ORk)-S-、-S-P(O)(ORk)-S-、-O-P(S)(ORk)-S-、-S-P(S)(ORk)-O-、-O-P(O)(Rk)-O-、-O-P(S)(Rk)-O-、-S-P(O)(Rk)-O-、-S-P(S)(Rk)-O-、-S-P(O)(Rk)-S-、-O-P(S)(Rk)-S-。具體實施方式包括 -O-P(O)(OH)-O-、-O-P(S)(OH)-O-、-O-P(S)(SH)-O-、-S-P(O)(OH)-O-、-O-P(O)(OH)-S-、-S-P(O)(OH)-S-、-O-P(S)(OH)-S-、-SP(S)(OH)-O-、-O-P(O)(H)-O-、-O-P(S)(H)-O-、-S-P(O)(H)-O-、-S-P(S)(H)-O-、-S-P(O)(H)-S-、-O-P(S)(H)-S-。另一具體實施方式為-O-P(O)(OH)-O-。該等候選連接子可以使用與上述彼等者類似之方法評估。
在其他實施方式中,連接子可包含酸可裂解基團,該等基團為在酸性條件下裂解之基團。在一些實施方式中,酸可裂解基團在pH為約6.5或更低(例如,約6.0、5.5、5.0或更低)之酸性環境中裂解,或藉由試劑諸如可充當一般酸之酶裂解。在細胞中,特定的低pH細胞器,例如內體及溶酶體,可以為酸可裂解基團提供裂解環境。酸可裂解連接基團之實例包括但不限於腙、酯及胺基酸酯。酸可裂解基團可具有通式-C=NN-、C(O)O或-OC(O)。一特定實施方式為當連接至酯(烷氧基)之氧之碳為芳基、經取代之烷基或第三烷基諸如二甲基、戊基或第三丁基時。該等候選物可以使用與上述彼等者類似之方法評估。
在其他實施方式中,連接子可包含基於酯之可裂解基團,該等基團藉由細胞中之酶,例如酯酶及醯胺酶來裂解。基於酯之可裂解基團的實例包括但不限於伸烷基、伸烯基及伸炔基之酯。酯可裂解基團具有通式-C(O)O-或-OC(O)-。該等候選連接子可以使用與上述彼等者類似之方法評估。
在其他實施方式中,連接子可以包含基於肽之可裂解基團,該等基團藉由細胞中之酶,例如肽酶及蛋白酶來裂解。基於肽之可裂解基團為在胺基酸之間形成之肽鍵,以產生寡肽(例如,二肽、三肽等)及多肽。基於肽之可裂解基團不包括醯胺基團(-C(O)NH-)。醯胺基團可以在任何伸烷基、伸烯基或伸烷基之間形成。肽鍵為在胺基酸之間形成之特殊類型的醯胺鍵,以產生肽及蛋白質。基於肽之裂解基團通常限於在產生肽及蛋白質之胺基酸之間形成之肽鍵(即醯胺鍵),且不包括完整醯胺官能基。基於肽之可裂解連接基團具有通式-NHCHRAC(O)NHCHRBC(O)-,其中RA及RB為兩個相鄰胺基酸之R基團。該等候選物可以使用與上述彼等者類似之方法評估。
適合於將配位基連接到本發明之RNAi構建體中之有義鏈或反義鏈的其他類型的連接子為此項技術已知的,且可以包括美國專利第7,723,509;8,017,762;8,828,956;8,877,917;及9,181,551號中描述之連接子,所有該等文獻均全文以引用之方式併入本文。
在某些實施方式中,共價連接至本發明之RNAi構建體之有義鏈或反義鏈的配位基包含GalNAc部分,例如多價GalNAc部分。在一些實施方式中,多價GalNAc部分為三價GalNAc部分且連接至有義鏈之3'端。在其他實施方式中,多價GalNAc部分為三價GalNAc部分且連接至有義鏈之5'端。在其他實施方式中,多價GalNAc部分為四價GalNAc部分且連接至有義鏈之3'端。在其他實施方式中,多價GalNAc部分為四價GalNAc部分且連接至有義鏈之5'端。
在一些實施方式中,可以藉由投與編碼及控制RNAi構建體之細胞內表現的載體,將本發明之RNAi構建體遞送至目標細胞或組織。「載體」(在本文中亦稱為「表現載體」)為物質組成物,其可用於將目標核酸遞送至細胞內部。此項技術已知許多載體,包括但不限於直鏈多核苷酸、與離子或兩親化合物締合之多核苷酸、質體、及病毒。因此,術語「載體」包括自主複製的質體或病毒。病毒載體之實例包括但不限於腺病毒載體、腺相關病毒載體、逆轉錄病毒載體、及類似載體。載體可以在活細胞中複製,或者可以合成製成。
通常,用於表現本發明之RNAi構建體之載體將包含一或多個與編碼RNAi構建體之序列可操作地連接之啟動子。本文所用片語「可操作地連接」或「在轉錄控制下」係指啟動子相對於多核苷酸序列處於正確位置及方向,以控制RNA聚合酶之轉錄起始及多核苷酸序列之表現。「啟動子」係指由啟動基因序列特異性轉錄所需之細胞合成機制或經引入之合成機制識別的序列。合適的啟動子包括但不限於RNA pol I、pol II、HI或U6 RNA pol III、及病毒啟動子(例如人巨細胞病毒(CMV)立即早期基因啟動子、SV40早期啟動子、及勞斯氏肉瘤病毒長終端重複)。在一些實施方式中,HI或U6RNA pol III啟動子為較佳的。啟動子可為組織特異性或誘導型啟動子。特別感興趣的是肝特異性啟動子,例如來自人α1-抗胰蛋白酶基因、白蛋白基因、血紅素結合蛋白基因、及肝脂酶基因之啟動子序列。誘導型啟動子包括由蛻皮激素、***、孕酮、四環素、及異丙基-PD1-硫代哌喃半乳糖苷(IPTG)調節之啟動子。
在RNAi構建體包含siRNA之一些實施方式中,兩條單獨鏈(有義鏈及反義鏈)可以自單個載體或兩個單獨載體表現。例如,在一個實施方式中,編碼有義鏈之序列與第一載體上之啟動子可操作地連接,且編碼反義鏈之序列與第二載體上之啟動子可操作地連接。在此實施方式中,第一載體及第二載體例如藉由感染或轉染共同引入靶細胞,使得有義鏈及反義鏈一旦轉錄,將在細胞內雜交以形成siRNA分子。在另一個實施方式中,有義鏈及反義鏈由位於單一載體中之兩個單獨啟動子轉錄。在一些此類實施方式中,編碼有義鏈之序列與第一啟動子可操作地連接,且編碼反義鏈之序列與第二啟動子可操作地連接,其中第一啟動子及第二啟動子位於單個載體中。在一個實施方式中,載體包含與編碼siRNA分子之序列可操作地連接之第一啟動子及在相反方向上與相同序列可操作地連接之第二啟動子,使得自第一啟動子轉錄序列使得siRNA分子之有義鏈得以合成,且自第二啟動子轉錄序列使得siRNA分子之反義鏈得以合成。
在RNAi構建體包含shRNA之其他實施方式中,編碼單個至少部分自身互補的RNA分子之序列與啟動子可操作地連接以產生單個轉錄物。在一些實施方式中,編碼shRNA之序列包含藉由連接子多核苷酸序列連接之反向重複序列,以在轉錄後產生shRNA之莖及環結構。
在一些實施方式中,編碼本發明之RNAi構建體之載體為是病毒載體。適用於表現本文所述RNAi構建體之各種病毒載體系統包括但不限於腺病毒載體、逆轉錄病毒載體(例如慢病毒載體、馬洛尼鼠白血病病毒)、腺相關病毒載體;單純皰疹病毒載體;SV 40載體;多瘤病毒載體;乳頭狀瘤病毒載體;小核糖核酸病毒載體;及痘病毒載體(例如痘苗病毒)。在某些實施方式中,病毒載體為逆轉錄病毒載體(例如慢病毒載體)。
適用於在本發明中使用之各種載體、用於將編碼siRNA或shRNA分子之核酸序列***載體中之方法、及將載體遞送至目標細胞之方法均處於熟習此項技術者之能力範圍內。參見例如Dornburg , Gene Therap., 第2卷: 301-310, 1995;Eglitis, Biotechniques, 第6卷: 608-614, 1988;Miller, HumGene Therap., 第1卷: 5-14, 1990;Anderson, Nature, 第392卷: 25-30, 1998;Rubinson D A等人, Nat. Genet., 第33卷: 401-406, 2003;Brummelkamp等人, Science, 第296卷: 550-553, 2002;Brummelkamp等人, Cancer Cell, 第2卷: 243-247, 2002;Lee等人, Nat Biotechnol, 第20卷:500-505, 2002;Miyagishi等人, Nat Biotechnol, 第20卷: 497-500, 2002;Paddison等人, GenesDev, 第16卷: 948-958, 2002;Paul等人, Nat Biotechnol, 第20卷: 505-508, 2002;Sui等人, ProcNatl Acad Sci USA, 第99卷: 5515-5520, 2002;及Yu等人, Proc Natl Acad Sci USA, 第99卷: 6047-6052, 2002,所有該等文獻均全文以引用之方式併入本文。
本發明亦包括藥物組成物及調配物,其包含本文所述之RNAi構建體及藥學上可接受之載體、賦形劑或稀釋劑。此類組成物及調配物適用於降低有需要的受試者中PNPLA3之表現。當考慮臨床應用時,將以適合於預期應用之形式製備藥物組成物及調配物。通常,這將需要製備基本上不含熱原以及可能對人或動物有害的其它雜質的組成物。
片語「藥學上可接受之」或「藥理學上可接受之」係指當投與動物或人時不產生不利、過敏或其他不良反應之分子實體及組成物。如本文所用,「藥學上可接受之載體、賦形劑或稀釋劑」包括可接受用於調配藥物,例如適合投與人類的藥物之溶劑、緩衝劑、溶液、分散介質、包衣、抗細菌及抗真菌劑、等滲及吸收延遲劑等。該等介質及試劑用於藥物活性物質之用途為此項技術熟知的。除非任何習知介質或試劑與本發明之RNAi構建體不相容,否則考慮其在治療組成物中之用途。補充活性成分亦可摻入組成物中,條件係它們不會使組成物之載體或RNAi構建體失活。
用於調配藥物組成物之組成物及方法取決於許多標準,包括但不限於投與途徑、待治療之疾病或病症之類型及程度、或投與劑量。在一些實施方式中,基於預期遞送途徑調配藥物組成物。例如,在某些實施方式中,調配藥物組成物以用於非經腸遞送。非經腸遞送形式包括靜脈內、動脈內、皮下、鞘內、腹膜內或肌內注射或輸注。在一個實施方式中,調配藥物組成物以用於靜脈內遞送。在此實施方式中,藥物組成物可包括基於脂質之遞送媒介物。在另一個實施方式中,調配藥物組成物以用於皮下遞送。在此實施方式中,藥物組成物可包括靶向配位基(例如,本文所述之含有GalNAc之配位基)。
在一些實施方式中,藥物組成物包含有效量之本文所述RNAi構建體。「有效量」為足以產生有益或期望的臨床結果之量。在一些實施方式中,有效量為足以降低受試者肝細胞中之PNPLA3表現之量。在一些實施方式中,有效量可為足以僅部分降低PNPLA3表現之量,例如,降低至與人雜合子中野生型PNPLA3等位基因之表現相當的水平。據報道,與非攜帶者相比,功能喪失的PNPLA3變異體等位基因的人雜合子攜帶者具有較低非HDL膽固醇血清水平及較低冠狀動脈疾病及心肌梗死風險(Nioi等人, New England Journal of Medicine, 第374卷第22期: 2131-2141, 2016)。因此,不受理論束縛,據信PNPLA3表現之部分降低可足以實現血清非HDL膽固醇之有益降低及冠狀動脈疾病及心肌梗死之風險降低。
本發明之RNAi構建體之有效量可為自約0.01 mg/kg體重至約100 mg/kg體重、約0.05 mg/kg體重至約75 mg/kg體重、約0.1 mg/kg體重至約50 mg/kg體重、約1 mg/kg至約30 mg/kg體重、約2.5 mg/kg體重至約20 mg/kg體重或約5 mg/kg體重至約15 mg/kg體重。在某些實施方式中,本發明之RNAi構建體之單一有效劑量可為約0.1 mg/kg、約0.5 mg/kg、約1 mg/kg、約2 mg/kg、約3 mg/kg、約4 mg/kg、約5 mg/kg、約6 mg/kg、約7 mg/kg、約8 mg/kg、約9 mg/kg或約10 mg/kg。包含有效量之RNAi構建體的藥物組成物可以每週、每兩週、每月、每季度或每半年投與。準確判定有效量及投與頻率可以基於若干因素,包括患者大小、年齡及一般狀況、待治療之病症類型(例如心肌梗死、心臟衰竭、冠狀動脈疾病、高膽固醇血症)、使用之特定RNAi構建體及投與途徑。可以使用習知方法及/或在合適的動物模型中測試來確定本發明之任何特定RNAi構建體之有效劑量及活體內半衰期的估計值。
投與本發明之藥物組成物可以經由任何常規途徑,只要靶組織可經由該途徑獲得。此類途徑包括但不限於非經腸(例如,皮下、肌內、腹膜內或靜脈內)、經口、經鼻、經頰、皮內、經皮、及舌下途徑,或藉由直接注射入肝臟組織或經由肝門靜脈遞送。在一些實施方式中,非經腸投與藥物組成物。例如,在某些實施方式中,靜脈內投與藥物組成物。在其他實施方式中,皮下投與藥物組成物。
膠體分散系統,例如大分子複合物、奈米膠囊、微球、珠粒、及基於脂質之系統,包括水包油乳液、膠束、混合膠束、及脂質體,可以用作本發明之RNAi構建體或編碼此類結構之載體的遞送媒介物。適用於遞送本發明之核酸之市售脂肪乳液包括Intralipid®、Liposyn®、Liposyn®II、Liposyn®III、Nutrilipid、及其他類似脂質乳液。用作活體內遞送媒介物之較佳膠體系統為脂質體(即人工膜囊泡)。本發明之RNAi構建體可以包封在脂質體內或可以與其形成複合物,特別是與陽離子脂質體形成複合物。或者,本發明之RNAi構建體可以與脂質複合,特別是與陽離子脂質複合。合適脂質及脂質體包括中性的(例如,二油醯磷脂醯乙醇胺(DOPE)、二肉豆蔻醯磷脂醯膽鹼(DMPC)、及二棕櫚醯磷脂醯膽鹼(DPPC))、二硬脂醯磷脂醯膽鹼)、陰性(例如,二肉豆蔻醯磷脂醯甘油(DMPG))、及陽性的(例如,二油醯四甲基胺基丙基(DOTAP)及二油醯磷脂醯乙醇胺(DOTMA))。此類膠體分散系統之製備及使用為此項技術熟知的。例示性調配物亦揭示於美國專利第5,981,505號;美國專利第6,217,900號;美國專利第6,383,512號;美國專利第5,783,565號;美國專利第7,202,227號;美國專利第6,379,965號;美國專利第6,127,170號;美國專利第5,837,533號;美國專利第6,747,014號;及W0 03/093449。
在一些實施方式中,本發明之RNAi構建體完全包封於脂質調配物中,例如,以形成SPLP、pSPLP、SNALP、或其他核酸 - 脂質顆粒。如本文所用,術語「SNALP」係指穩定的核酸-脂質顆粒,包括SPLP。如本文所用,術語「SPLP」係指包含包封於脂質囊泡內之質體DNA的核酸-脂質顆粒。SNALP及SPLP通常含有陽離子脂質、非陽離子脂質及防止顆粒聚集之脂質(例如,PEG-脂質綴合物)。SNALP及SPLP特別適用於全身應用,因為它們在靜脈內注射後表現出延長的循環壽命,且在遠端部位(例如,與投與部位物理分離之部位)處積聚。SPLP包括「pSPLP」,其包括PCT公佈第WO 00/03683號中所述之經包封縮合劑 - 核酸複合物。核酸 - 脂質顆粒通常具有約50 nm至約150 nm、約60 nm至約130 nm、約70 nm至約110 nm、或約70 nm至約90 nm之平均直徑,且基本上無毒。另外,核酸當存在於核酸 - 脂質顆粒中時在水溶液中對用核酸酶之降解具有抗性。核酸-脂質顆粒及其製備方法揭示於例如美國專利第5,976,567;5,981,501;6,534,484;6,586,410;6,815,432號;及PCT公佈第WO 96/40964號。
適於可注射使用之藥物組成物包括例如無菌水溶液或分散液及用於臨時製備無菌可注射溶液或分散液之無菌粉末。通常,該等製劑為無菌的且流動到易於注射之程度。製劑在製造及儲存條件下應保持穩定,且應防止微生物諸如細菌及真菌之污染作用。合適的溶劑或分散介質可包含例如水、乙醇、多元醇(例如,甘油、丙二醇、及液體聚乙二醇、及類似物)、其合適的混合物、及植物油。恰當流動性可例如藉由使用衣料諸如卵磷脂、在分散液情況下藉由保持所需粒度且藉由使用界面活性劑來保持。可以藉由各種抗細菌抗真菌劑,例如對羥基苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞、及類似試劑來防止微生物之作用。在許多情況下,較佳包括等滲劑,例如糖或氯化鈉。藉由在組成物中使用延遲吸收試劑,例如單硬脂酸鋁及明膠,可以實現可注射組成物之延長吸收。
無菌可注射溶液可以藉由將活性化合物按需要以適當量與任何其他成分(例如以上列舉者)一起摻入溶劑中,然後過濾滅菌來製備。通常,藉由將各種滅菌活性成分摻入無菌媒介物中來製備分散液,該無菌載體含有基礎分散介質及所要其它成分,例如,如上所列舉者。在用於製備無菌可注射溶液之無菌粉末的情況下,較佳製備方法包括真空乾燥及冷凍乾燥技術,其產生一或多種活性成分加上來自其先前無菌過濾溶液之任何其他所要成分之粉末
本發明之組成物通常可以調配成中性或鹽形式。藥學上可接受之鹽包括例如衍生自無機酸(例如鹽酸或磷酸)或衍生自有機酸(例如乙酸、草酸、酒石酸、扁桃酸及類似酸)之酸加成鹽(由游離胺基形成)。用游離羧基形成之鹽亦可衍生自無機鹼(例如氫氧化鈉、氫氧化鉀、氫氧化銨、氫氧化鈣或氫氧化鐵)或有機鹼(例如異丙胺、三甲胺、組胺酸、普魯卡因、及類似鹼)。
例如,對於在水溶液中之非經腸投與,通常將溶液適當地緩衝,且首先例如用足夠鹽水或葡萄糖使液體稀釋劑等滲。此類水溶液可用於例如靜脈內、肌肉內、皮下及腹膜內投與。較佳地,如熟習此項技術者已知的,特別是根據本發明,使用無菌含水介質。舉例說明,可將單劑量溶於1 ml等滲NaCl溶液中,且加入到1000 ml皮下注射液中或在所建議輸注部位注射(參見例如「Remington's Pharmaceutical Sciences」第15版, 第1035-1038頁及第1570-1580頁)。對於人類投與,製劑應符合FDA標準所要求之無菌、致熱原性、一般安全性及純度標準。在某些實施方式中,本發明之藥物組成物包含無菌鹽水溶液及本文所述之RNAi構建體或由其組成。在其他實施方式中,本發明之藥物組成物包含本文所述之RNAi構建體及無菌水(例如注射用水,WFI)或由其組成。在其他實施方式中,本發明之藥物組成物包含本文所述之RNAi構建體及磷酸鹽緩衝鹽水(PBS)或由其組成。
在一些實施方式中,本發明之藥物組成物與用於投與之裝置一起包裝或儲存於該裝置內。用於可注射調配物之裝置包括但不限於注射口、預填充注射器、自動注射器、注射泵、體內注射器、及注射筆。用於霧化或粉末調配物之裝置包括但不限於吸入器、吹入器、吸氣器、及類似裝置。因此,本發明包括投與裝置,其包含本發明的用於治療或預防一或多種本文所述病症之藥物組成物。抑制 PNPLA3 表現之方法
本發明亦提供抑制細胞中之PNPLA3基因表現的方法。該等方法包括使細胞與有效抑制細胞中之PNPLA3表現之量的RNAi劑(例如雙鏈RNAi劑)接觸,從而抑制細胞中之PNPLA3表現。細胞與RNAi劑(例如雙鏈RNAi劑)之接觸可以在活體外或活體內進行。使活體內細胞與RNAi劑接觸包括使受試者(例如人受試者)體內之細胞或細胞群與RNAi劑接觸。活體外及活體內接觸細胞之方法的組合亦為可能。
本發明提供用於在有需要之受試者中降低或抑制PNPLA3表現的方法以及治療或預防與PNPLA3表現或活性相關之病狀、疾病或病症的方法。「與PNPLA3表現相關之病狀、疾病或病症」係指其中PNPLA3表現水平發生改變或PNPLA3表現水平升高與發展病狀、疾病或病症之風險增加相關之病狀、疾病或病症。
如上所述,接觸細胞可為直接的或間接的。此外,可以經由靶向配位基實現與細胞接觸,該靶向配位基包括本文所述或此項技術已知之任何配位基。在較佳實施方式中,靶向配位基為碳水化合物部分,例如GalNAc3配位基或將RNAi劑引導至目標位點的任何其他配位基。
在一個實施方式中,使細胞與RNAi接觸包括經由促進或實現攝取或吸收至細胞中來「引入」或「將RNAi遞送到細胞中」。RNAi之吸收或攝取可以藉由無輔助擴散或活性細胞過程或藉由輔助劑或裝置發生。將RNAi引入細胞可為活體外及/或活體內。例如,對於活體內引入,可以將RNAi注射到組織部位或全身投與。活體外引入到細胞包括此項技術已知之方法,例如電穿孔及脂質轉染。其他方法在下文中描述及/或在此項技術中為已知的。
如本文所用,術語「抑制」可與「降低」、「緘默」、「下調」、「抑制」及其他類似術語互換使用,且包括任何水平的抑制。
片語「抑制PNPLA3表現」旨在係指抑制任何PNPLA3基因(例如小鼠PNPLA3基因、大鼠PNPLA3基因、猴PNPLA3基因、或人PNPLA3基因)以及PNPLA3基因之變異體或突變體的表現。因此,PNPLA3基因可為野生型PNPLA3基因、突變體PNPLA3基因(例如產生澱粉樣蛋白沈積之突變體PNPLA3基因)、或遺傳操作細胞、細胞群、或有機體背景下之轉基因PNPLA3基因。
「抑制PNPLA3基因之表現」包括任何水平的PNPLA3基因抑制,例如,至少部分抑制PNPLA3基因之表現。可以基於與PNPLA3基因表現相關之任何變量的水平或水平變化,例如PNPLA3 mRNA水平、PNPLA3蛋白水平、或澱粉樣沈積物之數量或程度來評估PNPLA3基因之表現。可以在單個細胞或細胞群中評估該水平,包括例如衍生自受試者之樣本。
可以藉由與PNPLA3表現相關之一或多個變量的絕對或相對水平與對照水平相比之降低來評估抑制。對照水平可為此項技術中使用之任何類型的對照水平,例如,給藥前基線水平、或由未經處理或用對照(例如僅緩衝對照或非活性劑對照)處理之類似受試者、細胞或樣本判定之水平。在本發明方法之一些實施方式中,PNPLA3基因之表現被抑制至少約5%、至少約10%、至少約15%、至少約20%、至少約25%、至少約30%、至少約35%、至少約40%、至少約45%、至少約50%、至少約55%、至少約60%、至少約65%、至少約70%、至少約75%、至少約80%、至少約85%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、或至少約99%。
PNPLA3基因表現之抑制可以藉由以下來證明:與第一細胞或細胞群基本相同但未進行過如此處理之第二細胞或細胞群(對照細胞)相比,由PNPLA3基因被轉錄且已經被處理(例如,藉由使一或多個細胞與本發明之RNAi劑接觸,或藉由將本發明之RNAi劑投與存在細胞之受試者)之第一細胞或細胞群(此類細胞可以存在於例如來自受試者之樣本中)表現之mRNA的量降低,使得PNPLA3基因之表現受到抑制。在較佳實施方式中,藉由使用下式將經處理細胞中之mRNA水平表示為對照細胞中之mRNA水平的百分比來評估抑制:
或者,可以根據與PNPLA3基因表現功能性相關之參數的減小來評估PNPLA3基因表現之抑制,該參數例如PNPLA3蛋白表現或Hedgehog途徑蛋白活性。PNPLA3基因緘默可以在表現PNPLA3之任何細胞中組成型或藉由基因組工程,且藉由此項技術已知之任何測定來判定。
PNPLA3蛋白表現之抑制可以藉由細胞或細胞群表現之PNPLA3蛋白水平(例如,來源於受試者之樣本中表現之蛋白水平)的降低來證明。如上所述,為評估mRNA抑制,經處理細胞或細胞群中蛋白質表現水平之抑制可以類似地表示為對照細胞或細胞群中蛋白質水平之百分比。
可用於評估PNPLA3基因表現抑制之對照細胞或細胞群包括尚未與本發明之RNAi劑接觸之細胞或細胞群。例如,對照細胞或細胞群可以源自在用RNAi劑治療受試者之前的個體受試者(例如,人或動物受試者)。
可以使用此項技術已知用於評估mRNA表現之任何方法判定由細胞或細胞群表現之PNPLA3 mRNA水平或循環PNPLA3 mRNA水平。在一個實施方式中,藉由偵測經轉錄多核苷酸或其部分,例如PNPLA3基因之mRNA來判定樣本中之PNPLA3表現水平。可以使用RNA提取技術自細胞中提取RNA,該等提取技術包括例如使用酸性酚/異硫氰酸胍提取(RNAzol B;生物起源公司(Biogenesis))、RNeasy RNA製備套組(kit)(凱傑公司(Qiagen))或PAXgene(瑞士普裡阿勒蒂克斯公司(PreAnalytix,Switzerland))。利用核糖核酸雜交之典型測定形式包括核連續測定、RT-PCR、RNA酶保護測定(Melton等人, Nuc. Acids Res. 12:7035)、北方墨點法、原位雜交、及微陣列分析。可以使用PCT/US 2012/043584中描述之方法偵測循環PNPLA3 mRNA,該專利全文以引用之方式併入本文。
在一個實施方式中,使用核酸探針判定PNPLA3表現水平。如本文所用,術語「探針」係指能夠選擇性結合特定PNPLA3的任何分子。探針可由熟習此項技術者合成,或衍生自適當生物製劑。探針可以經專門設計以用於標記。可用作探針之分子的實例包括但不限於RNA、DNA、蛋白質、抗體、及有機分子。
經分離之mRNA可用於雜交或擴增測定,該等測定包括但不限於南方或北方分析、聚合酶鏈反應(PCR)分析及探針陣列。一種用於判定mRNA水平之方法包括使經分離之mRNA與可與PNPLA3 mRNA雜交之核酸分子(探針)接觸。在一個實施方式中,將mRNA固定於固體表面上且與探針接觸,例如藉由在瓊脂糖凝膠上運行經分離之mRNA且將mRNA自凝膠轉移至膜,例如硝酸纖維素。在另一個替代性實施方式中,將一或多個探針固定於固體表面上,且使mRNA與一或多個探針接觸,例如在Affymetrix基因芯片陣列中。技術人員可以容易地使用適用於判定PNPLA3 mRNA水平之已知mRNA偵測方法。
用於判定樣本中之PNPLA3表現水平的另一種方法包括對樣本中之例如mRNA進行核酸擴增及/或逆轉錄酶(以製備cDNA)的過程,例如藉由RT-PCR(實驗實施方式闡述於Mullis, 1987, 美國專利第4,683,202號中)、連接酶鏈反應(Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193)、自我持續序列複製(Guatelli 等人 (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878)、轉錄擴增系統(Kwoh 等人(1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177)、Q-β複製酶(Lizardi 等人 (1988) Bio/Technology 6: 1197)、滾環複製(Lizardi 等人, 美國專利第5,854,033號)或者任何其他核酸擴增方法,然後使用熟習此項技術者熟知之技術偵測經擴增之分子。若該等分子以極低數量存在,則該等偵測方案尤其適用於偵測核酸分子。在本發明之特定態樣中,PNPLA3表現水平藉由定量螢光RT-PCR(即TaqMan™系統)判定。可以使用膜墨點(例如用於雜交分析,例如北方、南方、斑點、及類似方法)或微孔、樣本管、凝膠、珠粒或纖維(或包含結合核酸之任何固體支持物)來監測PNPLA3 mRNA之表現水平。參見美國專利第5,770,722號、第5,874,219號、第5,744,305號、第5,677,195號及第5,445,934號,該等專利全文以引用之方式併入本文。PNPLA3表現水平之判定亦可包括在溶液中使用核酸探針。
在較佳實施方式中,使用分支DNA(bDNA)測定或即時PCR(qPCR)評估mRNA表現水平。在本文提供之實例中描述且舉例說明該等方法之用途。
可以使用此項技術已知用於量測蛋白質水平之任何方法來判定PNPLA3蛋白質表現水平。該等方法包括例如電泳、毛細管電泳、高效液相層析(HPLC)、薄層層析(TLC)、超擴散層析、流體或凝膠沈澱素反應、吸收光譜、比色測定、分光光度測定、流式細胞術、免疫擴散(單或雙)、免疫電泳、西方墨點、放射免疫測定(RIA)、酶聯免疫吸附測定(ELISA)、免疫螢光測定、電化學發光測定、及類似測定。
在一些實施方式中,可以藉由偵測或監測PNPLA3疾病症狀之減輕來監測本發明之方法之功效,例如四肢、面部、喉、上呼吸道、腹部、軀幹、及生殖器之水腫腫脹,前驅症狀;喉腫脹;非瘙癢性皮疹;噁心;嘔吐;或腹痛之減輕。可以使用此項技術已知之任何方法在活體外或活體內評估該等症狀。
在本發明之方法之一些實施方式中,將RNAi劑投與受試者,使得RNAi劑遞送至受試者體內之特定部位。可以使用來自受試者內特定部位之液體或組織的樣本中之PNPLA3 mRNA或PNPLA3蛋白水平或水平變化的測量結果來評估PNPLA3表現之抑制。在較佳實施方式中,該等部位選自由肝、脈絡叢、視網膜、及胰腺所組成之群。該部位亦可為來自任一上述部位之細胞的小部分或子組。該部位亦可包括表現特定類型受體之細胞。治療或預防 PNPLA3 相關疾病之方法
本發明提供治療及預防方法,其包括向患有PNPLA3相關疾病、病症及/或病狀或易於發展PNPLA3相關疾病、病症及/或病狀之受試者投與包含RNAi劑之組成物、包含RNAi劑之藥物組成物、或包含本發明之RNAi之載體。PNPLA3相關疾病之非限制性實例包括例如脂肪肝(脂肪變性)、非酒精性脂肪性肝炎(NASH)、肝硬化、肝臟中之脂肪積累、肝臟炎症、肝細胞壞死、肝纖維化、肥胖症、或非酒精性脂肪性肝病(NAFLD)。在一個實施方式中,PNPLA3相關疾病為NAFLD。在另一個實施方式中,PNPLA3相關疾病為NASH。在另一個實施方式中,PNPLA3相關疾病為脂肪肝(脂肪變性)。在另一個實施方式中,PNPLA3相關疾病為胰島素抗性。在另一個實施方式中,PNPLA3相關疾病並非胰島素抗性。
在某些實施方式中,本發明提供一種用於在有需要患者中降低PNPLA3表現之方法,其包括向患者投與本文所述之任何RNAi構建體。如本文所用,術語「患者」係指哺乳動物,包括人,且可與術語「受試者」互換使用。較佳地,與未接受RNAi構建體之患者中之PNPLA3表現水平相比,患者肝細胞中之PNPLA3表現水平在投與RNAi構建體後降低。
本發明之方法適用於治療患有PNPLA3相關疾病之受試者,例如受益於PNPLA3基因表現及/或PNPLA3蛋白產生得以降低之受試者。在一態樣中,本發明提供降低患有非酒精性脂肪性肝病(NAFLD)之受試者中含有Patatin樣磷脂酶結構域3(PNPLA3)基因表現水平之方法。在另一態樣中,本發明提供降低患有NAFLD之受試者中之PNPLA3蛋白水平的方法。本發明亦提供降低患有NAFLD之受試者中之hedgehog途徑的活性水平的方法。
在另一態樣中,本發明提供治療患有NAFLD之受試者的方法。在一態樣中,本發明提供治療患有PNPLA3相關疾病之受試者的方法,該等疾病例如脂肪肝(脂肪變性)、非酒精性脂肪性肝炎(NASH)、肝硬化、肝臟中之脂肪積累、肝臟炎症、肝細胞壞死、肝纖維化、肥胖症、或非酒精性脂肪性肝病(NAFLD)。本發明之治療方法(及用途)包括向受試者例如人投與治療有效量的本發明的靶向PNPLA3基因之RNAi劑、或包含本發明的靶向PNPLA3基因之RNAi劑之藥物組成物、或包含靶向PNPLA3基因之RNAi劑的本發明的載體。
在一態樣,本發明提供預防患有NAFLD之受試者中之至少一種症狀的方法,該等症狀例如存在hedgehog信號傳導途徑升高、疲勞、虛弱、體重減輕、食欲不振、噁心、腹痛、蜘蛛樣血管、皮膚及眼睛發黃(黃疸)、瘙癢、腿部液體積聚及腫脹(水腫)、腹部腫脹(腹水)、及精神錯亂。該等方法包括向受試者投與治療有效量之RNAi劑,例如本發明之dsRNA、藥物組成物或載體,從而預防患有將受益於PNPLA3基因表現降低之病症之受試者中的至少一種症狀。
在另一態樣中,本發明提供治療有效量的本發明之RNAi劑用於治療受試者之用途,該受試者例如受益於PNPLA3基因表現之降低及/或抑制之受試者。在另一態樣中,本發明提供本發明的靶向PNPLA3基因之RNAi劑例如dsRNA或包含靶向PNPLA3基因之RNAi劑的藥物組成物在製備用於治療受試者之藥物中的用途,該受試者例如受益於PNPLA3基因表現及/或PNPLA3蛋白產生之降低及/或抑制的受試者,例如患有將受益於PNPLA3基因表現降低之病症例如PNPLA3相關疾病的受試者。
在另一態樣中,本發明提供本發明之RNAi(例如dsRNA)用於預防患有可受益於PNPLA3基因表現及/或PNPLA3蛋白質產生之降低及/或抑制之病症的受試者中的至少一種症狀的用途。
在另一態樣中,本發明提供本發明之RNAi劑在製備藥物中之用途,該藥物用於預防患有受益於PNPLA3基因表現及/或PNPLA3蛋白質產生之降低及/或抑制之病症例如PNPLA3相關疾病之受試者中的至少一種症狀。
在一個實施方式中,將靶向PNPLA3之RNAi劑投與患有PNPLA3相關疾病(例如非酒精性脂肪性肝病(NAFLD))之受試者,使得當將dsRNA劑投與受試者時,PNPLA3基因例如在受試者之細胞、組織、血液或其他組織或液體中之表現降低至少約10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、62%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或至少約99%、或更多。
本發明之方法及用途包括投與本文所述之組成物,以使靶PNPLA3基因之表現降低,例如持續約1、2、3、4、5、6、7、8、12、16、18、24、28、32、36、40、44、48、52、56、60、64、68、72、76或約80小時。在一個實施方式中,靶PNPLA3基因之表現降低延長的持續時間,例如至少約兩天、三天、四天、五天、六天、七天或更多天,例如約一週、兩週、三週、或約四週或更長時間。
根據本發明之方法及用途投與dsRNA可使得患有PNPLA3相關疾病(例如,非酒精性脂肪性肝病(NAFLD))之患者中此類疾病或病症之嚴重性、體征、症狀及/或標誌物降低。在這種情況下,「降低」係指此水平之統計學顯著降低。降低可為例如至少約5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、或約100%。可以例如藉由量測疾病進展、疾病緩解、症狀嚴重程度、疼痛減輕、生活質量、維持治療效果所需之藥物劑量、疾病標記物或適用於所治療或靶向預防之特定疾病的任何其他可量測參數的水平來評估疾病的治療或預防功效。藉由量測該等參數中之任一個或任何參數組合來監測治療或預防功效為在熟習此項技術者的能力範圍內。例如,可以例如藉由定期監測NAFLD症狀、肝臟脂肪水平或下游基因之表現來評估NAFLD治療功效。後續讀數與初始讀數之比較為醫生提供治療是否有效的指示。藉由量測該等參數中之任一個或任何參數組合來監測治療或預防功效為在熟習此項技術者的能力範圍內。與靶向PNPLA3之RNAi或其藥物組成物之投與結合,「有效對抗」PNPLA3相關疾病表明以臨床上適當的方式投與至少對患者之統計學顯著部分產生有益效果,例如改善症狀、治癒、疾病減輕、生命延長、生活質量改善、或熟悉治療NAFLD及/或PNPLA3相關疾病及相關病因的醫生普遍認為是積極的其他效果。
當疾病狀態之一或多個參數得到統計學顯著改善時,或者由於未能惡化或在其他情況下預期會出現症狀時,治療或預防效果明顯。例如,在可量測疾病參數中,至少10%,較佳至少20%、30%、40%、50%或更多有利變化可以指示有效治療。亦可以使用此項技術已知之針對給定疾病之實驗動物模型來判斷給定RNAi藥物或該藥物之調配物的功效。當使用實驗動物模型時,當觀察到標誌物或症狀之統計學顯著降低時,證明了治療功效。
可以向受試者投與治療量之RNAi,例如約0.01 mg/kg、0.02 mg/kg、0.03 mg/kg、0.04 mg/kg、0.05 mg/kg、0.1 mg/kg、0.15 mg/kg、0.2 mg/kg、0.25 mg/kg、0.3 mg/kg、0.35 mg/kg、0.4 mg/kg、0.45 mg/kg、0.5 mg/kg、0.55 mg/kg、0.6 mg/kg、0.65 mg/kg、0.7 mg/kg、0.75 mg/kg、0.8 mg/kg、0.85 mg/kg、0.9 mg/kg、0.95 mg/kg、1.0 mg/kg、1.1 mg/kg、1.2 mg/kg、1.3 mg/kg、1.4 mg/kg、1.5 mg/kg、1.6 mg/kg、1.7 mg/kg、1.8 mg/kg、1.9 mg/kg、2.0 mg/kg、2.1 mg/kg、2.2 mg/kg、2.3 mg/kg、2.4 mg/kg、2.5 mg/kg dsRNA、2.6 mg/kg dsRNA、2.7 mg/kg dsRNA、2.8 mg/kg dsRNA、2.9 mg/kg dsRNA、3.0 mg/kg dsRNA、3.1 mg/kg dsRNA、3.2 mg/kg dsRNA、3.3 mg/kg dsRNA、3.4 mg/kg dsRNA、3.5 mg/kg dsRNA、3.6 mg/kg dsRNA、3.7 mg/kg dsRNA、3.8 mg/kg dsRNA、3.9 mg/kg dsRNA、4.0 mg/kg dsRNA、4.1 mg/kg dsRNA、4.2 mg/kg dsRNA、4.3 mg/kg dsRNA、4.4 mg/kg dsRNA、4.5 mg/kg dsRNA、4.6 mg/kg dsRNA、4.7 mg/kg dsRNA、4.8 mg/kg dsRNA、4.9 mg/kg dsRNA、5.0 mg/kg dsRNA、5.1 mg/kg dsRNA、5.2 mg/kg dsRNA、5.3 mg/kg dsRNA、5.4 mg/kg dsRNA、5.5 mg/kg dsRNA、5.6 mg/kg dsRNA、5.7 mg/kg dsRNA、5.8 mg/kg dsRNA、5.9 mg/kg dsRNA、6.0 mg/kg dsRNA、6.1 mg/kg dsRNA、6.2 mg/kg dsRNA、6.3 mg/kg dsRNA、6.4 mg/kg dsRNA、6.5 mg/kg dsRNA、6.6 mg/kg dsRNA、6.7 mg/kg dsRNA、6.8 mg/kg dsRNA、6.9 mg/kg dsRNA、7.0 mg/kg dsRNA、7.1 mg/kg dsRNA、7.2 mg/kg dsRNA、7.3 mg/kg dsRNA、7.4 mg/kg dsRNA、7.5 mg/kg dsRNA、7.6 mg/kg dsRNA、7.7 mg/kg dsRNA、7.8 mg/kg dsRNA、7.9 mg/kg dsRNA、8.0 mg/kg dsRNA、8.1 mg/kg dsRNA、8.2 mg/kg dsRNA、8.3 mg/kg dsRNA、8.4 mg/kg dsRNA、8.5 mg/kg dsRNA、8.6 mg/kg dsRNA、8.7 mg/kg dsRNA、8.8 mg/kg dsRNA、8.9 mg/kg dsRNA、9.0 mg/kg dsRNA、9.1 mg/kg dsRNA、9.2 mg/kg dsRNA、9.3 mg/kg dsRNA、9.4 mg/kg dsRNA、9.5 mg/kg dsRNA、9.6 mg/kg dsRNA、9.7 mg/kg dsRNA、9.8 mg/kg dsRNA、9.9 mg/kg dsRNA、9.0 mg/kg dsRNA、10 mg/kg dsRNA、15 mg/kg dsRNA、20 mg/kg dsRNA、25 mg/kg dsRNA、30 mg/kg dsRNA、35 mg/kg dsRNA、40 mg/kg dsRNA、45 mg/kg dsRNA或約50 mg/kg dsRNA。在一個實施方式中,可以向受試者投與0.5 mg/kg之dsRNA。值及所述值之中間範圍亦為本發明之一部分。
投與RNAi可以將例如在患者之細胞、組織、血液、尿液或其他隔室中之PNPLA3蛋白水平的存在降低至少約5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、或至少約99%或更多。
在投與全劑量RNAi之前,可以向患者投與較小劑量,例如5%輸注,且監測不良反應,例如過敏反應。在另一個實例中,可以監測患者之不需要的免疫刺激作用,例如增加的細胞介素(例如,TNF-α或INF-α)水平。
由於對PNPLA3表現之抑制作用,根據本發明之組成物或由其製備之藥物組成物可以提高生活質量。
本發明之RNAi可以「裸」形式投與,其中經修飾或未經修飾之RNAi劑直接懸浮在水性或合適緩衝溶劑中,作為「游離RNAi」。在不存在藥物組成物之情況下投與游離RNAi。游離RNAi可以處於合適的緩衝溶液中。緩衝溶液可包含乙酸鹽、檸檬酸鹽、穀醇溶蛋白、碳酸鹽或磷酸鹽、或其任何組合。在一個實施方式中,緩衝溶液為磷酸鹽緩衝鹽水(PBS)。可以調節含有RNAi之緩衝溶液的pH及重量莫耳滲透壓濃度,使其適合投與受試者。
或者,本發明之RNAi可以作為藥物組成物,例如dsRNA脂質體調配物投與。
受益於PNPLA3基因表現之降低及/或抑制之受試者為患有如本文所述之非酒精性脂肪性肝病(NAFLD)及/或PNPLA3相關疾病或病症之受試者。
對受益於PNPLA3基因表現之降低及/或抑制之受試者的治療包括治療性及預防性治療。
本發明進一步提供用於治療受益於PNPLA3基因表現之降低及/或抑制之受試者(例如,患有PNPLA3相關疾病之受試者)的方法及RNAi劑或其藥物組成物用於治療受益於PNPLA3基因表現之降低及/或抑制之受試者的用途,與其他藥物及/或其他治療方法,例如與已知藥物及/或已知治療方法,例如目前用於治療該等病症之彼等藥物及/或方法之組合。
例如,在某些實施方式中,靶向PNPLA3基因之RNAi與例如適用於治療如本文其他地方所述之PNPLA3相關疾病之藥劑組合投與。例如,適用於治療受益於PNPLA3表現降低之受試者,例如患有PNPLA3相關疾病之受試者的其他治療劑及治療方法,包括靶向PNPLA3基因之不同部分的RNAi劑、治療劑、及/或用於治療PNPLA3相關疾病之程序、或任何前述之組合。
在某些實施方式中,靶向PNPLA3基因之第一RNAi劑與靶向PNPLA3基因之不同部分之第二RNAi劑組合投與。例如,第一RNAi劑包含形成雙鏈區之第一有義鏈及第一反義鏈,其中該第一有義鏈之基本上所有核苷酸及該第一反義鏈之基本上所有核苷酸均為經修飾之核苷酸,其中該第一有義鏈與連接在3'端之配位基綴合,且其中該配位基為藉由二價或三價支鏈連接子連接之一或多個GalNAc衍生物;且第二RNAi劑包含形成雙鏈區之第二有義鏈及第二反義鏈,其中第二有義鏈之基本上所有核苷酸及第二反義鏈之基本上所有核苷酸均為經修飾之核苷酸,其中第二有義鏈與連接在3'-端之配位基綴合,且其中配位基為藉由二價或三價支鏈連接子連接之一或多個GalNAc衍生物。
在一個實施方式中,第一及第二有義鏈之所有核苷酸及/或第一及第二反義鏈之所有核苷酸均包含修飾。
在一個實施方式中,至少一個經修飾之核苷酸選自由以下所組成之群:3'-端去氧胸腺嘧啶(dT)核苷酸、經2'-O-甲基修飾之核苷酸、經2'-氟修飾之核苷酸、經2'-去氧修飾之核苷酸、鎖定核苷酸、解鎖核苷酸、構象限制性核苷酸、受約束乙基核苷酸、無鹼基核苷酸、經2'-胺基修飾之核苷酸、經2'-O-烯丙基修飾之核苷酸、經2'-C-烷基修飾之核苷酸、經2'-羥基修飾之核苷酸、經2'-甲氧基乙基修飾之核苷酸、經2'-O-烷基修飾之核苷酸、𠰌啉基核苷酸、胺基磷酸酯、包含非天然鹼基之核苷酸、經四氫哌喃修飾之核苷酸、經1,5-脫水己糖醇修飾之核苷酸、經環己烯基修飾之核苷酸、包含硫代磷酸酯基團之核苷酸、包含甲基膦酸酯基團之核苷酸、包含5'-磷酸之核苷酸、及包含5'-磷酸模擬物之核苷酸。
在某些實施方式中,靶向PNPLA3基因之第一RNAi劑與靶向不同於PNPLA3基因之基因的第二RNAi劑組合投與。例如,靶向PNPLA3基因之RNAi劑可以與靶向SCAP基因之RNAi劑組合投與。靶向PNPLA3基因之第一RNAi劑及靶向不同於PNPLA3基因之基因(例如SCAP基因)之第二RNAi劑可以作為同一藥物組成物之一部分投與。或者,靶向PNPLA3基因之第一RNAi劑及靶向不同於PNPLA3基因之基因(例如SCAP基因)之第二RNAi劑可以作為不同藥物組成物之部分投與。
RNAi劑及另外的治療劑及/或治療可以同時及/或以相同組合投與,例如非經腸,或另外的治療劑可以作為單獨組成物之一部分或在不同時間及/或藉由此項技術已知或本文描述之另一種方法投與。
本發明亦提供使用本發明之RNAi劑及/或含有本發明之RNAi劑的組成物來降低及/或抑制細胞中之PNPLA3表現的方法。在其他態樣,本發明提供本發明之RNAi及/或包含本發明之RNAi的組成物,其用於降低及/或抑制細胞中之PNPLA3基因之表現。在其他態樣,提供本發明之RNAi及/或包含本發明之RNAi的組成物在製備用於降低及/或抑制細胞中之PNPLA3基因表現之藥物中的用途。在其他態樣,本發明提供本發明之RNAi及/或包含本發明之RNAi的組成物,其用於降低及/或抑制細胞中之PNPLA3蛋白產生。在其他態樣,提供本發明之RNAi及/或包含本發明之RNAi的組成物在製備用於降低及/或抑制細胞中之PNPLA3蛋白質產生的藥物中的用途。該等方法及用途包括使細胞與本發明之RNAi(例如dsRNA)接觸,且將細胞維持足夠時間以獲得PNPLA3基因之mRNA轉錄物的降解,從而抑制PNPLA3基因之表現或抑制細胞中之PNPLA3蛋白質產生。
可以藉由此項技術已知之任何方法評估基因表現之降低。例如,PNPLA3表現之降低可以藉由使用熟習此項技術者的常規方法,例如北方墨點、qRT-PCR判定PNPLA3之mRNA表現水平;藉由使用熟習此項技術者的常規方法,例如蛋白質墨點、免疫學技術、流式細胞術方法、ELISA判定PNPLA3之蛋白質水平;及/或藉由判定PNPLA3之生物學活性來判定。
在本發明之方法及用途中,細胞可以在活體外或活體內接觸,即細胞可以處於受試者體內。
適用於使用本發明之方法進行處理之細胞可為表現PNPLA3基因之任何細胞,例如來自患有NAFLD之受試者的細胞或包含含有PNPLA3基因或PNPLA3基因之部分之表現載體的細胞。適用於本發明之方法及用途的細胞可為哺乳動物細胞,例如靈長類動物細胞(例如人細胞或非人靈長類動物細胞,例如猴細胞或黑猩猩細胞)、非靈長類細胞(如牛細胞、豬細胞、駱駝細胞、美洲駝細胞、馬細胞、山羊細胞、兔細胞、綿羊細胞、倉鼠、豚鼠細胞、貓細胞、狗細胞、大鼠細胞、小鼠細胞、獅子細胞、老虎細胞、熊細胞、或水牛細胞)、鳥類細胞(例如鴨細胞或鵝細胞)、或鯨細胞。在一個實施方式中,細胞為人細胞。
PNPLA3基因表現可在細胞中被抑制至少約5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或約100%。
PNPLA3蛋白產生可以在細胞中被抑制至少約5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、或約100%。
本發明之活體內方法及用途可包括向受試者投與含有RNAi之組成物,其中RNAi包括與待治療之哺乳動物之PNPLA3基因之RNA轉錄物的至少一部分互補的核苷酸序列。當待治療的生物體為人時,該組成物可以藉由此項技術已知之任何方式投與,包括但不限於皮下、靜脈內、口服、腹膜內或非經腸途徑,包括顱內(例如,腦室內、實質內及鞘內)、肌內、透皮、氣道(氣溶膠)、鼻腔、直腸及局部(包括口腔及舌下)投與。在某些實施方式中,組成物藉由皮下或靜脈內輸注或注射投與。在一個實施方式中,組成物藉由皮下注射投與。
在一些實施方式中,經由長效注射投與。長效注射可以在延長的時間段內以一致方式釋放RNAi。因此,長效注射可減少獲得所需效果,例如所需PNPLA3抑制或治療或預防效果所需要的給藥頻率。長效注射亦可以提供更一致的血清濃度。長效注射可包括皮下注射或肌內注射。在較佳實施方式中,長效注射為皮下注射。
在一些實施方式中,藉由泵投與。泵可為外部泵或外科植入泵。在某些實施方式中,泵為皮下植入滲透泵。在其他實例中,泵為輸液泵。輸液泵可用於靜脈內、皮下、動脈或硬膜外輸注。在較佳實施方式中,輸液泵為皮下輸注泵。在其他實施方式中,泵為將RNAi遞送至受試者之外科植入泵。
可以基於是否需要局部或全身治療且基於待治療之區域來選擇投與方式。可以選擇投與途徑及投與部位以增強靶向。
在一態樣中,本發明亦提供用於抑制哺乳動物(例如人)中之PNPLA3基因表現的方法。本發明亦提供一種包含靶向哺乳動物細胞中之PNPLA3基因之RNAi(例如dsRNA)的組成物,其用於抑制哺乳動物中PNPLA3基因的表現。在另一態樣中,本發明提供靶向哺乳動物細胞中之PNPLA3基因之RNAi(例如dsRNA)在製備用於抑制哺乳動物中之PNPLA3基因表現的藥物中的用途。
該等方法及用途包括向哺乳動物(例如人)投與包含靶向哺乳動物細胞中之PNPLA3基因之RNAi(例如dsRNA)之組成物且維持哺乳動物足夠時間以獲得PNPLA3基因之mRNA轉錄物的降解,從而抑制哺乳動物中之PNPLA3基因之表現。
可以藉由此項技術已知的任何方法(例如本文所述之qRT-PCR)在經RNAi投與之受試者的外周血樣本中評估基因表現之降低。可以藉由此項技術已知的任何方法及藉由本文所述的方法(例如ELISA或西方墨點)評估蛋白質產生之減少。在一個實施方式中,組織樣本用作組織材料,用於監測PNPLA3基因及/或蛋白質表現之減少。在另一個實施方式中,血液樣本用作組織材料,用於監測PNPLA3基因及/或蛋白質表現之減少。
在一個實施方式中,在投與RNAi劑後靶標在活體內進行RISC介導之裂解的驗證藉由進行5'-RACE或此項技術已知之方案的改進來完成(Lasham A 等人, (2010) Nucleic Acid Res., 38 (3) p-el9)(Zimmermann 等人 (2006) Nature 441: 111-4)。
應當理解,本文揭示之所有核糖核酸序列可以藉由用胸腺嘧啶鹼基取代序列中之尿嘧啶鹼基而轉化為去氧核糖核酸序列。同樣地,本文揭示之所有去氧核糖核酸序列可以藉由用尿嘧啶鹼基取代序列中之胸腺嘧啶鹼基而轉化為核糖核酸序列。本發明包括去氧核糖核酸序列、核糖核酸序列及含有本文揭示之所有序列之去氧核糖核苷酸及核糖核苷酸之混合物的序列。
另外,本文揭示之任何核酸序列可以用化學修飾之任何組合進行修飾。熟習此項技術者將容易理解,在某些情況下,描述經修飾之多核苷酸的命名諸如「RNA」或「DNA」為任意的。例如,包含在核糖上具有2'-OH取代基且具有胸腺嘧啶鹼基之核苷酸的多核苷酸可以描述為具有經修飾之糖的DNA分子(2'-OH取代DNA的天然2'-H)或描述為具有經修飾之鹼基(胸腺嘧啶(甲基化尿嘧啶)取代RNA的天然尿嘧啶)的RNA分子。
因此,本文提供之核酸序列(包括但不限於序列表中之核酸序列)意欲涵蓋含有天然或經修飾RNA及/或DNA之任何組合之核酸,包括但不限於具有經修飾核苷鹼基之此等核酸。作為另一個實例且為非限制性,具有序列「ATCGATCG」 之多核苷酸包括具有經修飾或未經修飾之此序列的任何多核苷酸,包括但不限於包含RNA鹼基之化合物,例如具有序列「AUCGAUCG」之彼等化合物、及具有一些DNA鹼基及一些RNA鹼基之彼等化合物(例如「AUCGATCG」)、及具有其他經修飾鹼基之多核苷酸(例如「ATmeCGAUCG」),其中meC表示在5-位包含甲基之胞嘧啶鹼基。
以下實例,包括所進行之實驗及所實現之結果,僅用於說明目的,且不應解釋為限制所附申請專利範圍之範疇。以引用之方式併入
本說明書中提及之所有公開案、專利及專利申請案均以引用之方式併入本文,達到如同明確及個別地指示將各個個別公開案、專利或專利申請案以引用之方式併入的相同程度。然而,本文對參考文獻之引用不應解釋為承認此類參考文獻為本發明之先前技術。在以引用之方式併入的參考文獻中所提供之任何定義或術語與在本文中所提供之術語及討論不同的情況下,以本發明術語及定義為準。等同物
認為前述書面說明係足以能夠使熟習此項技術者來實踐本發明。前述描述及實例詳述本發明之某些較佳實施方式,且描述由發明人所考慮的最佳模式。然而,應當理解,無論前述以文本形式表現得如何詳細,本發明仍可以許多方式實踐,且應根據隨附申請專利範圍及其任何等同物解釋本發明。
僅出於說明目的提供以下實例(包括進行之實驗及達成之結果)且其不欲解釋為限制本發明。
本文所述的所有動物實驗均由美商安進公司研究動物護理及使用委員會(IACUC)批准,且根據實驗室動物護理及使用指南第8版(國家研究委員會(美國))、實驗室動物護理及使用指南更新委員會、實驗室動物研究所(美國)、及國家科學院出版社(美國)(2011) 實驗室動物護理及使用指南第8版(國家科學院出版社(華盛頓特區))來進行護理。將小鼠單獨圈養在22°C ± 2°C、具有12小時光照;12小時黑暗週期之空調房間內(0600-1800小時)。除非另有說明,否則動物可隨意獲得常規食物(Envigo公司,2920X;或另外規定的食物)且藉由自動澆水系統獲得水(反滲透純化)。在結束時,在深度麻醉下藉由心臟穿刺收集血液,且然後根據實驗室動物護理評估及認證協會(AAALAC)指南,藉由二次物理方法實施安樂死。實例 1 :經修飾之 PNPLA3 siRNA 分子之選擇、設計及合成
使用人PNPLA3轉錄物(NM_025225.2)之生物信息學分析鑒定靶向含有patatin樣磷脂酶結構域3(PNPLA3)之治療性siRNA分子的最佳序列之鑒定及選擇。表1顯示鑒定為具有治療特性之序列。在整個各種序列中,{INVAB }為倒A鹼性,{INVDA}為反向去氧胸苷,GNA為乙二醇核酸,dT為去氧胸苷,且dC為去氧胞嘧啶。
[表1]. 針對PNPLA3之siRNA序列
雙鏈體編號 | 有義序列 (5’-3’) | SEQ ID NO: (有義) | 反義序列 (5’-3’) | SEQ ID NO: (反義) |
D-1000 | GGGCAAUAAAGUACCUGCUUU | 1 | AGCAGGUACUUUAUUGCCCUU | 2 |
D-1001 | CGGCCAAUGUCCACCAGCUUU | 3 | AGCUGGUGGACAUUGGCCGUU | 4 |
D-1002 | GGUCCAGCCUGAACUUCUUUU | 5 | AAGAAGUUCAGGCUGGACCUU | 6 |
D-1003 | GCUUCAUCCCCUUCUACAGUU | 7 | CUGUAGAAGGGGAUGAAGCUU | 8 |
D-1004 | GCGGCUUCCUGGGCUUCUAUU | 9 | UAGAAGCCCAGGAAGCCGCUU | 10 |
D-1005 | GCCUCUGAGCUGAGUUGGUUU | 11 | ACCAACUCAGCUCAGAGGCUU | 12 |
D-1006 | GUGACAACGUACCCUUCAUUU | 13 | AUGAAGGGUACGUUGUCACUU | 14 |
D-1007 | CCCGCCUCCAGGUCCCAAAUU | 15 | UUUGGGACCUGGAGGCGGGUU | 16 |
D-1008 | CUUCAUCCCCUUCUACAGUUU | 17 | ACUGUAGAAGGGGAUGAAGUU | 18 |
D-1009 | GGUAUGUUCCUGCUUCAUGUU | 19 | CAUGAAGCAGGAACAUACCUU | 20 |
D-1010 | GUAUGUUCCUGCUUCAUGCUU | 21 | GCAUGAAGCAGGAACAUACUU | 22 |
D-1011 | UAUGUUCCUGCUUCAUGCCUU | 23 | GGCAUGAAGCAGGAACAUAUU | 24 |
D-1012 | AUGUUCCUGCUUCAUGCCCUU | 25 | GGGCAUGAAGCAGGAACAUUU | 26 |
D-1013 | UGUUCCUGCUUCAUGCCCUUU | 27 | AGGGCAUGAAGCAGGAACAUU | 28 |
D-1014 | GUUCCUGCUUCAUGCCCUUUU | 29 | AAGGGCAUGAAGCAGGAACUU | 30 |
D-1015 | UUCCUGCUUCAUGCCCUUCUU | 31 | GAAGGGCAUGAAGCAGGAAUU | 32 |
D-1016 | UCCUGCUUCAUGCCCUUCUUU | 33 | AGAAGGGCAUGAAGCAGGAUU | 34 |
D-1017 | CCUGCUUCAUGCCCUUCUAUU | 35 | UAGAAGGGCAUGAAGCAGGUU | 36 |
D-1018 | CUGCUUCAUGCCCUUCUACUU | 37 | GUAGAAGGGCAUGAAGCAGUU | 38 |
D-1019 | UGCUUCAUGCCCUUCUACAUU | 39 | UGUAGAAGGGCAUGAAGCAUU | 40 |
D-1020 | GCUUCAUGCCCUUCUACAGUU | 41 | CUGUAGAAGGGCAUGAAGCUU | 42 |
D-1021 | CUUCAUGCCCUUCUACAGUUU | 43 | ACUGUAGAAGGGCAUGAAGUU | 44 |
D-1022 | UUCAUGCCCUUCUACAGUGUU | 45 | CACUGUAGAAGGGCAUGAAUU | 46 |
D-1023 | UCAUGCCCUUCUACAGUGGUU | 47 | CCACUGUAGAAGGGCAUGAUU | 48 |
D-1024 | CAUGCCCUUCUACAGUGGCUU | 49 | GCCACUGUAGAAGGGCAUGUU | 50 |
D-1025 | AUGCCCUUCUACAGUGGCCUU | 51 | GGCCACUGUAGAAGGGCAUUU | 52 |
D-1026 | UGCCCUUCUACAGUGGCCUUU | 53 | AGGCCACUGUAGAAGGGCAUU | 54 |
D-1027 | GCCCUUCUACAGUGGCCUUUU | 55 | AAGGCCACUGUAGAAGGGCUU | 56 |
D-1028 | GGUAUGUUCCUGCUUCAUCUU | 57 | GAUGAAGCAGGAACAUACCUU | 58 |
D-1029 | GUAUGUUCCUGCUUCAUCCUU | 59 | GGAUGAAGCAGGAACAUACUU | 60 |
D-1030 | UAUGUUCCUGCUUCAUCCCUU | 61 | GGGAUGAAGCAGGAACAUAUU | 62 |
D-1031 | AUGUUCCUGCUUCAUCCCCUU | 63 | GGGGAUGAAGCAGGAACAUUU | 64 |
D-1032 | UGUUCCUGCUUCAUCCCCUUU | 65 | AGGGGAUGAAGCAGGAACAUU | 66 |
D-1033 | GUUCCUGCUUCAUCCCCUUUU | 67 | AAGGGGAUGAAGCAGGAACUU | 68 |
D-1034 | UUCCUGCUUCAUCCCCUUCUU | 69 | GAAGGGGAUGAAGCAGGAAUU | 70 |
D-1035 | UCCUGCUUCAUCCCCUUCUUU | 71 | AGAAGGGGAUGAAGCAGGAUU | 72 |
D-1036 | CCUGCUUCAUCCCCUUCUAUU | 73 | UAGAAGGGGAUGAAGCAGGUU | 74 |
D-1037 | CUGCUUCAUCCCCUUCUACUU | 75 | GUAGAAGGGGAUGAAGCAGUU | 76 |
D-1038 | UGCUUCAUCCCCUUCUACAUU | 77 | UGUAGAAGGGGAUGAAGCAUU | 78 |
D-1039 | UUCAUCCCCUUCUACAGUGUU | 79 | CACUGUAGAAGGGGAUGAAUU | 80 |
D-1040 | UCAUCCCCUUCUACAGUGGUU | 81 | CCACUGUAGAAGGGGAUGAUU | 82 |
D-1041 | CAUCCCCUUCUACAGUGGCUU | 83 | GCCACUGUAGAAGGGGAUGUU | 84 |
D-1042 | UCCCCUUCUACAGUGGCCUUU | 85 | AGGCCACUGUAGAAGGGGAUU | 86 |
D-1043 | GAUCAGGACCCGAGCCGAUUU | 87 | AUCGGCUCGGGUCCUGAUCUU | 88 |
D-1044 | UGGGCUUCUACCACGUCGUUU | 89 | ACGACGUGGUAGAAGCCCAUU | 90 |
D-1045 | GAGCGAGCACGCCCCGCAUUU | 91 | AUGCGGGGCGUGCUCGCUCUU | 92 |
D-1046 | UGCACUGCGUCGGCGUCCUUU | 93 | AGGACGCCGACGCAGUGCAUU | 94 |
D-1047 | UGGAGCAGACUCUGCAGGUUU | 95 | ACCUGCAGAGUCUGCUCCAUU | 96 |
D-1048 | UGCAGGUCCUCUCAGAUCUUU | 97 | AGAUCUGAGAGGACCUGCAUU | 98 |
D-1049 | CCCGGCCAAUGUCCACCAUUU | 99 | AUGGUGGACAUUGGCCGGGUU | 100 |
D-1050 | UUCUACAGUGGCCUUAUCUUU | 101 | AGAUAAGGCCACUGUAGAAUU | 102 |
D-1051 | UCUACAGUGGCCUUAUCCUUU | 103 | AGGAUAAGGCCACUGUAGAUU | 104 |
D-1052 | CUUCCUUCAGAGGCGUGCUUU | 105 | AGCACGCCUCUGAAGGAAGUU | 106 |
D-1053 | UUCCUUCAGAGGCGUGCGAUU | 107 | UCGCACGCCUCUGAAGGAAUU | 108 |
D-1054 | GCGUGCGAUAUGUGGAUGUUU | 109 | ACAUCCACAUAUCGCACGCUU | 110 |
D-1055 | CGUGCGAUAUGUGGAUGGAUU | 111 | UCCAUCCACAUAUCGCACGUU | 112 |
D-1056 | UGGAUGGAGGAGUGAGUGAUU | 113 | UCACUCACUCCUCCAUCCAUU | 114 |
D-1057 | ACGUACCCUUCAUUGAUGUUU | 115 | ACAUCAAUGAAGGGUACGUUU | 116 |
D-1058 | UGGACAUCACCAAGCUCAUUU | 117 | AUGAGCUUGGUGAUGUCCAUU | 118 |
D-1059 | CACCUGCGUCUCAGCAUCUUU | 119 | AGAUGCUGAGACGCAGGUGUU | 120 |
D-1060 | ACCUGCGUCUCAGCAUCCUUU | 121 | AGGAUGCUGAGACGCAGGUUU | 122 |
D-1061 | CCAGAGACUGGUGACAUGUUU | 123 | ACAUGUCACCAGUCUCUGGUU | 124 |
D-1062 | AUGGCUUCCAGAUAUGCCUUU | 125 | AGGCAUAUCUGGAAGCCAUUU | 126 |
D-1063 | CCGCCUCCAGGUCCCAAAUUU | 127 | AUUUGGGACCUGGAGGCGGUU | 128 |
D-1064 | UACCUGCUGGUGCUGAGGUUU | 129 | ACCUCAGCACCAGCAGGUAUU | 130 |
D-1065 | ACCUGCUGGUGCUGAGGGUUU | 131 | ACCCUCAGCACCAGCAGGUUU | 132 |
D-1066 | CUCUCCACCUUUCCCAGUUUU | 133 | AACUGGGAAAGGUGGAGAGUU | 134 |
D-1067 | UUUUUCACCUAACUAAAAUUU | 135 | AUUUUAGUUAGGUGAAAAAUU | 136 |
D-1068 | CGGCCAAUGUCCACCAGCUUU | 137 | AGCUGGUGGACAUUGGCCGUU | 138 |
D-1069 | GGUCCAGCCUGAACUUCUUUU | 139 | AAGAAGUUCAGGCUGGACCUU | 140 |
D-1070 | GCGGCUUCCUGGGCUUCUAUU | 141 | UAGAAGCCCAGGAAGCCGCUU | 142 |
D-1071 | GUGACAACGUACCCUUCAUUU | 143 | AUGAAGGGUACGUUGUCACUU | 144 |
D-1072 | GGUAUGUUCCUGCUUCAUGUU | 145 | CAUGAAGCAGGAACAUACCUU | 146 |
D-1073 | GUAUGUUCCUGCUUCAUGCUU | 147 | GCAUGAAGCAGGAACAUACUU | 148 |
D-1074 | UGUUCCUGCUUCAUGCCCUUU | 149 | AGGGCAUGAAGCAGGAACAUU | 150 |
D-1075 | GUUCCUGCUUCAUGCCCUUUU | 151 | AAGGGCAUGAAGCAGGAACUU | 152 |
D-1076 | CCUGCUUCAUGCCCUUCUAUU | 153 | UAGAAGGGCAUGAAGCAGGUU | 154 |
D-1077 | GCUUCAUGCCCUUCUACAGUU | 155 | CUGUAGAAGGGCAUGAAGCUU | 156 |
D-1078 | CUUCAUGCCCUUCUACAGUUU | 157 | ACUGUAGAAGGGCAUGAAGUU | 158 |
D-1079 | UUCAUGCCCUUCUACAGUGUU | 159 | CACUGUAGAAGGGCAUGAAUU | 160 |
D-1080 | AUGGCUUCCAGAUAUGCCUUU | 161 | AGGCAUAUCUGGAAGCCAUUU | 162 |
D-1081 | AUGCCCUUCUACAGUGGCCUU | 163 | GGCCACUGUAGAAGGGCAUUU | 164 |
D-1082 | GCUUCAUGCCCUUCUACAUUU | 165 | AUGUAGAAGGGCAUGAAGCUU | 166 |
D-1083 | GGAAAGACUGUUCCAAAAAUU | 333 | UUUUUGGAACAGUCUUUCCUU | 334 |
D-1084 | GGUAUGUUCCUGCUUCAUGUU | 335 | CAUGAAGCAGGAACAUACCUU | 336 |
D-1085 | GUAUGUUCCUGCUUCAUGCUU | 337 | GCAUGAAGCAGGAACAUACUU | 338 |
D-1086 | UGUUCCUGCUUCAUGCCCUUU | 339 | AGGGCAUGAAGCAGGAACAUU | 340 |
D-1087 | GCUUCAUGCCCUUCUACAGUU | 341 | CUGUAGAAGGGCAUGAAGCUU | 342 |
D-1088 | CUUCAUGCCCUUCUACAGUUU | 343 | ACUGUAGAAGGGCAUGAAGUU | 344 |
D-1089 | GCGGCUUCCUGGGCUUCUAUU | 345 | UAGAAGCCCAGGAAGCCGCUU | 346 |
D-1090 | GUUCCUGCUUCAUGCCCUUUU | 347 | AAGGGCAUGAAGCAGGAACUU | 348 |
D-1091 | AUGGCUUCCAGAUAUGCCUUU | 349 | AGGCAUAUCUGGAAGCCAUUU | 350 |
D-1092 | CCUGCUUCAUGCCCUUCUAUU | 351 | UAGAAGGGCAUGAAGCAGGUU | 352 |
D-1093 | UUCAUGCCCUUCUACAGUUUU | 353 | AACUGUAGAAGGGCAUGAAUU | 354 |
D-1094 | UUCAUGCCCUUCUACAGUGUU | 355 | CACUGUAGAAGGGCAUGAAUU | 356 |
D-1095 | GCUUCAUGCCCUUCUACAUUU | 357 | AUGUAGAAGGGCAUGAAGCUU | 358 |
D-1096 | GGUCCAGCCUGAACUUCUUUU | 359 | AAGAAGUUCAGGCUGGACCUU | 360 |
D-1097 | GCGGCUUCCUGGGCUUCUAUU | 361 | UAGAAGCCCAGGAAGCCGCUU | 362 |
D-1098 | GCGGCUUCCUGGGCUUCUAUU | 363 | UAGAAGCCCAGGAAGCCGCUU | 364 |
D-1099 | GUUCCUGCUUCAUGCCCUUUU | 365 | AAGGGCAUGAAGCAGGAACUU | 366 |
D-1100 | GUUCCUGCUUCAUGCCCUUUU | 367 | AAGGGCAUGAAGCAGGAACUU | 368 |
D-1101 | CCUGCUUCAUGCCCUUCUAUU | 369 | UAGAAGGGCAUGAAGCAGGUU | 370 |
D-1102 | CCUGCUUCAUGCCCUUCUAUU | 371 | UAGAAGGGCAUGAAGCAGGUU | 372 |
D-1103 | AUGCCCUUCUACAGUGGCCUU | 373 | GGCCACUGUAGAAGGGCAUUU | 374 |
D-1104 | AUGGCUUCCAGAUAUGCCUUU | 375 | AGGCAUAUCUGGAAGCCAUUU | 376 |
D-1105 | GUGACAACGUACCCUUCAUUU | 377 | AUGAAGGGUACGUUGUCACUU | 378 |
D-1106 | GUAUGUUCCUGCUUCAUGCUU | 379 | GCAUGAAGCAGGAACAUACUU | 380 |
D-1107 | GUAUGUUCCUGCUUCAUGCUU | 381 | GCAUGAAGCAGGAACAUACUU | 382 |
D-1108 | GUAUGUUCCUGCUUCAUGCUU | 383 | GCAUGAAGCAGGAACAUACUU | 384 |
D-1109 | GUAUGUUCCUGCUUCAUGCCU | 385 | AGGCAUGAAGCAGGAACAUACUU | 386 |
D-1110 | UGGUAUGUUCCUGCUUCAUGU | 387 | GCAUGAAGCAGGAACAUACCAUU | 388 |
D-1111 | GUAUGUUCCUGCUUCAUGU | 389 | GCAUGAAGCAGGAACAUACUU | 390 |
D-1112 | GUAUGUUCCUGCUUCAUGC{INVAB} | 391 | GCAUGAAGCAGGAACAUACUU | 392 |
D-1113 | GCUUCAUGCCCUUCUACAUUU | 393 | AUGUAGAAGGGCAUGAAGCUU | 394 |
D-1114 | GCUUCAUGCCCUUCUACAUUU | 395 | AUGUAGAAGGGCAUGAAGCUU | 396 |
D-1115 | GCUUCAUGCCCUUCUACAUUU | 397 | AUGUAGAAGGGCAUGAAGCUU | 398 |
D-1116 | GCUUCAUGCCCUUCUACAUUU | 399 | AUGUAGAAGGGCAUGAAGCUU | 400 |
D-1117 | GCUUCAUGCCCUUCUACAUUU | 401 | AUGUAGAAGGGCAUGAAGCUU | 402 |
D-1118 | GCUUCAUGCCCUUCUACAUUU | 403 | AUGUAGAAGGGCAUGAAGCUU | 404 |
D-1119 | GCUUCAUGCCCUUCUACAUUU | 405 | AUGUAGAAGGGCAUGAAGCUU | 406 |
D-1120 | GCUUCAUGCCCUUCUACAUUU | 407 | AUGUAGAAGGGCAUGAAGCUU | 408 |
D-1121 | GCUUCAUGCCCUUCUACAUUU | 409 | AUGUAGAAGGGCAUGAAGCUU | 410 |
D-1122 | GCUUCAUGCCCUUCUACAUUU | 411 | AUGUAGAAGGGCAUGAAGCUU | 412 |
D-1123 | GCUUCAUGCCCUUCUACAUUU | 413 | AUGUAGAAGGGCAUGAAGCUU | 414 |
D-1124 | GCUUCAUGCCCUUCUACAUUU | 415 | AUGUAGAAGGGCAUGAAGCUU | 416 |
D-1125 | GCUUCAUGCCCUUCUACAUUU | 417 | AUGUAGAAGGGCAUGAAGCUU | 418 |
D-1126 | GCUUCAUGCCCUUCUACAUUU | 419 | AUGUAGAAGGGCAUGAAGCUU | 420 |
D-1127 | GCUUCAUGCCCUUCUACAUUU | 421 | AUGUAGAAGGGCAUGAAGCUU | 422 |
D-1128 | GCUUCAUGCCCUUCUACAUUU | 423 | AUGUAGAAGGGCAUGAAGCUU | 424 |
D-1129 | GCUUCAUG[DC]CCUUCUACAUUU | 425 | AUGUAGAAGGGCAUGAAGCUU | 426 |
D-1130 | GCUUCAUGCC[DC]UUCUACAUUU | 427 | AUGUAGAAGGGCAUGAAGCUU | 428 |
D-1131 | GCUUCAUGC[DC]CUUCUACAUUU | 429 | AUGUAGAAGGGCAUGAAGCUU | 430 |
D-1132 | GCUUCAUGCCCUUCUACAUUU | 431 | AUGUAGAAGGGCAUGAAGCUU | 432 |
D-1133 | GCUUCAUGCCCUUCUACAUUU | 433 | AUGUAGAAGGGCAUGAAGCUU | 434 |
D-1134 | GCUUCAUGCCCUUCUACAUUU | 435 | AUGUAGAAGGGCAUGAAGCUU | 436 |
D-1135 | GCUUCAUGCCCUUCUACAUUU | 437 | AUGUAGAAGGGCAUGAAGCUU | 438 |
D-1136 | GCUUCAUGCCCUUCUACAUUU | 439 | AUGUAGAAGGGCAUGAAGCUU | 440 |
D-1137 | GCUUCAUGCCCUUCUACAGUU | 441 | AACUGUAGAAGGGCAUGAAGCUU | 442 |
D-1138 | CUGCUUCAUGCCCUUCUACAU | 443 | AUGUAGAAGGGCAUGAAGCAGUU | 444 |
D-1139 | GCUUCAUGCCCUUCUACAU | 445 | AUGUAGAAGGGCAUGAAGCUU | 446 |
D-1140 | GCUUCAUGCCCUUCUACAU{INVAB} | 447 | AUGUAGAAGGGCAUGAAGCUU | 448 |
D-1141 | GCUUCAUGCCCUUCUACAUUU{INVAB} | 449 | AUGUAGAAGGGCAUGAAGCUU | 450 |
D-1142 | GCUUCAUGCCCUUCUACAUUU | 451 | AUGUAGAAGGGCAUGAAGCUU | 452 |
D-1143 | GCUUCAUGCCCUUCUACAUUU | 453 | AUGUAGAAGGGCAUGAAGCUU | 454 |
D-1144 | GCUUCAUGCCCUUCUACAUUU | 455 | AUGUAGAAGGGCAUGAAGCUU | 456 |
D-1145 | GCUUCAUGCCCUUCUACAUUU | 457 | AUGUAGAAGGGCAUGAAGCUU | 458 |
D-1146 | GCUUCAUGCCCUUCUACAUUU | 459 | AUGUAGAAGGGCAUGAAGCUU | 460 |
D-1147 | GCUUCAUGCCCUUCUACAUUU | 461 | AUGUAGAAGGGCAUGAAGCUU | 462 |
D-1148 | GCUUCAUGCCCUUCUACAUUU | 463 | AUGUAGAAGGGCAUGAAGCUU | 464 |
D-1149 | GCUUCAUGCCCUUCUACAUUU | 465 | AUGUAGAAGGGCAUGAAGCUU | 466 |
D-1150 | GCUUCAUGCCCUUCUACAUUU | 467 | AUGUAGAAGGGCAUGAAGCUU | 468 |
D-1151 | GUAUGUUCCUGCUUCAUGCUU{INVAB} | 469 | GCAUGAAGCAGGAACAUACUU | 470 |
D-1152 | GGUAUGUUCCUGCUUCAUUUU | 471 | AAUGAAGCAGGAACAUACCUU | 472 |
D-1153 | GUAUGUUCCUGCUUCAUGUUU | 473 | ACAUGAAGCAGGAACAUACUU | 474 |
D-1154 | CGGCCAAUGUCCACCAGCUUU | 475 | AGCUGGUGGACAUUGGCCGUU | 476 |
D-1155 | UGGAGCAGACUCUGCAGGUUU | 477 | ACCUGCAGAGUCUGCUCCAUU | 478 |
D-1156 | ACGUACCCUUCAUUGAUGUUU | 479 | ACAUCAAUGAAGGGUACGUUU | 480 |
D-1157 | CCAGAGACUGGUGACAUGUUU | 481 | ACAUGUCACCAGUCUCUGGUU | 482 |
D-1158 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 483 | AUGUAGAAAGGCAUGAAGCUU | 484 |
D-1159 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 485 | AAAUGUAGAAAGGCAUGAAGCUU | 486 |
D-1160 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 487 | AAAUGUAGAAAGGCAUGAAGCUU | 488 |
D-1161 | UGCUUCAUGCCUUUCUACAUU | 489 | UGUAGAAAGGCAUGAAGCAUU | 490 |
D-1162 | UAUGUUCCUGCUUCAUGCUUU | 491 | AGCAUGAAGCAGGAACAUAUU | 492 |
D-1163 | UUCCUGCUUCAUGCCUUUUUU | 493 | AAAAGGCAUGAAGCAGGAAUU | 494 |
D-1164 | UCAUGCCUUUCUACAGUGUUU | 495 | ACACUGUAGAAAGGCAUGAUU | 496 |
D-1165 | CAUGCCUUUCUACAGUGGUUU | 497 | ACCACUGUAGAAAGGCAUGUU | 498 |
D-1166 | AUGCCUUUCUACAGUGGCUUU | 499 | AGCCACUGUAGAAAGGCAUUU | 500 |
D-1167 | GGUAUGUUCCUGCUUCAUAUU | 501 | UAUGAAGCAGGAACAUACCUU | 502 |
D-1168 | GUAUGUUCCUGCUUCAUGAUU | 503 | UCAUGAAGCAGGAACAUACUU | 504 |
D-1169 | UAUGUUCCUGCUUCAUGCAUU | 505 | UGCAUGAAGCAGGAACAUAUU | 506 |
D-1170 | UUCCUGCUUCAUGCCUUUAUU | 507 | UAAAGGCAUGAAGCAGGAAUU | 508 |
D-1171 | CUGCUUCAUGCCUUUCUAAUU | 509 | UUAGAAAGGCAUGAAGCAGUU | 510 |
D-1172 | GCUUCAUGCCUUUCUACAAUU | 511 | UUGUAGAAAGGCAUGAAGCUU | 512 |
D-1173 | UUCAUGCCUUUCUACAGUAUU | 513 | UACUGUAGAAAGGCAUGAAUU | 514 |
D-1174 | UCAUGCCUUUCUACAGUGAUU | 515 | UCACUGUAGAAAGGCAUGAUU | 516 |
D-1175 | CAUGCCUUUCUACAGUGGAUU | 517 | UCCACUGUAGAAAGGCAUGUU | 518 |
D-1176 | AUGCCUUUCUACAGUGGCAUU | 519 | UGCCACUGUAGAAAGGCAUUU | 520 |
D-1177 | ACGUACCCUUCAUUGAUGAUU | 521 | UCAUCAAUGAAGGGUACGUUU | 522 |
D-1178 | CCAGAGACUGGUGACAUGAUU | 523 | UCAUGUCACCAGUCUCUGGUU | 524 |
D-1179 | AUGGCUUCCAGAUAUGCCAUU | 525 | UGGCAUAUCUGGAAGCCAUUU | 526 |
D-1180 | GUUCCUGCUUCAUGCCUUUUU | 527 | AAAGGCAUGAAGCAGGAACUU | 528 |
D-1181 | CCUGCUUCAUGCCUUUCUAUU | 529 | UAGAAAGGCAUGAAGCAGGUU | 530 |
D-1182 | GCUUCAUGCCUUUCUACAUUU | 531 | AUGUAGAAAGGCAUGAAGCUU | 532 |
D-1183 | CUUCAUGCCUUUCUACAGUUU | 533 | ACUGUAGAAAGGCAUGAAGUU | 534 |
D-1184 | UUCAUGCCUUUCUACAGUUUU | 535 | AACUGUAGAAAGGCAUGAAUU | 536 |
D-1185 | GCUUCAUCCCUUUCUACAUUU | 537 | AUGUAGAAAGGGAUGAAGCUU | 538 |
D-1186 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 539 | AUGUAGAAGGGCAUGAAGCUU | 540 |
D-1187 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 541 | AAAUGUAGAAGGGCAUGAAGCUU | 542 |
D-1188 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 543 | AAAUGUAGAAGGGCAUGAAGCUU | 544 |
D-1189 | [INVAB]GCGGCUUCCUGGGCUUCUAUU | 545 | UAGAAGCCCAGGAAGCCGCUU | 546 |
D-1190 | [INVAB]CUGCGGCUUCCUGGGCUUCUA | 547 | UAGAAGCCCAGGAAGCCGCAGUU | 548 |
D-1191 | CUGCGGCUUCCUGGGCUUCU{INVAB} | 549 | UAGAAGCCCAGGAAGCCGCAGUU | 550 |
D-1192 | [INVAB]CUGCGGCUUCCUGGGCUUCUA | 551 | UAGAAGCCCAGGAAGCCGCAGUU | 552 |
D-1193 | [INVAB]GCGGCUUCCUGGGCUUCUAUU | 553 | UAGAAGCCCAGGAAGCCGCUU | 554 |
D-1194 | CUGCGGCUUCCUGGGCUUCU{INVAB} | 555 | UAGAAGCCCAGGAAGCCGCAGUU | 556 |
D-1195 | [INVAB]CUGCGGCUUCCUGGGCUUCUA | 557 | UAGAAGCCCAGGAAGCCGCAGUU | 558 |
D-1196 | [INVAB]GCGGCUUCCUGGGCUUCUAUU | 559 | UAGAAGCCCAGGAAGCCGCUU | 560 |
D-1197 | CUGCGGCUUCCUGGGCUUCU{INVAB} | 561 | UAGAAGCCCAGGAAGCCGCAGUU | 562 |
D-1198 | [INVAB]CUGCGGCUUCCUGGGCUUCUA | 563 | UAGAAGCCCAGGAAGCCGCAGUU | 564 |
D-1199 | [INVAB]GCGGCUUCCUGGGCUUCUAUU | 565 | UAGAAGCCCAGGAAGCCGCUU | 566 |
D-1200 | [INVAB]CUGCGGCUUCCUGGGCUUCUA | 567 | UAGAAGCCCAGGAAGCCGCAGUU | 568 |
D-1201 | [INVAB]CUGCGGCUUCCUGGGCUUCUA | 569 | UAGAAGCCCAGGAAGCCGCAGUU | 570 |
D-1202 | [INVAB]CUGCGGCUUCCUGGGCUUCUA | 571 | UAGAAGCCCAGGAAGCCGCAGUU | 572 |
D-1203 | [INVAB]AUGGCUUCCAGAUAUGCCUUU | 573 | AGGCAUAUCUGGAAGCCAUUU | 574 |
D-1204 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 575 | AGGCAUAUCUGGAAGCCAUGUUU | 576 |
D-1205 | ACAUGGCUUCCAGAUAUGCC{INVAB} | 577 | AGGCAUAUCUGGAAGCCAUGUUU | 578 |
D-1206 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 579 | AGGCAUAUCUGGAAGCCAUGUUU | 580 |
D-1207 | [INVAB]AUGGCUUCCAGAUAUGCCUUU | 581 | AGGCAUAUCUGGAAGCCAUUU | 582 |
D-1208 | ACAUGGCUUCCAGAUAUGCC{INVAB} | 583 | AGGCAUAUCUGGAAGCCAUGUUU | 584 |
D-1209 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 585 | AGGCAUAUCUGGAAGCCAUGUUU | 586 |
D-1210 | [INVAB]AUGGCUUCCAGAUAUGCCUUU | 587 | AGGCAUAUCUGGAAGCCAUUU | 588 |
D-1211 | ACAUGGCUUCCAGAUAUGCC{INVAB} | 589 | AGGCAUAUCUGGAAGCCAUGUUU | 590 |
D-1212 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 591 | AGGCAUAUCUGGAAGCCAUGUUU | 592 |
D-1213 | [INVAB]AUGGCUUCCAGAUAUGCCUUU | 593 | AGGCAUAUCUGGAAGCCAUUU | 594 |
D-1214 | ACAUGGCUUCCAGAUAUGCC{INVAB} | 595 | AGGCAUAUCUGGAAGCCAUGUUU | 596 |
D-1215 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 597 | AGGCAUAUCUGGAAGCCAUGUUU | 598 |
D-1216 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 599 | AGGCAUAUCUGGAAGCCAUGUUU | 600 |
D-1217 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 601 | AGGCAUAUCUGGAAGCCAUGUUU | 602 |
D-1218 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 603 | AGGCAUAUCUGGAAGCCAUGUUU | 604 |
D-1219 | [INVAB]ACGUACCCUUCAUUGAUGUUU | 605 | ACAUCAAUGAAGGGUACGUUU | 606 |
D-1220 | [INVAB]CAACGUACCCUUCAUUGAUGU | 607 | ACAUCAAUGAAGGGUACGUUGUU | 608 |
D-1221 | [INVAB]CAACGUACCCUUCAUUGAUGU | 609 | ACAUCAAUGAAGGGUACGUUGUU | 610 |
D-1222 | [INVAB]ACGUACCCUUCAUUGAUGUUU | 611 | ACAUCAAUGAAGGGUACGUUU | 612 |
D-1223 | CAACGUACCCUUCAUUGAUG{INVAB} | 613 | ACAUCAAUGAAGGGUACGUUGUU | 614 |
D-1224 | [INVAB]ACGUACCCUUCAUUGAUGUUU | 615 | ACAUCAAUGAAGGGUACGUUU | 616 |
D-1225 | CAACGUACCCUUCAUUGAUG{INVAB} | 617 | ACAUCAAUGAAGGGUACGUUGUU | 618 |
D-1226 | [INVAB]CAACGUACCCUUCAUUGAUGU | 619 | ACAUCAAUGAAGGGUACGUUGUU | 620 |
D-1227 | [INVAB]ACGUACCCUUCAUUGAUGUUU | 621 | ACAUCAAUGAAGGGUACGUUU | 622 |
D-1228 | CAACGUACCCUUCAUUGAUG{INVAB} | 623 | ACAUCAAUGAAGGGUACGUUGUU | 624 |
D-1229 | [INVAB]CAACGUACCCUUCAUUGAUGU | 625 | ACAUCAAUGAAGGGUACGUUGUU | 626 |
D-1230 | [INVAB]CAACGUACCCUUCAUUGAUGU | 627 | ACAUCAAUGAAGGGUACGUUGUU | 628 |
D-1231 | [INVAB]CAACGUACCCUUCAUUGAUGU | 629 | ACAUCAAUGAAGGGUACGUUGUU | 630 |
D-1232 | [INVAB]CAACGUACCCUUCAUUGAUGU | 631 | ACAUCAAUGAAGGGUACGUUGUU | 632 |
D-1233 | CUGCUUCAUGCCUUUCUACAU | 633 | AUGUAGAAAGGCAUGAAGCAGUU | 634 |
D-1234 | CUGCUUCAUGCCUUUCUACAU | 635 | AUGUAGAAAGGCAUGAAGCAGUU | 636 |
D-1235 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 637 | AUGUAGAAAGGCAUGAAGCUU | 638 |
D-1236 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 639 | AUGUAGAAAGGCAUGAAGCUU | 640 |
D-1237 | CUGCUUCAUGCCUUUCUACAU | 641 | AUGUAGAAAGGCAUGAAGCAGUU | 642 |
D-1238 | CUGCUUCAUGCCUUUCUACAU | 643 | AUGUAGAAAGGCAUGAAGCAGUU | 644 |
D-1239 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 645 | AUGUAGAAAGGCAUGAAGCAGUU | 646 |
D-1240 | CUGCUUCAUGCCUUUCUACA{INVAB} | 647 | AUGUAGAAAGGCAUGAAGCAGUU | 648 |
D-1241 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 649 | AUGUAGAAAGGCAUGAAGCAGUU | 650 |
D-1242 | [INVAB]CUGCUUCAUGC[DC]UUUCUACAU | 651 | AUGUAGAAAGGCAUGAAGCAGUU | 652 |
D-1243 | CUGCUUCAUGCCUUUCUACAU | 653 | AUGUAGAAAGGCAUGAAGCAGUU | 654 |
D-1244 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 655 | AUGUAGAAAGGCAUGAAGCAGUU | 656 |
D-1245 | CUGCUUCAUGCCUUUCUACAU | 657 | AUGUAGAAAGGCAUGAAGCAGUU | 658 |
D-1246 | CUGCUUCAUGCCUUUCUACA{INVAB} | 659 | AUGUAGAAAGGCAUGAAGCAGUU | 660 |
D-1247 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 661 | AUGUAGAAAGGCAUGAAGCAGUU | 662 |
D-1248 | CUGCUUCAUGCCUUUCUACAU | 663 | AUGUAGAAAGGCAUGAAGCAGUU | 664 |
D-1249 | CUGCUUCAUGCCUUUCUACA{INVAB} | 665 | AUGUAGAAAGGCAUGAAGCAGUU | 666 |
D-1250 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 667 | AUGUAGAAAGGCAUGAAGCAGUU | 668 |
D-1251 | CUGCUUCAUGCCUUUCUACA{INVAB} | 669 | AUGUAGAAAGGCAUGAAGCAGUU | 670 |
D-1252 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 671 | AUGUAGAAAGGCAUGAAGCAGUU | 672 |
D-1253 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 673 | AUGUAGAAAGGCAUGAAGCUU | 674 |
D-1254 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 675 | AUGUAGAAAGGCAUGAAGCUU | 676 |
D-1255 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 677 | AUGUAGAAAGGCAUGAAGCUU | 678 |
D-1256 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 679 | AUGUAGAAAGGCAUGAAGCUU | 680 |
D-1257 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 681 | AUGUAGAAAGGCAUGAAGCUU | 682 |
D-1258 | CUGCUUCAUGCCCUUCUACAU | 683 | AUGUAGAAGGGCAUGAAGCAGUU | 684 |
D-1259 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 685 | AUGUAGAAGGGCAUGAAGCUU | 686 |
D-1260 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 687 | AUGUAGAAGGGCAUGAAGCUU | 688 |
D-1261 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 689 | AUGUAGAAGGGCAUGAAGCUU | 690 |
D-1262 | CUGCUUCAUGCCCUUCUACA{INVAB} | 691 | AUGUAGAAGGGCAUGAAGCAGUU | 692 |
D-1263 | [INVAB]CUGCUUCAUGCCCUUCUACAU | 693 | AUGUAGAAGGGCAUGAAGCAGUU | 694 |
D-1264 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 695 | AUGUAGAAGGGCAUGAAGCUU | 696 |
D-1265 | AUGUUCCUGCUUCAUGCCUUU | 697 | AGGCAUGAAGCAGGAACAUUU | 698 |
D-1266 | UGUUCCUGCUUCAUGCCUUUU | 699 | AAGGCAUGAAGCAGGAACAUU | 700 |
D-1267 | UGCCUUUCUACAGUGGCCUUU | 701 | AGGCCACUGUAGAAAGGCAUU | 702 |
D-1268 | CGUACUUCGUCCUUGUAUGUU | 703 | CAUACAAGGACGAAGUACGUU | 704 |
D-1269 | [INVAB]GCUUCAUGC[DC]CUUCUACAUUU | 705 | AUGUAGAAGGGCAUGAAGCUU | 706 |
D-1270 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 707 | AUGUAGAAGGGCAUGAAGCUU | 708 |
D-1271 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 709 | AUGUAGAAGGGCAUGAAGCUU | 710 |
D-1272 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 711 | AUGUAGAAGGGCAUGAAGCUU | 712 |
D-1273 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 713 | AUGUAGAAGGGCAUGAAGCUU | 714 |
D-1274 | [INVAB]GCUUCAUGCCCUUCUACAUUU | 715 | AUGUAGAAGGGCAUGAAGCUU | 716 |
D-1275 | [INVAB]CCUGCUUCAUGCCUUUCUAUU | 717 | UAGAAAGGCAUGAAGCAGGUU | 718 |
D-1276 | [INVAB]CCUGCUUCAUGCCUUUCUAUU | 719 | UAGAAAGGCAUGAAGCAGGUU | 720 |
D-1277 | UUCCUGCUUCAUGCCUUUCU{INVAB} | 721 | UAGAAAGGCAUGAAGCAGGAAUU | 722 |
D-1278 | [INVAB]CCUGCUUCAUGCCUUUCUAUU | 723 | UAGAAAGGCAUGAAGCAGGUU | 724 |
D-1279 | UUCCUGCUUCAUGCCUUUCU{INVAB} | 725 | UAGAAAGGCAUGAAGCAGGAAUU | 726 |
D-1280 | [INVAB]AUGCCUUUCUACAGUGGCUUU | 727 | AGCCACUGUAGAAAGGCAUUU | 728 |
D-1281 | [INVAB]AUGCCUUUCUACAGUGGCUUU | 729 | AGCCACUGUAGAAAGGCAUUU | 730 |
D-1282 | [INVAB]AUGCCUUUCUACAGUGGCUUU | 731 | AGCCACUGUAGAAAGGCAUUU | 732 |
D-1283 | UCAUGCCUUUCUACAGUGGC{INVAB} | 733 | AGCCACUGUAGAAAGGCAUGAUU | 734 |
D-1284 | [INVAB]AUGCCUUUCUACAGUGGCUUU | 735 | AGCCACUGUAGAAAGGCAUUU | 736 |
D-1285 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 737 | AGCCACUGUAGAAAGGCAUGAUU | 738 |
D-1286 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 739 | ACUGUAGAAAGGCAUGAAGCAUU | 740 |
D-1287 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 741 | ACUGUAGAAAGGCAUGAAGUU | 742 |
D-1289 | UGCUUCAUGCCUUUCUACAG{INVAB} | 743 | ACUGUAGAAAGGCAUGAAGCAUU | 744 |
D-1290 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 745 | ACUGUAGAAAGGCAUGAAGUU | 746 |
D-1291 | UGCUUCAUGCCUUUCUACAG{INVAB} | 747 | ACUGUAGAAAGGCAUGAAGCAUU | 748 |
D-1292 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 749 | ACUGUAGAAAGGCAUGAAGCAUU | 750 |
D-1293 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 751 | ACUGUAGAAAGGCAUGAAGCAUU | 752 |
D-1294 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 753 | ACUGUAGAAAGGCAUGAAGCAUU | 754 |
D-1295 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 755 | ACUGUAGAAAGGCAUGAAGCAUU | 756 |
D-1296 | [INVAB]GUUCCUGCUUCAUGCCUUUUU | 757 | AAAGGCAUGAAGCAGGAACUU | 758 |
D-1297 | [INVAB]CCUGCUUCAUGCCUUUCUAUU | 759 | UAGAAAGGCAUGAAGCAGGUU | 760 |
D-1298 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 761 | ACUGUAGAAAGGCAUGAAGUU | 762 |
D-1299 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 763 | UAGAAAGGCAUGAAGCAGGAAUU | 764 |
D-1300 | UUCCUGCUUCAUGCCUUUCU{INVAB} | 765 | UAGAAAGGCAUGAAGCAGGAAUU | 766 |
D-1301 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 767 | UAGAAAGGCAUGAAGCAGGAAUU | 768 |
D-1302 | UUCCUGCUUCAUGCCUUUCU{INVAB} | 769 | UAGAAAGGCAUGAAGCAGGAAUU | 770 |
D-1303 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 771 | UAGAAAGGCAUGAAGCAGGAAUU | 772 |
D-1304 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 773 | UAGAAAGGCAUGAAGCAGGAAUU | 774 |
D-1305 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 775 | UAGAAAGGCAUGAAGCAGGAAUU | 776 |
D-1306 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 777 | UAGAAAGGCAUGAAGCAGGAAUU | 778 |
D-1307 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 779 | UAGAAAGGCAUGAAGCAGGAAUU | 780 |
D-1308 | [INVAB]UUCCUGCUUCAUGCCUUUCUA | 781 | UAGAAAGGCAUGAAGCAGGAAUU | 782 |
D-1309 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 783 | AGCCACUGUAGAAAGGCAUGAUU | 784 |
D-1310 | UCAUGCCUUUCUACAGUGGC{INVAB} | 785 | AGCCACUGUAGAAAGGCAUGAUU | 786 |
D-1311 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 787 | AGCCACUGUAGAAAGGCAUGAUU | 788 |
D-1312 | UCAUGCCUUUCUACAGUGGC{INVAB} | 789 | AGCCACUGUAGAAAGGCAUGAUU | 790 |
D-1313 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 791 | AGCCACUGUAGAAAGGCAUGAUU | 792 |
D-1314 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 793 | AGCCACUGUAGAAAGGCAUGAUU | 794 |
D-1315 | UCAUGCCUUUCUACAGUGGC{INVAB} | 795 | AGCCACUGUAGAAAGGCAUGAUU | 796 |
D-1316 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 797 | AGCCACUGUAGAAAGGCAUGAUU | 798 |
D-1317 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 799 | AGCCACUGUAGAAAGGCAUGAUU | 800 |
D-1318 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 801 | AGCCACUGUAGAAAGGCAUGAUU | 802 |
D-1319 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 803 | ACUGUAGAAAGGCAUGAAGCAUU | 804 |
D-1320 | UGCUUCAUGCCUUUCUACAG{INVAB} | 805 | ACUGUAGAAAGGCAUGAAGCAUU | 806 |
D-1321 | UGCUUCAUGCCUUUCUACAG{INVAB} | 807 | ACUGUAGAAAGGCAUGAAGCAUU | 808 |
D-1322 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 809 | ACUGUAGAAAGGCAUGAAGCAUU | 810 |
D-1323 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 811 | ACUGUAGAAAGGCAUGAAGUU | 812 |
D-1324 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 813 | ACUGUAGAAAGGCAUGAAGCAUU | 814 |
D-1325 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 815 | AUGUAGAAAGGCAUGAAGCUU | 816 |
D-1326 | [INVAB]CAUGCCUUUCUACAGUGGUUU | 817 | ACCACUGUAGAAAGGCAUGUU | 818 |
D-1327 | [INVAB]CUGCUUCAUGCCUUUCUAAUU | 819 | UUAGAAAGGCAUGAAGCAGUU | 820 |
D-1328 | [INVAB]GCUUCAUGCCUUUCUACAAUU | 821 | UUGUAGAAAGGCAUGAAGCUU | 822 |
D-1329 | [INVAB]UUCAUGCCUUUCUACAGUAUU | 823 | UACUGUAGAAAGGCAUGAAUU | 824 |
D-1330 | [INVAB]GUUCCUGCUUCAUGCCUUUUU | 825 | AAAGGCAUGAAGCAGGAACUU | 826 |
D-1331 | AUGUUCCUGCUUCAUGCCUU{INVAB} | 827 | AAAGGCAUGAAGCAGGAACAUUU | 828 |
D-1332 | [INVAB]AUGUUCCUGCUUCAUGCCUUU | 829 | AAAGGCAUGAAGCAGGAACAUUU | 830 |
D-1333 | [INVAB]GUUCCUGCUUCAUGCCUUUUU | 831 | AAAGGCAUGAAGCAGGAACUU | 832 |
D-1334 | AUGUUCCUGCUUCAUGCCUU{INVAB} | 833 | AAAGGCAUGAAGCAGGAACAUUU | 834 |
D-1335 | [INVAB]AUGUUCCUGCUUCAUGCCUUU | 835 | AAAGGCAUGAAGCAGGAACAUUU | 836 |
D-1336 | [INVAB]GUUCCUGCUUCAUGCCUUUUU | 837 | AAAGGCAUGAAGCAGGAACUU | 838 |
D-1337 | AUGUUCCUGCUUCAUGCCUU{INVAB} | 839 | AAAGGCAUGAAGCAGGAACAUUU | 840 |
D-1338 | [INVAB]AUGUUCCUGCUUCAUGCCUUU | 841 | AAAGGCAUGAAGCAGGAACAUUU | 842 |
D-1339 | [INVAB]AUGUUCCUGCUUCAUGCCUUU | 843 | AAAGGCAUGAAGCAGGAACAUUU | 844 |
D-1340 | [INVAB]AUGUUCCUGCUUCAUGCCUUU | 845 | AAAGGCAUGAAGCAGGAACAUUU | 846 |
D-1341 | [INVAB]GCUUCAUGCCUUUCUACAGUA | 847 | UACUGUAGAAAGGCAUGAAGCUU | 848 |
D-1342 | [INVAB]GCUUCAUGCCUUUCUACAGUA | 849 | UACUGUAGAAAGGCAUGAAGCUU | 850 |
D-1343 | [INVAB]UUCAUGCCUUUCUACAGUAUU | 851 | UACUGUAGAAAGGCAUGAAUU | 852 |
D-1344 | [INVAB]GCUUCAUGCCUUUCUACAGUA | 853 | UACUGUAGAAAGGCAUGAAGCUU | 854 |
D-1345 | GCUUCAUGCCUUUCUACAGU{INVAB} | 855 | UACUGUAGAAAGGCAUGAAGCUU | 856 |
D-1346 | [INVAB]GCGGCUUCCUGGGCUUCUAUU | 857 | UAGAAGCCCAGGAAGCCGCUU | 858 |
D-1347 | [INVAB]AUGGCUUCCAGAUAUGCCUUU | 859 | AGGCAUAUCUGGAAGCCAUUU | 860 |
D-1348 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 861 | AGGCAUAUCUGGAAGCCAUGUUU | 862 |
D-1349 | [INVAB]ACAUGGCUUCCAGAUAUGCCU | 863 | AGGCAUAUCUGGAAGCCAUGUUU | 864 |
D-1350 | [INVAB]CAACGUACCCUUCAUUGAUGU | 865 | ACAUCAAUGAAGGGUACGUUGUU | 866 |
D-1351 | [INVAB]CAACGUACCCUUCAUUGAUGU | 867 | ACAUCAAUGAAGGGUACGUUGUU | 868 |
D-1352 | [INVAB]ACGUACCCUUCAUUGAUGUUU | 869 | ACAUCAAUGAAGGGUACGUUU | 870 |
D-1353 | CUGCUUCAUGCCUUUCUACA{INVAB} | 871 | AUGUAGAAAGGCAUGAAGCAGUU | 872 |
D-1354 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 873 | AUGUAGAAAGGCAUGAAGCAGUU | 874 |
D-1355 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 875 | AUGUAGAAAGGCAUGAAGCUU | 876 |
D-1356 | CUGCUUCAUGCCUUUCUACA{INVAB} | 877 | AUGUAGAAAGGCAUGAAGCAGUU | 878 |
D-1357 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 879 | AUGUAGAAAGGCAUGAAGCAGUU | 880 |
D-1358 | CUGCUUCAUGCCUUUCUACA{INVAB} | 881 | AUGUAGAAAGGCAUGAAGCAGUU | 882 |
D-1359 | [INVAB]CUGCUUCAUGCCUUUCUACA{INVAB} | 883 | AUGUAGAAAGGCAUGAAGCAGUU | 884 |
D-1360 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 885 | AUGUAGAAAGGCAUGAAGCUU | 886 |
D-1361 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 887 | ACUGUAGAAAGGCAUGAAGUU | 888 |
D-1362 | UGCUUCAUGCCUUUCUACAG{INVAB} | 889 | ACUGUAGAAAGGCAUGAAGCAUU | 890 |
D-1363 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 891 | AUGUAGAAAGGCAUGAAGCAGUU | 892 |
D-1364 | CUGCUUCAUGCCUUUCUACA{INVAB} | 893 | AUGUAGAAAGGCAUGAAGCAGUU | 894 |
D-1365 | CUGCUUCAUGCCUUUCUACA{INVAB} | 895 | AUGUAGAAAGGCAUGAAGCAGUU | 896 |
D-1366 | [INVAB]CUUCAUCCCUUUCUACAGUUU | 897 | ACUGUAGAAAGGGAUGAAGUU | 898 |
D-1367 | [INVAB]GCUUCAUCCCUUUCUACAUUU | 899 | AUGUAGAAAGGGAUGAAGCUU | 900 |
D-1368 | UGCUUCAUCCCUUUCUACAG{INVAB} | 901 | ACUGUAGAAAGGGAUGAAGCAUU | 902 |
D-1369 | CUGCUUCAUCCCUUUCUACA{INVAB} | 903 | AUGUAGAAAGGGAUGAAGCAGUU | 904 |
D-1370 | [INVAB]ACAUUGCUCUUUCACCUGAUU | 905 | UCAGGUGAAAGAGCAAUGUUU | 906 |
D-1371 | CUGCUUCAUGCCUUUCUACA{INVAB} | 907 | AUGUAGAAAGGCAUGAAGCAGUU | 908 |
D-1372 | CUGCUUCAUGCCUUUCUACA{INVAB} | 909 | AUGUAGAAAGGCAUGAAGCAGUU | 910 |
D-1373 | CUGCUUCAUGCCUUUCUACA{INVAB} | 911 | AUGUAGAAAGGCAUGAAGCAGUU | 912 |
D-1374 | CUGCUUCAUGCCUUUCUACA{INVAB} | 913 | AUGUAGAAAGGCAUGAAGCAGUU | 914 |
D-1375 | CUGCUUCAUGCCUUUCUACA{INVAB} | 915 | AUGUAGAAAGGCAUGAAGCAGUU | 916 |
D-1376 | CUGCUUCAUGCCUUUCUACA{INVAB} | 917 | AUGUAGAAAGGCAUGAAGCAGUU | 918 |
D-1377 | CUGCUUCAUGCCUUUCUACA{INVAB} | 919 | AUGUAGAAAGGCAUGAAGCAGUU | 920 |
D-1378 | CUGCUUCAUGCCUUUCUACA{INVAB} | 921 | AUGUAGAAAGGCAUGAAGCAGUU | 922 |
D-1379 | CUGCUUCAUGCCUUUCUACA{INVAB} | 923 | AUGUAGAAAGGCAUGAAGCAGUU | 924 |
D-1380 | CUGCUUCAUGCCUUUCUACA{INVAB} | 925 | AUGUAGAAAGGCAUGAAGCAGUU | 926 |
D-1381 | CUGCUUCAUGCCUUUCUACA{INVAB} | 927 | AUGUAGAAAGGCAUGAAGCAGUU | 928 |
D-1381 | [INVAB]CUUCAUGCC[DT]UUCUACAGUUU | 929 | ACUGUAGAAAGGCAUGAAGUU | 930 |
D-1382 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 931 | ACUGUAGAAAGGCAUGAAGUU | 932 |
D-1383 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 933 | ACUGUAGAAAGGCAUGAAGUU | 934 |
D-1384 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 935 | ACUGUAGAAAGGCAUGAAGUU | 936 |
D-1385 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 937 | ACUGUAGAAAGGCAUGAAGUU | 938 |
D-1386 | [INVAB]CUUCAUGC[DC]UUUCUACAGUUU | 939 | ACUGUAGAAAGGCAUGAAGUU | 940 |
D-1387 | [INVAB]CUUCAUGCCU[DT]UCUACAGUUU | 941 | ACUGUAGAAAGGCAUGAAGUU | 942 |
D-1388 | [INVAB]GCUUCAUGGGAUUCUACAUUU | 943 | AUGUAGAAUCCCAUGAAGCUU | 944 |
D-1389 | [INVAB]CUUCAUGCGAAUCUACAGUUU | 945 | ACUGUAGAUUCGCAUGAAGUU | 946 |
D-1390 | CUGCUUCAUGCCUUUCUACA{INVAB} | 947 | UUGUAGAAAGGCAUGAAGCAGUU | 948 |
D-1391 | [INVAB]CUGCUUCAUGCCUUUCUACAA | 949 | UUGUAGAAAGGCAUGAAGCAGUU | 950 |
D-1392 | CUGCUUCAUGCCUUUCUACA{INVDA} | 951 | UUGUAGAAAGGCAUGAAGCAGUU | 952 |
D-1393 | CUGCUUCAUGGGAUUCUACA{INVAB} | 953 | AUGUAGAAUCCCAUGAAGCAGUU | 954 |
D-1394 | [INVAB]CUGCUUCAUGGGAUUCUACAU | 955 | AUGUAGAAUCCCAUGAAGCAGUU | 956 |
D-1395 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 957 | ACUGUAGAAAGGCAUGAAGUU | 958 |
D-1396 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 959 | ACUGUAGAAAGGCAUGAAGUU | 960 |
D-1397 | [INVAB]GCUUCAUGCCUUUCUACAUUU | 961 | AUGUAGAAAGGCAUGAAGCUU | 962 |
D-1398 | [INVAB]UGCUUCAUGCCUUUCUACAG{INVAB} | 963 | ACUGUAGAAAGGCAUGAAGCAUU | 964 |
D-1399 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 965 | ACUGUAGAAAGGCAUGAAGUU | 966 |
D-1400 | UGCUUCAUGCCUUUCUACAG{INVAB} | 967 | ACUGUAGAAAGGCAUGAAGCAUU | 968 |
D-1401 | UGCUUCAUGCCUUUCUACAG{INVAB} | 969 | ACUGUAGAAAGGCAUGAAGCAUU | 970 |
D-1402 | UGCUUCAUGCCUUUCUACAG{INVAB} | 971 | ACUGUAGAAAGGCAUGAAGCAUU | 972 |
D-1403 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 973 | ACUGUAGAAAGGCAUGAAGUU | 974 |
D-1404 | UGCUUCAUGCCUUUCUACAG{INVAB} | 975 | ACUGUAGAAAGGCAUGAAGCAUU | 976 |
D-1405 | AUGCCUUUCUACAGUGGCUU{INVAB} | 977 | AGCCACUGUAGAAAGGCAUGAUU | 978 |
D-1406 | UCAUGCCUUUCUACAGUGGC{INVAB} | 979 | AGCCACUGUAGAAAGGCAUGAUU | 980 |
D-1407 | [INVAB]AUGCCUUUCUACAGUGGCUUU | 981 | AGCCACUGUAGAAAGGCAUUU | 982 |
D-1408 | [INVAB]AUGCCUUUCUACAGUGGCUUU | 983 | AGCCACUGUAGAAAGGCAUUU | 984 |
D-1409 | [INVAB]UCAUGCCUUUCUACAGUGGCU | 985 | AGCCACUGUAGAAAGGCAUGAUU | 986 |
D-1410 | UCAUGCCUUUCUACAGUGGC{INVAB} | 987 | AGCCACUGUAGAAAGGCAUGAUU | 988 |
D-1411 | [INVAB]AUGCCUUUCUACAGUGGCUUU | 989 | AGCCACUGUAGAAAGGCAUUU | 990 |
D-1412 | UCAUGCCUUUCUACAGUGGC{INVAB} | 991 | AGCCACUGUAGAAAGGCAUGAUU | 992 |
D-1413 | UCAUGCCUUUCUACAGUGGC{INVAB} | 993 | AGCCACUGUAGAAAGGCAUGAUU | 994 |
D-1414 | [INVAB]UCAUGCCUUUCUACAGUGGC{INVAB} | 995 | AGCCACUGUAGAAAGGCAUGAUU | 996 |
D-1415 | [INVAB]AUGCCUUUCUACAGUGGCAUU | 997 | UGCCACUGUAGAAAGGCAUUU | 998 |
D-1416 | UCAUGCCUUUCUACAGUGGC{INVAB} | 999 | UGCCACUGUAGAAAGGCAUGAUU | 1000 |
D-1417 | [INVAB]UCAUGCCUUUCUACAGUGGCA | 1001 | UGCCACUGUAGAAAGGCAUGAUU | 1002 |
D-1418 | UCAUGCCUUUCUACAGUGGC{INVDA} | 1003 | UGCCACUGUAGAAAGGCAUGAUU | 1004 |
D-1419 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1005 | AUGUAGAAAGGCAUGAAGCAGUU | 1006 |
D-1420 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1007 | UUGUAGAAAGGCAUGAAGCAGUU | 1008 |
D-1421 | CUGCUUCAUGCCUUUCUACA{INVDA} | 1009 | UUGUAGAAAGGCAUGAAGCAGUU | 1010 |
D-1422 | UCAUGCCUUUCUACAGUGGC{INVAB} | 1011 | UGCCACUGUAGAAAGGCAUGAUU | 1012 |
D-1423 | UCAUGCCUUUCUACAGUGGC{INVDA} | 1013 | UGCCACUGUAGAAAGGCAUGAUU | 1014 |
D-1424 | [INVAB]CUUCAUGCCUUUCUACAGUUU | 1015 | ACUGUAGAAAGGCAUGAAGUU | 1016 |
D-1425 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1017 | AUGUA[AB]AAAGGCAUGAAGCAGUU | 1018 |
D-1426 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1019 | AUGUA[AB]AAAGGCAUGAAGCAGUU | 1020 |
D-1427 | UGCUUCAUGCCUUUCUACAG{INVAB} | 1021 | ACUGU[AB]GAAAGGCAUGAAGCAUU | 1022 |
D-1428 | UGCUUCAUGCCUUUCUACAG{INVAB} | 1023 | ACUGU[AB]GAAAGGCAUGAAGCAUU | 1024 |
D-1429 | UCAUGCCUUUCUACAGUGGC{INVAB} | 1025 | AGCCA[AB]UGUAGAAAGGCAUGAUU | 1026 |
D-1430 | UCAUGCCUUUCUACAGUGGC{INVAB} | 1027 | AGCCA[AB]UGUAGAAAGGCAUGAUU | 1028 |
D-1431 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1029 | AU[GNA-G]UAGAAAGGCAUGAAGCAGUU | 1030 |
D-1432 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1031 | AUG[GNA-U]AGAAAGGCAUGAAGCAGUU | 1032 |
D-1433 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1033 | AUGU[GNA-A]GAAAGGCAUGAAGCAGUU | 1034 |
D-1434 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1035 | AUGUA[GNA-G]AAAGGCAUGAAGCAGUU | 1036 |
D-1435 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1037 | AUGUAG[GNA-A]AAGGCAUGAAGCAGUU | 1038 |
D-1436 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1039 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1040 |
D-1437 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1041 | AUGUAGAAAGGCAUGAAGCAGUU | 1042 |
D-1438 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 1043 | AUGUAGAAAGGCAUGAAGCAGUU | 1044 |
D-1439 | [INVAB]CUGCUUCAUGCCUUUCUACA{INVAB} | 1045 | AUGUAGAAAGGCAUGAAGCAGUU | 1046 |
D-1440 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1047 | AUGUAGAAAGGCAUGAAGCAGUU | 1048 |
D-1441 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1049 | AUGUAGAAAGGCAUGAAGCAGUU | 1050 |
D-1442 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 1051 | AUGUAGAAAGGCAUGAAGCAGUU | 1052 |
D-1443 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1053 | AUGUAGAAAGGCAUGAAGCAGUU | 1054 |
D-1444 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1055 | AUGUAGAAAGGCAUGAAGCAGUU | 1056 |
D-1445 | [INVAB]CUGCUUCAUGCCUUUCUACAU | 1057 | AUGUAGAAAGGCAUGAAGCAGUU | 1058 |
D-1446 | [INVAB]CUGCUUCAUGCCUUUCUACA{INVAB} | 1059 | AUGUAGAAAGGCAUGAAGCAGUU | 1060 |
D-1447 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1061 | A[AB]GUAGAAAGGCAUGAAGCAGUU | 1062 |
D-1448 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1063 | AU[AB]UAGAAAGGCAUGAAGCAGUU | 1064 |
D-1449 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1065 | AUG[AB]AGAAAGGCAUGAAGCAGUU | 1066 |
D-1450 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1067 | AUGU[AB]GAAAGGCAUGAAGCAGUU | 1068 |
D-1451 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1069 | AUGUAG[AB]AAGGCAUGAAGCAGUU | 1070 |
D-1452 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1071 | AUGUAGA[AB]AGGCAUGAAGCAGUU | 1072 |
D-1453 | CAACGUACCCUUCAUUGAUG{INVAB} | 1073 | ACAUCAAUGAAGGGUACGUUGUU | 1074 |
D-1454 | CAACGUACCCUUCAUUGAUG{INVAB} | 1075 | ACAUCAAUGAAGGGUACGUUGUU | 1076 |
D-1455 | ACAUGGCUUCCAGAUAUGCC{INVAB} | 1077 | AGGCAUAUCUGGAAGCCAUGUUU | 1078 |
D-1456 | ACAUGGCUUCCAGAUAUGCC{INVAB} | 1079 | AGGCAUAUCUGGAAGCCAUGUUU | 1080 |
D-1457 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1081 | AUGUAGAAAGGCAUGAAGCAGUU | 1082 |
D-1458 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1083 | AUGUAGAAAGGCAUGAAGCAGUU | 1084 |
D-1459 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1085 | AUGUAGAAAGGCAUGAAGCAGUU | 1086 |
D-1460 | CUGCGGCUUCCUGGGCUUCU{INVAB} | 1087 | UAGAAGCCCAGGAAGCCGCAGUU | 1088 |
D-1461 | CUGCGGCUUCCUGGGCUUCU{INVAB} | 1089 | UAGAAGCCCAGGAAGCCGCAGUU | 1090 |
D-1462 | UGCUUCAUGCCUUUCUACAG{INVAB} | 1091 | ACUGUAGAAAGGCAUGAAGCAUU | 1092 |
D-1463 | [INVAB]UGCUUCAUGCCUUUCUACAGU | 1093 | ACUGUAGAAAGGCAUGAAGCAUU | 1094 |
D-1464 | [INVAB]UGCUUCAUGCCUUUCUACAG{INVAB} | 1095 | ACUGUAGAAAGGCAUGAAGCAUU | 1096 |
D-1465 | UGCUUCAUGCCUUUCUACAG{INVAB} | 1097 | ACUGUAGAAAGGCAUGAAGCAUU | 1098 |
D-1466 | UGCUUCAUGCCUUUCUACAG{INVAB} | 1099 | ACUGUAGAAAGGCAUGAAGCAUU | 1100 |
D-1467 | UGCUUCAUGCCUUUCUACAG{INVAB} | 1101 | ACUGUAGAAAGGCAUGAAGCAUU | 1102 |
D-1468 | CUGCUUCAUGCCUUUCUACAU | 1103 | AUGUAGAAAGGCAUGAAGCAGUU | 1104 |
D-1469 | CUGCUUCAUGCCUUUCUACAU | 1105 | AUGUAGAAAGGCAUGAAGCAGUU | 1106 |
D-1470 | CUGCUUCAUGCCUUUCUACAU | 1107 | AUGUAGAAAGGCAUGAAGCAGUU | 1108 |
D-1471 | UGCUUCAUGCCUUUCUACAG{INVAB} | 1109 | UCUGUAGAAAGGCAUGAAGCAUU | 1110 |
D-1472 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1111 | UCUGUAGAAAGGCAUGAAGCAUU | 1112 |
D-1473 | CUGCUUCAUGCCUUUCUACA{INVDT} | 1113 | AUGUAGAAAGGCAUGAAGCAGUU | 1114 |
D-1474 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1115 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1116 |
D-1475 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1117 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1118 |
D-1476 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1119 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1120 |
D-1477 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1121 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1122 |
D-1478 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1123 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1124 |
D-1479 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1125 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1126 |
D-1480 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1127 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1128 |
D-1481 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1129 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1130 |
D-1482 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1131 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1132 |
D-1483 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1133 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1134 |
D-1484 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1135 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1136 |
D-1485 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1137 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1138 |
D-1486 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1139 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1140 |
D-1487 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1141 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1142 |
D-1488 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1143 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1144 |
D-1489 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1145 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1146 |
D-1490 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1147 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1148 |
D-1491 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1149 | AUGUAGA[GNA-A]A[DG]GCAUGAAGCAGUU | 1150 |
D-1492 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1151 | AUGUAGA[GNA-A][DA]GGCAUGAAGCAGUU | 1152 |
D-1493 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1153 | AUGUAGA[GNA-A]AG[DG]CAUGAAGCAGUU | 1154 |
D-1494 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1155 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1156 |
D-1495 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1157 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1158 |
D-1496 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1159 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1160 |
D-1497 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1161 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1162 |
D-1498 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1163 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1164 |
D-1499 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1165 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1166 |
D-1500 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1167 | AUGUAGAAAGGCAUGAAGCAGUU | 1168 |
D-1501 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1169 | U[GNA-C]UGUAGAAAGGCAUGAAGCAUU | 1170 |
D-1502 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1171 | UC[GNA-U]GUAGAAAGGCAUGAAGCAUU | 1172 |
D-1503 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1173 | UCUG[GNA-U]AGAAAGGCAUGAAGCAUU | 1174 |
D-1504 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1175 | UCUGU[GNA-A]GAAAGGCAUGAAGCAUU | 1176 |
D-1505 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1177 | UCUGUAG[GNA-A]AAGGCAUGAAGCAUU | 1178 |
D-1506 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1179 | U[AB]UGUAGAAAGGCAUGAAGCAUU | 1180 |
D-1507 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1181 | UC[AB]GUAGAAAGGCAUGAAGCAUU | 1182 |
D-1508 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1183 | UCU[AB]UAGAAAGGCAUGAAGCAUU | 1184 |
D-1509 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1185 | UCUG[AB]AGAAAGGCAUGAAGCAUU | 1186 |
D-1510 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1187 | UCUGU[AB]GAAAGGCAUGAAGCAUU | 1188 |
D-1511 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1189 | UCUGUA[AB]AAAGGCAUGAAGCAUU | 1190 |
D-1512 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1191 | UCUGUAG[AB]AAGGCAUGAAGCAUU | 1192 |
D-1513 | UCAUGCCUUUCUACAGUGGC{INVDT} | 1193 | AGCCACUGUAGAAAGGCAUGAUU | 1194 |
D-1514 | UCAUGCCUUUCUACAGUGGC{INVAB} | 1195 | UGCCACUGUAGAAAGGCAUGAUU | 1196 |
D-1515 | UCAUGCCUUUCUACAGUGGC{INVDA} | 1197 | UGCCACUGUAGAAAGGCAUGAUU | 1198 |
D-1516 | UCCUGCUUCAUGCCUUUCUA{INVDT} | 1199 | AUAGAAAGGCAUGAAGCAGGAUU | 1200 |
D-1517 | UCCUGCUUCAUGCCUUUCUA{INVAB} | 1201 | UUAGAAAGGCAUGAAGCAGGAUU | 1202 |
D-1518 | UCCUGCUUCAUGCCUUUCUA{INVDA} | 1203 | UUAGAAAGGCAUGAAGCAGGAUU | 1204 |
D-1519 | UAUGUUCCUGCUUCAUGCCU{INVDT} | 1205 | AAGGCAUGAAGCAGGAACAUAUU | 1206 |
D-1520 | UAUGUUCCUGCUUCAUGCCU{INVAB} | 1207 | UAGGCAUGAAGCAGGAACAUAUU | 1208 |
D-1521 | UAUGUUCCUGCUUCAUGCCU{INVDA} | 1209 | UAGGCAUGAAGCAGGAACAUAUU | 1210 |
D-1522 | UCCUGCUUCAUGCCUUUCUA{INVAB} | 1211 | AUAGAAAGGCAUGAAGCAGGAUU | 1212 |
D-1523 | UAUGUUCCUGCUUCAUGCCU{INVAB} | 1213 | AAGGCAUGAAGCAGGAACAUAUU | 1214 |
D-1524 | UCAUGCCUUUCUACAGUGGC{INVDT} | 1215 | AGCCACUGUAGAAAGGCAUGAUU | 1216 |
D-1525 | UCCUGCUUCAUGCCUUUCUA{INVDT} | 1217 | AUAGAAAGGCAUGAAGCAGGAUU | 1218 |
D-1526 | UCCUGCUUCAUGCCUUUCUA{INVDA} | 1219 | UUAGAAAGGCAUGAAGCAGGAUU | 1220 |
D-1527 | UAUGUUCCUGCUUCAUGCCU{INVDA} | 1221 | UAGGCAUGAAGCAGGAACAUAUU | 1222 |
D-1528 | UGCUUCAUGCCUUUCUACAG{INVDA} | 1223 | UCUGUA[GNA-G]AAAGGCAUGAAGCAUU | 1224 |
D-1529 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1225 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1226 |
D-1530 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1227 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1228 |
D-1531 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1229 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1230 |
D-1532 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1231 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1232 |
D-1533 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1233 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1234 |
D-1534 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1235 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1236 |
D-1535 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1237 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1238 |
D-1536 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1239 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1240 |
D-1537 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1241 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1242 |
D-1538 | CUGCUUCAUGCCU[LNA-T]UCUACA{INVAB} | 1243 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1244 |
D-1539 | CUGCUUCAUGCC[LNA-T]UUCUACA{INVAB} | 1245 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1246 |
D-1540 | CUGCUUCAUGCCUU[LNA-T]CUACA{INVAB} | 1247 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1248 |
D-1541 | CUGCUUCAUGCCUUUC[LNA-T]ACA{INVAB} | 1249 | AUGU[GNA-A]GAAAGGCAUGAAGCAGUU | 1250 |
D-1542 | CUGCUUCAUGCCUUUCU[LNA-A]CA{INVAB} | 1251 | AUG[GNA-U]AGAAAGGCAUGAAGCAGUU | 1252 |
D-1543 | CUGCUUCAUGCCUUUCUAC[LNA-A]{INVAB} | 1253 | A[GNA-U]GUAGAAAGGCAUGAAGCAGUU | 1254 |
D-1544 | CUGCUUCAUGCCUU[LNA-T]CUACA{INVAB} | 1255 | AUGUAG[AB]AAGGCAUGAAGCAGUU | 1256 |
D-1545 | CUGCUUCAUGCCUUUC[LNA-T]ACA{INVAB} | 1257 | AUGU[AB]GAAAGGCAUGAAGCAGUU | 1258 |
D-1546 | CUGCUUCAUGCCUUUCU[LNA-A]CA{INVAB} | 1259 | AUG[AB]AGAAAGGCAUGAAGCAGUU | 1260 |
D-1547 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1261 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1262 |
D-1548 | CUGCUUCAUGCCUUUCUACA{INVAB} | 1263 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1264 |
D-1549 | CUGCUUCAUGCCU[LNA-T]UCUACA{INVAB} | 1265 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1266 |
D-1550 | CUGCUUCAUGCCU[LNA-T]UCUACA{INVAB} | 1267 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1268 |
D-1551 | CUGCUUCAUGCCU[LNA-T]UCUACA{INVAB} | 1269 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1270 |
D-1552 | CUGCUUCAUGCCU[LNA-T]UCUACA{INVAB} | 1271 | AUGUAGA[GNA-A]AGGCAUGAAGCAGUU | 1272 |
D-1553 | UGCUUCAUGCCUUUCU[LNA-A]CAG{INVDA} | 1273 | UCUG[AB]AGAAAGGCAUGAAGCAUU | 1274 |
D-1554 | UGCUUCAUGCCUUUC[LNA-T]ACAG{INVDA} | 1275 | UCUGU[AB]GAAAGGCAUGAAGCAUU | 1276 |
D-1555 | UGCUUCAUGCCUU[LNA-T]CUACAG{INVDA} | 1277 | UCUGUAG[AB]AAGGCAUGAAGCAUU | 1278 |
D-1556 | UGCUUCAUGCCUUUCUAC[LNA-A]G{INVDA} | 1279 | UC[GNA-U]GUAGAAAGGCAUGAAGCAUU | 1280 |
D-1557 | UGCUUCAUGCCUUUCU[LNA-A]CAG{INVDA} | 1281 | UCUG[GNA-U]AGAAAGGCAUGAAGCAUU | 1282 |
D-1558 | UGCUUCAUGCCUUUC[LNA-T]ACAG{INVDA} | 1283 | UCUGU[GNA-A]GAAAGGCAUGAAGCAUU | 1284 |
D-1559 | UGCUUCAUGCCUUUCUACA[LNA-G]{INVDA} | 1285 | U[AB]UGUAGAAAGGCAUGAAGCAUU | 1286 |
D-1560 | UGCUUCAUGCCUUUCUAC[LNA-A]G{INVDA} | 1287 | UC[AB]GUAGAAAGGCAUGAAGCAUU | 1288 |
D-1561 | UCAUGCCUUUCUACA[LNA-G]UGGC{INVAB} | 1289 | AGCCA[AB]UGUAGAAAGGCAUGAUU | 1290 |
D-1562 | UCAUGCCUUUCUACAG[LNA-T]GGC{INVAB} | 1291 | AGCCA[AB]UGUAGAAAGGCAUGAUU | 1292 |
D-1563 | UCAUGCCUUUCUACAGUGGC{INVAB} | 1293 | AGCCA[AB]UGUAGAAAGGCAUGAUU | 1294 |
D-1564 | GGUAUGUUCCUGCUUCAUUUU | 2257 | AAUGAAGCAGGAACAUACCUU | 2258 |
D-1565 | GGUAUGUUCCUGCUUCAUAUU | 2259 | UAUGAAGCAGGAACAUACCUU | 2260 |
D-1566 | GUAUGUUCCUGCUUCAUGUUU | 2261 | ACAUGAAGCAGGAACAUACUU | 2262 |
D-1567 | UAUGUUCCUGCUUCAUGCAUU | 2263 | UGCAUGAAGCAGGAACAUAUU | 2264 |
D-1568 | AUGUUCCUGCUUCAUGCCUUU | 2265 | AGGCAUGAAGCAGGAACAUUU | 2266 |
D-1569 | UGUUCCUGCUUCAUGCCUUUU | 2267 | AAGGCAUGAAGCAGGAACAUU | 2268 |
D-1570 | GUUCCUGCUUCAUGCCUUUUU | 2269 | AAAGGCAUGAAGCAGGAACUU | 2270 |
D-1571 | UUCCUGCUUCAUGCCUUUUUU | 2271 | AAAAGGCAUGAAGCAGGAAUU | 2272 |
D-1572 | UUCCUGCUUCAUGCCUUUAUU | 2273 | UAAAGGCAUGAAGCAGGAAUU | 2274 |
D-1573 | UCCUGCUUCAUGCCUUUCUUU | 2275 | AGAAAGGCAUGAAGCAGGAUU | 2276 |
D-1574 | CCUGCUUCAUGCCUUUCUAUU | 2277 | UAGAAAGGCAUGAAGCAGGUU | 2278 |
D-1575 | CUGCUUCAUGCCUUUCUAUUU | 2279 | AUAGAAAGGCAUGAAGCAGUU | 2280 |
D-1576 | CUGCUUCAUGCCUUUCUAAUU | 2281 | UUAGAAAGGCAUGAAGCAGUU | 2282 |
D-1577 | UGCUUCAUGCCUUUCUACAUU | 2283 | UGUAGAAAGGCAUGAAGCAUU | 2284 |
D-1578 | GCUUCAUGCCUUUCUACAUUU | 2285 | AUGUAGAAAGGCAUGAAGCUU | 2286 |
D-1579 | GCUUCAUGCCUUUCUACAAUU | 2287 | UUGUAGAAAGGCAUGAAGCUU | 2288 |
D-1580 | CUUCAUGCCUUUCUACAGUUU | 2289 | ACUGUAGAAAGGCAUGAAGUU | 2290 |
D-1581 | UUCAUGCCUUUCUACAGUUUU | 2291 | AACUGUAGAAAGGCAUGAAUU | 2292 |
D-1582 | UUCAUGCCUUUCUACAGUAUU | 2293 | UACUGUAGAAAGGCAUGAAUU | 2294 |
D-1583 | UCAUGCCUUUCUACAGUGUUU | 2295 | ACACUGUAGAAAGGCAUGAUU | 2296 |
D-1584 | UCAUGCCUUUCUACAGUGAUU | 2297 | UCACUGUAGAAAGGCAUGAUU | 2298 |
D-1585 | CAUGCCUUUCUACAGUGGUUU | 2299 | ACCACUGUAGAAAGGCAUGUU | 2300 |
D-1586 | CAUGCCUUUCUACAGUGGAUU | 2301 | UCCACUGUAGAAAGGCAUGUU | 2302 |
D-1587 | AUGCCUUUCUACAGUGGCUUU | 2303 | AGCCACUGUAGAAAGGCAUUU | 2304 |
D-1588 | AUGCCUUUCUACAGUGGCAUU | 2305 | UGCCACUGUAGAAAGGCAUUU | 2306 |
D-1589 | UGCCUUUCUACAGUGGCCUUU | 2307 | AGGCCACUGUAGAAAGGCAUU | 2308 |
D-1590 | GCCUUUCUACAGUGGCCUUUU | 2309 | AAGGCCACUGUAGAAAGGCUU | 2310 |
D-1591 | GGUAUGUUCCUGCUUCAUCUU | 2311 | GAUGAAGCAGGAACAUACCUU | 2312 |
D-1592 | GUAUGUUCCUGCUUCAUCCUU | 2313 | GGAUGAAGCAGGAACAUACUU | 2314 |
D-1593 | UAUGUUCCUGCUUCAUCCCUU | 2315 | GGGAUGAAGCAGGAACAUAUU | 2316 |
D-1594 | AUGUUCCUGCUUCAUCCCCUU | 2317 | GGGGAUGAAGCAGGAACAUUU | 2318 |
D-1595 | UGUUCCUGCUUCAUCCCCUUU | 2319 | AGGGGAUGAAGCAGGAACAUU | 2320 |
D-1596 | GUUCCUGCUUCAUCCCCUUUU | 2321 | AAGGGGAUGAAGCAGGAACUU | 2322 |
D-1597 | UUCCUGCUUCAUCCCCUUCUU | 2323 | GAAGGGGAUGAAGCAGGAAUU | 2324 |
D-1598 | UCCUGCUUCAUCCCCUUCUUU | 2325 | AGAAGGGGAUGAAGCAGGAUU | 2326 |
D-1599 | CCUGCUUCAUCCCCUUCUAUU | 2327 | UAGAAGGGGAUGAAGCAGGUU | 2328 |
D-1600 | CUGCUUCAUCCCCUUCUACUU | 2329 | GUAGAAGGGGAUGAAGCAGUU | 2330 |
D-1601 | UGCUUCAUCCCCUUCUACAUU | 2331 | UGUAGAAGGGGAUGAAGCAUU | 2332 |
D-1602 | GCUUCAUCCCCUUCUACAGUU | 2333 | CUGUAGAAGGGGAUGAAGCUU | 2334 |
D-1603 | CUUCAUCCCCUUCUACAGUUU | 2335 | ACUGUAGAAGGGGAUGAAGUU | 2336 |
D-1604 | UUCAUCCCCUUCUACAGUGUU | 2337 | CACUGUAGAAGGGGAUGAAUU | 2338 |
D-1605 | UCAUCCCCUUCUACAGUGGUU | 2339 | CCACUGUAGAAGGGGAUGAUU | 2340 |
D-1606 | CAUCCCCUUCUACAGUGGCUU | 2341 | GCCACUGUAGAAGGGGAUGUU | 2342 |
D-1607 | AUCCCCUUCUACAGUGGCCUU | 2343 | GGCCACUGUAGAAGGGGAUUU | 2344 |
D-1608 | UCCCCUUCUACAGUGGCCUUU | 2345 | AGGCCACUGUAGAAGGGGAUU | 2346 |
D-1609 | CCCCUUCUACAGUGGCCUUUU | 2347 | AAGGCCACUGUAGAAGGGGUU | 2348 |
D-1610 | cugcuucaUgCCUUucuacaA | 2441 | aUguagAAaggCaUgaagcaguu | 2442 |
D-1611 | cugcuucaUgCCUUucuacaA | 2443 | aUguagAAaggCaUgaagcaguu | 2444 |
D-1612 | cugcuucaUgCCUUucuacaA | 2445 | aUguagAAaggCaUgaagcaguu | 2446 |
D-1613 | cugcuucaUgCCUUucuacaA | 2447 | aUguagAAaggCaUgaagcaguu | 2448 |
D-1614 | cugcuucaUgCCUUucuacaA | 2449 | aUguagAAAggCaUgaagcaguu | 2450 |
D-1615 | cugcuucaUgCCUUucuacaA | 2451 | aUguagaAaggCaUgaagcaguu | 2452 |
D-1616 | cugcuucaUgCCUuucuacaA | 2453 | aUguagAAaggCaUgaagcaguu | 2454 |
D-1617 | cugcuucaUgCCUuucuacaA | 2455 | aUguagaAaggCaUgaagcaguu | 2456 |
D-1618 | cugcuucaUgCCUUucuacaA | 2457 | aUguagaAAggCaUgaagcaguu | 2458 |
D-1619 | cugcuucaUgCCUuucuacaA | 2459 | aUguagAAAggCaUgaagcaguu | 2460 |
D-1620 | cugcuucaUgCCUuucuacaA | 2461 | aUguagaAAggCaUgaagcaguu | 2462 |
D-1621 | cugcuucaUgCCUUucuacaA | 2463 | aUgUaGAAaggCaUgaagcaguu | 2464 |
D-1622 | cugcuucaUgCCUUucuacaA | 2465 | aUgUagAAaGgCaUgaagcaguu | 2466 |
D-1623 | cugcuucaUgCCUUucuacaA | 2467 | aUgUagAAaggCaUgaagcaguu | 2468 |
D-1624 | cugcuucaUgCCUUucuacaA | 2469 | aUgUaGAAaGgCaUgaagcaguu | 2470 |
D-1625 | cugcuucaUgCCUUucuacaA | 2471 | aUguagAAaGgCaUgaagcaguu | 2472 |
D-1626 | cugcuucaUgCCUUucUaCaA | 2473 | aUguagAAaGgCaUgaagcaguu | 2474 |
D-1627 | cugcuucaUgCCUUucuacaA | 2475 | aUguagAAaGgCaUgaagcaguu | 2476 |
D-1628 | cugcuucaUgCCUUucuacaA | 2477 | aUguagAAAggCaUgaagcaguu | 2478 |
D-1629 | cugcuucaUgCCUUucuacaA | 2479 | aUguagAAagGCaUgaagcaguu | 2480 |
D-1630 | cugcuucaUgCCUUucuacaA | 2481 | aUguaGAAaggCaUgaagcaguu | 2482 |
D-1631 | cugcuucaUgCCUUucuacaA | 2483 | aUguaGaAaggCaUgaagcaguu | 2484 |
D-1632 | cugcuucaUgCCUUucuacaA | 2485 | aUguaGaAAggCaUgaagcaguu | 2486 |
D-1633 | cugcuuCaUGCcuuucuacaA | 2487 | aUguaGaAaggcaUgAagcaguu | 2488 |
D-1634 | cugcuuCaUGCcuuucuacaA | 2489 | aUguaGaAAggcaUgAagcaguu | 2490 |
D-1635 | cugcuuCaUgCcuuucuacaA | 2491 | aUguagaAaggCaUgAagcaguu | 2492 |
D-1636 | cugcuuCaUgCcuuucuacaA | 2493 | aUguagaaaggCaUgAagcaguu | 2494 |
D-1637 | ugcuucauGcCUUUcuacagA | 2495 | uCuguaGaaagGcAugaagcauu | 2496 |
D-1638 | ugcuucauGcCUUUcuacagA | 2497 | uCUguaGaaagGcAugaagcauu | 2498 |
D-1639 | ugcuucauGcCUUUcuacagA | 2499 | uCugUaGaaagGcAugaagcauu | 2500 |
D-1640 | ugcuucauGcCUUUcuacagA | 2501 | uCuguAGaaagGcAugaagcauu | 2502 |
D-1641 | ugcuucauGcCUUUcuacagA | 2503 | uCuguaGAaagGcAugaagcauu | 2504 |
D-1642 | ugcuucauGcCUUUcuacagA | 2505 | uAuguaGaaagGcAugaagcauu | 2506 |
D-1643 | ugcuucauGcCUUUcuacagA | 2507 | uCAguaGaaagGcAugaagcauu | 2508 |
D-1644 | ugcuucauGcCUUUcuacagA | 2509 | uCuAuaGaaagGcAugaagcauu | 2510 |
D-1645 | ugcuucauGcCUUUcuacagA | 2511 | uCugAaGaaagGcAugaagcauu | 2512 |
D-1646 | ugcuucauGcCUUUcuacagA | 2513 | uCuguAGaaagGcAugaagcauu | 2514 |
D-1647 | ugcuucauGcCUUUcuacagA | 2515 | uCuguaAaaagGcAugaagcauu | 2516 |
D-1648 | ugcuucauGcCUUUcuacagA | 2517 | uCuguaGAaagGcAugaagcauu | 2518 |
D-1649 | ucaugccuUuCUACaguggcT | 2519 | aGccacUguagAaAggcaugauu | 2520 |
D-1650 | ucaugccuUuCUACaguggcA | 2521 | uGccacUguagAaAggcaugauu | 2522 |
D-1651 | ucaugccuUuCUACaguggcA | 2523 | uGccacUguagAaAggcaugauu | 2524 |
D-1652 | uccugcuuCaUGCCuuucuaT | 2525 | aUagaaAggcaUgAagcaggauu | 2526 |
D-1653 | uccugcuuCaUGCCuuucuaA | 2527 | uUagaaAggcaUgAagcaggauu | 2528 |
D-1654 | uccugcuuCaUGCCuuucuaA | 2529 | uUagaaAggcaUgAagcaggauu | 2530 |
D-1655 | uauguuccUgCUUCaugccuT | 2531 | aAggcaUgaagCaGgaacauauu | 2532 |
D-1656 | uauguuccUgCUUCaugccuA | 2533 | uAggcaUgaagCaGgaacauauu | 2534 |
D-1657 | uauguuccUgCUUCaugccuA | 2535 | uAggcaUgaagCaGgaacauauu | 2536 |
D-1658 | uccugcuuCaUGCCuuucuaA | 2537 | aUagaaAggcaUgAagcaggauu | 2538 |
D-1659 | uauguuccUgCUUCaugccuA | 2539 | aAggcaUgaagCaGgaacauauu | 2540 |
D-1660 | uauguuccUgCUUCaugccuA | 2541 | aAggcaUgaagCaGgaacauauu | 2542 |
D-1661 | ucaugccuUuCUACaguggcT | 2543 | aGccacUguagAaAggcaugauu | 2544 |
D-1662 | uccugcuuCaUGCCuuucuaT | 2545 | aUagaaAggcaUgAagcaggauu | 2546 |
D-1663 | uccugcuuCaUGCCuuucuaA | 2547 | uUagaaAggcaUgAagcaggauu | 2548 |
D-1664 | uauguuccUgCUUCaugccuA | 2549 | uAggcaUgaagCaGgaacauauu | 2550 |
D-1665 | ugcuucauGcCUUUcuacagA | 2551 | uCuguaGaaagGcAugaagcauu | 2552 |
D-1666 | cugcuucaUgCCUUucuacaA | 2553 | aUguagAAaggCaUgaagcaguu | 2554 |
D-1667 | cugcuucaUgCCUUucuacaA | 2555 | aUguagAAaggCaUgaagcaguu | 2556 |
D-1668 | cugcuucaUgCCUUucuacaA | 2557 | aUguagAAaggCaUgaagcaguu | 2558 |
D-1669 | cugcuucaUgCCUUucuacaA | 2559 | aUguagAAaggCaUgaagcaguu | 2560 |
D-1670 | cugcuucaUgCCUUucuacaA | 2561 | aUguagAAaggCaUgaagcaguu | 2562 |
D-1671 | cugcuucaUgCCUUucuacaA | 2563 | aUguagAAaggCaUgaagcaguu | 2564 |
D-1672 | cugcuucaUgCCUUucuacaA | 2565 | aUguagAAaggCaUgaagcaguu | 2566 |
D-1673 | cugcuucaUgCCUUucuacaA | 2567 | aUguagAAaggCaUgaagcaguu | 2568 |
D-1674 | cugcuucaUgCCUUucuacaA | 2569 | aUguagAAaggCaUgaagcaguu | 2570 |
D-1675 | cugcuucaUgCCUTucuacaA | 2571 | aUguagAAaggCaUgaagcaguu | 2572 |
D-1676 | cugcuucaUgCCTUucuacaA | 2573 | aUguagAAaggCaUgaagcaguu | 2574 |
D-1677 | cugcuucaUgCCUUTcuacaA | 2575 | aUguagAAaggCaUgaagcaguu | 2576 |
D-1678 | cugcuucaUgCCUUucTacaA | 2577 | aUguAgAaaggCaUgaagcaguu | 2578 |
D-1679 | cugcuucaUgCCUUucuAcaA | 2579 | aUgUagAaaggCaUgaagcaguu | 2580 |
D-1680 | cugcuucaUgCCUUucuacAA | 2581 | aUguagAaaggCaUgaagcaguu | 2582 |
D-1681 | cugcuucaUgCCUUTcuacaA | 2583 | aUguagAaaggCaUgaagcaguu | 2584 |
D-1682 | cugcuucaUgCCUUucTacaA | 2585 | aUguAgAaaggCaUgaagcaguu | 2586 |
D-1683 | cugcuucaUgCCUUucuAcaA | 2587 | aUgAagAaaggCaUgaagcaguu | 2588 |
D-1684 | cugcuucaUgCCUUucUaCaA | 2589 | aUgUagAAaggCaUgaagcaguu | 2590 |
D-1685 | cugcuucaUgCCUUucUaCaA | 2591 | aUguagAAaggCaUgaagcaguu | 2592 |
D-1686 | cugcuucaUgCCUTucuacaA | 2593 | aUguagAAaggCaUgaagcaguu | 2594 |
D-1687 | cugcuucaUgCCUTucuacaA | 2595 | aUguagAAaggCaUgaagcaguu | 2596 |
D-1688 | cugcuucaUgCCUTucuacaA | 2597 | aUguagAAaggCaUgaagcaguu | 2598 |
D-1689 | cugcuucaUgCCUTucuacaA | 2599 | aUguagAAaggCaUgaagcaguu | 2600 |
D-1690 | ugcuucauGcCUUUcuAcagA | 2601 | uCugAaGaaagGcAugaagcauu | 2602 |
D-1691 | ugcuucauGcCUUUcTacagA | 2603 | uCuguAGaaagGcAugaagcauu | 2604 |
D-1692 | ugcuucauGcCUUTcuacagA | 2605 | uCuguaGAaagGcAugaagcauu | 2606 |
D-1693 | ugcuucauGcCUUUcuacAgA | 2607 | uCUguaGaaagGcAugaagcauu | 2608 |
D-1694 | ugcuucauGcCUUUcuAcagA | 2609 | uCugUaGaaagGcAugaagcauu | 2610 |
D-1695 | ugcuucauGcCUUUcTacagA | 2611 | uCuguAGaaagGcAugaagcauu | 2612 |
D-1696 | ugcuucauGcCUUUcuacaGA | 2613 | uAuguaGaaagGcAugaagcauu | 2614 |
D-1697 | ugcuucauGcCUUUcuacAgA | 2615 | uCAguaGaaagGcAugaagcauu | 2616 |
D-1698 | ucaugccuUuCUACaGuggcA | 2617 | aGccaAUguagAaAggcaugauu | 2618 |
D-1699 | ucaugccuUuCUACagTggcA | 2619 | aGccaAUguagAaAggcaugauu | 2620 |
D-1700 | ucaugccuUuCUACaguggcA | 2621 | aGccaAUguagAaAggcaugauu | 2622 |
D-1701 | cugcuucaUgCCUUucuaCaA | 2623 | aUGuagAaaggCaUgaagcaguu | 2624 |
D-1702 | cugcuucaUgCCUUucuaCaA | 2625 | aUAuagAaaggCaUgaagcaguu | 2626 |
D-1703 | ugcuucauGcCUUUcuaCagA | 2627 | uCuAuaGaaagGcAugaagcauu | 2628 |
D-1704 | ugcuucauGcCUUUcuaCagA | 2629 | uCuGuaGaaagGcAugaagcauu | 2630 |
D-1705 | ucaugccuUuCUACaguggcA | 2631 | aGccaAUguagAaAggcaugauu | 2632 |
D-1706 | ucaugccuUuCUACaguggcA | 2633 | aGccaAUguagAaAggcaugauu | 2634 |
D-1707 | ucaugccuUuCUACaGuggcA | 2635 | aGccaAUguagAaAggcaugauu | 2636 |
D-1708 | ucaugccuUuCUACaGuggcA | 2637 | aGccaAUguagAaAggcaugauu | 2638 |
D-1709 | cugcuucaUgCCUUucuacaA | 2639 | a[MeO-I]guagAaaggCaUgaagcaguu | 2640 |
D-1710 | cugcuucaUgCCUUucuacaA | 2641 | aUg[MeO-I]agAaaggCaUgaagcaguu | 2642 |
D-1711 | cugcuucaUgCCUUucuacaA | 2643 | aUgu[MeO-I]gAaaggCaUgaagcaguu | 2644 |
D-1712 | cugcuucaUgCCUUucuacaA | 2645 | aUgua[MeO-I]AaaggCaUgaagcaguu | 2646 |
D-1713 | cugcuucaUgCCUUucuacaA | 2647 | aUguagA[MeO-I]aggCaUgaagcaguu | 2648 |
D-1714 | cugcuucaUgCCUUucuaccA | 2649 | a[MeO-I]guagAaaggCaUgaagcaguu | 2650 |
D-1715 | cugcuucaUgCCUUucuccaA | 2651 | aUg[MeO-I]agAaaggCaUgaagcaguu | 2652 |
D-1716 | cugcuucaUgCCUUccuacaA | 2653 | aUguag[MeO-I]aaggCaUgaagcaguu | 2654 |
D-1717 | cugcuucaUgCCUCucuacaA | 2655 | aUguagA[MeO-I]aggCaUgaagcaguu | 2656 |
D-1718 | cugcuucaUgCCUUucuacuA | 2657 | aAguagAaaggCaUgaagcaguu | 2658 |
D-1719 | cugcuucaUgCCUUucuucaA | 2659 | aUgaagAaaggCaUgaagcaguu | 2660 |
D-1720 | cugcuucaUgCCUUucaacaA | 2661 | aUguugAaaggCaUgaagcaguu | 2662 |
D-1721 | cugcuucaUgCCUAucuacaA | 2663 | aUguagAuaggCaUgaagcaguu | 2664 |
D-1722 | ugcuucauGcCUUUcuacacA | 2665 | a[MeO-I]uguaGaaagGcAugaagcauu | 2666 |
D-1723 | ugcuucauGcCUUUcuacagA | 2667 | aC[MeO-I]guaGaaagGcAugaagcauu | 2668 |
D-1724 | ugcuucauGcCUUUcuacagA | 2669 | aCug[MeO-I]aGaaagGcAugaagcauu | 2670 |
D-1725 | ugcuucauGcCUUUcuacagA | 2671 | aCugu[MeO-I]GaaagGcAugaagcauu | 2672 |
D-1726 | ugcuucauGcCUUUcuacagA | 2673 | aCugua[MeO-I]aaagGcAugaagcauu | 2674 |
D-1727 | ugcuucauGcCUUUcuaccgA | 2675 | aC[MeO-I]guaGaaagGcAugaagcauu | 2676 |
D-1728 | ugcuucauGcCUUUcuccagA | 2677 | aCug[MeO-I]aGaaagGcAugaagcauu | 2678 |
D-1729 | ugcuucauGcCUUUccacagA | 2679 | aCugu[MeO-I]GaaagGcAugaagcauu | 2680 |
D-1730 | cugcuucaUgCCUUucuaaaA | 2681 | aUuuagAaaggCaUgaagcaguu | 2682 |
D-1731 | cugcuucaUgCCUUuauacaA | 2683 | aUguauAaaggCaUgaagcaguu | 2684 |
D-1732 | cugcuucaUgCCUUucuacaA | 2685 | aUguagAAaggCaUgaagcaguu | 2686 |
D-1733 | cugcuucaUgCCUTucuacaA | 2687 | aUguagAAaggCaUgaagcaguu | 2688 |
D-1734 | ugcuucauGcCUUUcuacaGA | 2689 | uCuguaGaaagGcAugaagcauu | 2690 |
D-1735 | ugcuucauGcCUUTcuacagA | 2691 | uCuguaGAaagGcAugaagcauu | 2692 |
D-1736 | ucaugccuUuCUACAguggcA | 2693 | aGccaAUguagAaAggcaugauu | 2694 |
D-1737 | cugcuucaUgCCUUuCuacaA | 2695 | aUguaAAaaggCaUgaagcaguu | 2696 |
D-1738 | ucaugccuUuCUACaGuggcA | 2697 | aGccaAUguagAaAggcaugauu | 2698 |
D-1739 | cugcuucaUgCCUUucuacaA | 2699 | aU[MeO-I]uagAaaggCaUgaagcaguu | 2700 |
D-1740 | cugcuucaUgCCUUucuagaA | 2701 | aUcuagAaaggCaUgaagcaguu | 2702 |
D-1741 | cugcuucaUgCCUUacuacaA | 2703 | aUguagUaaggCaUgaagcaguu | 2704 |
D-1742 | ugcuucauGcCUUUcuacagA | 2705 | aCu[MeO-I]uaGaaagGcAugaagcauu | 2706 |
D-1743 | ugcuucauGcCUUUcuacagA | 2707 | aCuguaG[MeO-I]aagGcAugaagcauu | 2708 |
D-1744 | ugcuucauGcCUUCcuacagA | 2709 | aCuguaG[MeO-I]aagGcAugaagcauu | 2710 |
D-1745 | cugcuucaUgCCUUucuauaA | 2711 | aUauagAaaggCaUgaagcaguu | 2712 |
D-1746 | cugcuucaUgCCUUuuuacaA | 2713 | aUguaaAaaggCaUgaagcaguu | 2714 |
D-1747 | cugcuucaUgCCUUucuacaA | 2715 | aUguagAAaggCaUgaagcaguu | 2716 |
D-1748 | cugcuucaUgCCUUucuacaA | 2717 | aUguagAAaggCaUgaagcaguu | 2718 |
D-1749 | cugcuucaUgCCUUTcuacaA | 2719 | aUguagAaaggCaUgaagcaguu | 2720 |
D-1750 | cugcuucaUgCCUTucuacaA | 2721 | aUguagAAaggCaUgaagcaguu | 2722 |
D-1751 | cugcuucaUgCCUUucuacAA | 2723 | aAguagAaaggCaUgaagcaguu | 2724 |
D-1752 | cugcuucaUgCCUUuCuacaA | 2725 | aUguaGAaaggCaUgaagcaguu | 2726 |
D-1753 | ugcuucauGcCUUUCuacagA | 2727 | uCuguaAaaagGcAugaagcauu | 2728 |
D-1754 | cugcuucaUgCCUUucuacaA | 2729 | aUguag[MeO-I]aaggCaUgaagcaguu | 2730 |
D-1755 | cugcuucaUgCCUUucuacaA | 2731 | aUguagAAaggCaUgaagcaguu | 2732 |
D-1756 | cugcuucaUgCCUTucuacaA | 2733 | aUguagAAaggCaUgaagcaguu | 2734 |
D-1757 | cugcuucaUgCCUUucUaCaA | 2735 | aUguagAAaGgCaUgaagcaguu | 2736 |
D-1758 | cugcuuCaUGCCuuucuacaA | 2737 | aUgUaGaAaggcaUgAagcaguu | 2738 |
D-1759 | cugcuuCaUGCCuuucuacaA | 2739 | aUgUaGaAAggcaUgAagcaguu | 2740 |
D-1760 | cugcuuCaUgCcuuucuacaA | 2741 | aUguagaAaggCaUgAagcaguu | 2742 |
D-1761 | cugcuucaUgCcUuucuacaA | 2743 | aUguagAAAggCaUgaagcaguu | 2744 |
D-1762 | cugcuucaUgCcUuucuacaA | 2745 | aUguagaAAggCaUgaagcaguu | 2746 |
D-1763 | cugcuucaUgCCUTucuacaA | 2747 | aUguagAAaggCaUgaagcaguu | 2748 |
D-1764 | cugcuucaUgCCUUucuaCaA | 2749 | aUGuagAaaggCaUgaagcaguu | 2750 |
D-1765 | ucaugccuUuCUACaGuggcA | 2751 | aGccaAUguagAaAggcaugauu | 2752 |
D-1766 | ucaugccuUuCUACaGuggcA | 2753 | aGccaAUguagAaAggcaugauu | 2754 |
D-1767 | cugcuucaUgGGAUucuacaA | 2755 | aUguagAauccCaUgaagcaguu | 2756 |
D-1768 | cugcuucaUgCCUUucuacaA | 2757 | aUguaGaAAggCaUgaagcaguu | 2758 |
D-1769 | guauguucCuGCUUcaugccA | 2759 | aGgcauGaagcAgGaacauacuu | 2760 |
D-1770 | uguccugcUuCAUGccuuucA | 2761 | aGaaagGcaugAaGcaggacauu | 2762 |
D-1771 | gcuucaugCcUUUCuacaguA | 2763 | aAcuguAgaaaGgCaugaagcuu | 2764 |
D-1772 | cuucaugcCuUUCUacagugA | 2765 | aCacugUagaaAgGcaugaaguu | 2766 |
D-1773 | uucaugccUuUCUAcaguggA | 2767 | aCcacuGuagaAaGgcaugaauu | 2768 |
D-1774 | cugcuucaUgCCUUucaacaA | 2769 | aUguugAaaggCaUgaagcaguu | 2770 |
D-1775 | cugcuucaUgCCUUucuccaA | 2771 | aUg[MeO-I]agAaaggCaUgaagcaguu | 2772 |
D-1776 | ugcuucauGcCUUUcuccagA | 2773 | aCug[MeO-I]aGaaagGcAugaagcauu | 2774 |
D-1777 | cugcuucaUgCCUUucuacaA | 2775 | aUgu[MeO-I]gAaaggCaUgaagcaguu | 2776 |
D-1778 | ugcuucauGcCUUUcuacagA | 2777 | aCugu[MeO-I]GaaagGcAugaagcauu | 2778 |
D-1779 | cugcuucaUgCCUUucuacaA | 2779 | aUgua[MeO-I]AaaggCaUgaagcaguu | 2780 |
D-1780 | ugcuucauGcCUUUcuacagA | 2781 | aCugua[MeO-I]aaagGcAugaagcauu | 2782 |
D-1781 | cugcuucaUgCCUUccuacaA | 2783 | aUguag[MeO-I]aaggCaUgaagcaguu | 2784 |
D-1782 | cugcuucaUgCCUCucuacaA | 2785 | aUguagA[MeO-I]aggCaUgaagcaguu | 2786 |
D-1783 | ugcuucauGcCUUUcuAcagA | 2787 | uCugAaGaaagGcAugaagcauu | 2788 |
D-1784 | ugcuucauGcCUUUcuacAgA | 2789 | uCUguaGaaagGcAugaagcauu | 2790 |
D-1785 | ucaugccuUuCUACaguggcA | 2791 | aGccaAUguagAaAggcaugauu | 2792 |
D-1786 | cugcuucaUgCCUUucTacaA | 2793 | aUguAgAaaggCaUgaagcaguu | 2794 |
D-1787 | cugcuucaUgCCUUucTacaA | 2795 | aUguAgAaaggCaUgaagcaguu | 2796 |
D-1788 | ugcuucauGcCUUUcTacagA | 2797 | uCuguAGaaagGcAugaagcauu | 2798 |
D-1789 | ugcuucauGcCUUUcuaCagA | 2799 | uCuGuaGaaagGcAugaagcauu | 2800 |
D-1790 | ucaugccuUuCUACaGuggcA | 2801 | aGccaAUguagAaAggcaugauu | 2802 |
D-1791 | cugcuucaUgCCUUucuacaA | 2803 | aU[MeO-I]uagAaaggCaUgaagcaguu | 2804 |
D-1792 | cugcuucaUgCCUUucuacaA | 2805 | aUguag[MeO-I]aaggCaUgaagcaguu | 2806 |
D-1793 | cugcuucaUgCCUUacuacaA | 2807 | aUguagUaaggCaUgaagcaguu | 2808 |
D-1794 | ugcuucauGcCUUUcuacagA | 2809 | aCuguaG[MeO-I]aagGcAugaagcauu | 2810 |
D-1795 | ugcuucauGcCUUCcuacagA | 2811 | aCuguaG[MeO-I]aagGcAugaagcauu | 2812 |
D-1796 | ugcuucauGcCUUUcuacagA | 2813 | aCu[MeO-I]uaGaaagGcAugaagcauu | 2814 |
D-1797 | cugcuucaUgCCUUucuacaA | 2815 | aUgu[MeO-I]gAaaggCaUgaagcaguu | 2816 |
D-1798 | cugcuucaUgCCUUucuacaA | 2817 | aUgu[MeO-I]gAaaggCaUgaagcaguu | 2818 |
D-1799 | cugcuucaUgCCUUucuacaA | 2819 | aUgu[MeO-I]gAaaggCaUgaagcaguu | 2820 |
D-1800 | cugcuucaUgCCUUucuacaA | 2821 | aUguag[MeO-I]aaggCaUgaagcaguu | 2822 |
D-1801 | ucaugccuUuCUACaGuggcA | 2823 | aGccaAUguagAaAggcaugauu | 2824 |
D-1802 | ucaugccuUuCUACAguggcA | 2825 | aGccaAUguagAaAggcaugauu | 2826 |
D-1803 | cugcuucaUgGGAUuguacaA | 2827 | aUguacAauccCaUgaagcaguu | 2828 |
D-1804 | cugcuucaUgCCUUucTacaA | 2829 | aUgu[MeO-I]gAaaggCaUgaagcaguu | 2830 |
D-1805 | ugcuucauGcCUUUCuacagA | 2831 | aCugua[MeO-I]aaagGcAugaagcauu | 2832 |
D-1806 | cugcuuCaUGCCuTucuacaA | 2833 | aUgUaGaAaggcaUgAagcaguu | 2834 |
D-1807 | cugcuuCaUGCCuTucuacaA | 2835 | aUgUaGaAAggcaUgAagcaguu | 2836 |
D-1808 | cugcuucaUgCCUUTcuacaA | 2837 | aUguag[MeO-I]aaggCaUgaagcaguu | 2838 |
D-1809 | ucaugcCuUuCuacaguggcA | 2839 | aGccacuguagAaAgGcaugauu | 2840 |
D-1810 | uauguuCcUgCuucaugccuA | 2841 | aAggcaugaagCaGgAacauauu | 2842 |
D-1811 | ugcuucAuGcCuuucuacagA | 2843 | aCuguagaaagGcAuGaagcauu | 2844 |
D-1812 | uguuccUgCUUCaugccuuuA | 2845 | aAggcaUgaagCaGgaacauu | 2846 |
D-1813 | cugcuuCaUgCcuuucuacaA | 2847 | aUguagaaaggCaUgAagcaguu | 2848 |
D-1814 | uauguuccUgCuUCaugccuA | 2849 | aAggcaUgaagCaGgaacauauu | 2850 |
D-1815 | uguuccUgCUUCaugccuA | 2851 | aAggcaUgaagCaGgaacauu | 2852 |
D-1816 | uauguuCcUgCuucaugccuuA | 2853 | aAggcaugaagCaGgAacauauu | 2854 |
D-1817 | uauguuCcUgCuucaugccuA | 2855 | aAggcaugaagCaGgAacauacc | 2856 |
D-1818 | uauguuCcUgCuucaugccuuA | 2857 | aAggcaugaagCaGgAacauacc | 2858 |
D-1819 | uauguuccUgCUUCaugccuA | 2859 | aAggcaUgaagCaGgaacauauu | 2860 |
D-1820 | uauguuccUgCUUCaugcguA | 2861 | aAcgcaUgaagCaGgaacauauu | 2862 |
D-1821 | uauguuccUgCUUCauggcuA | 2863 | aAgccaUgaagCaGgaacauauu | 2864 |
D-1822 | uauguuccUgCUUCaagccuA | 2865 | aAggcuUgaagCaGgaacauauu | 2866 |
D-1823 | uauguuccUgCUUCuugccuA | 2867 | aAggcaAgaagCaGgaacauauu | 2868 |
D-1824 | uauguuccUgCUUGaugccuA | 2869 | aAggcaUcaagCaGgaacauauu | 2870 |
D-1825 | uauguuccUgCUACaugccuA | 2871 | aAggcaUguagCaGgaacauauu | 2872 |
D-1826 | uauguuccUgCAUCaugccuA | 2873 | aAggcaUgaugCaGgaacauauu | 2874 |
D-1827 | uauguuccUgGUUCaugccuA | 2875 | aAggcaUgaacCaGgaacauauu | 2876 |
D-1828 | uauguuccUcCUUCaugccuA | 2877 | aAggcaUgaagGaGgaacauauu | 2878 |
D-1829 | uauguuccAgCUUCaugccuA | 2879 | aAggcaUgaagCuGgaacauauu | 2880 |
D-1830 | uauguucgUgCUUCaugccuA | 2881 | aAggcaUgaagCaCgaacauauu | 2882 |
D-1831 | uauguugcUgCUUCaugccuA | 2883 | aAggcaUgaagCaGcaacauauu | 2884 |
D-1832 | uauguaccUgCUUCaugccuA | 2885 | aAggcaUgaagCaGguacauauu | 2886 |
D-1833 | uaugauccUgCUUCaugccuA | 2887 | aAggcaUgaagCaGgaucauauu | 2888 |
D-1834 | uaucuuccUgCUUCaugccuA | 2889 | aAggcaUgaagCaGgaagauauu | 2890 |
D-1835 | uaaguuccUgCUUCaugccuA | 2891 | aAggcaUgaagCaGgaacuuauu | 2892 |
D-1836 | uuuguuccUgCUUCaugccuA | 2893 | aAggcaUgaagCaGgaacaaauu | 2894 |
D-1837 | aauguuccUgCUUCaugccuA | 2895 | aAggcaUgaagCaGgaacauuuu | 2896 |
D-1838 | ucaugccuUuCUACaguggcA | 2897 | aGccacUguagAaAggcaugauu | 2898 |
D-1839 | ucaugccuUuCUACagugggA | 2899 | aCccacUguagAaAggcaugauu | 2900 |
D-1840 | ucaugccuUuCUACagugccA | 2901 | aGgcacUguagAaAggcaugauu | 2902 |
D-1841 | ucaugccuUuCUACagucgcA | 2903 | aGcgacUguagAaAggcaugauu | 2904 |
D-1842 | ucaugccuUuCUACagaggcA | 2905 | aGccucUguagAaAggcaugauu | 2906 |
D-1843 | ucaugccuUuCUACacuggcA | 2907 | aGccagUguagAaAggcaugauu | 2908 |
D-1844 | ucaugccuUuCUACuguggcA | 2909 | aGccacAguagAaAggcaugauu | 2910 |
D-1845 | ucaugccuUuCUAGaguggcA | 2911 | aGccacUcuagAaAggcaugauu | 2912 |
D-1846 | ucaugccuUuCUUCaguggcA | 2913 | aGccacUgaagAaAggcaugauu | 2914 |
D-1847 | ucaugccuUuCAACaguggcA | 2915 | aGccacUguugAaAggcaugauu | 2916 |
D-1848 | ucaugccuUuGUACaguggcA | 2917 | aGccacUguacAaAggcaugauu | 2918 |
D-1849 | ucaugccuAuCUACaguggcA | 2919 | aGccacUguagAuAggcaugauu | 2920 |
D-1850 | ucaugcguUuCUACaguggcA | 2921 | aGccacUguagAaAcgcaugauu | 2922 |
D-1851 | ucauggcuUuCUACaguggcA | 2923 | aGccacUguagAaAgccaugauu | 2924 |
D-1852 | ucaagccuUuCUACaguggcA | 2925 | aGccacUguagAaAggcuugauu | 2926 |
D-1853 | ucuugccuUuCUACaguggcA | 2927 | aGccacUguagAaAggcaagauu | 2928 |
D-1854 | ugaugccuUuCUACaguggcA | 2929 | aGccacUguagAaAggcaucauu | 2930 |
D-1855 | acaugccuUuCUACaguggcA | 2931 | aGccacUguagAaAggcauguuu | 2932 |
D-1856 | ucaugccuUaCUACaguggcA | 2933 | aGccacUguagUaAggcaugauu | 2934 |
D-1857 | augccuUuCUACaguggcuuA | 2935 | aGccacUguagAaAggcauuu | 2936 |
D-1858 | augccuUuCUACaguggcA | 2937 | aGccacUguagAaAggcauuu | 2938 |
D-1859 | ucaugccuUuCuACaguggcA | 2939 | aGccacUguagAaAggcaugauu | 2940 |
D-1860 | AaugccuUUCUacaguggcu | 2941 | aGccacUguagaaAgGcauuu | 2942 |
D-1861 | ucaugcCuUuCuacaguggcuA | 2943 | aGccacuguagAaAgGcaugauu | 2944 |
D-1862 | ucaugcCuUuCuacaguggcA | 2945 | aGccacuguagAaAgGcaugaag | 2946 |
D-1863 | ucaugcCuUuCuacaguggcuA | 2947 | aGccacuguagAaAgGcaugaag | 2948 |
D-1864 | ucaugccuUuCUACaguggcuA | 2949 | aGccacUguagAaAggcaugauu | 2950 |
D-1865 | ucaugccuUuCUACaguggcA | 2951 | aGccacUguagAaAggcaugaag | 2952 |
D-1866 | ucaugccuUuCUACaguggcuA | 2953 | aGccacUguagAaAggcaugaag | 2954 |
D-1867 | cugcuucaUgCCUUuCuacaA | 2955 | aUgua[MeO-I]AaaggCaUgaagcaguu | 2956 |
D-1868 | gcggcuUcGACGgcuucuauu | 2957 | uAgAaGCcgucGaAgccgcuu | 2958 |
D-1869 | gcggcuUcGACGgguucuauu | 2959 | uAgAaCCcgucGaAgccgcuu | 2960 |
D-1870 | gcggcuUcGACGccuucuauu | 2961 | uAgAaGGcgucGaAgccgcuu | 2962 |
D-1871 | gcggcuUcGACCgcuucuauu | 2963 | uAgAaGCggucGaAgccgcuu | 2964 |
D-1872 | gcggcuUcCUGGgcuucuauu | 2965 | AuAgAaGCccagGaAgccgcuu | 2966 |
D-1873 | AgcuucaUgGGAUuguacauuu | 2967 | aUgUaCAauccCaUgaagcuu | 2968 |
D-1874 | ucaugccuUuGAUCacuggcA | 2969 | aGccagUgaucAaAggcaugauu | 2970 |
D-1875 | gcggcuUcCUGGgcuucuauu | 2971 | uAgAaGCccagGaAgccgcuu | 2972 |
D-1876 | ugcuucauGcCUUUcuacagA | 2973 | aCuguaGaaagGcAugaagcauu | 2974 |
D-1877 | ucaugccuUuCUACaguggcA | 2975 | aGccacUguagAaAggcaugauu | 2976 |
D-1878 | cugcuucaUgCCUUucuacaA | 2977 | aUguagAaaggCaUgaagcaguu | 2978 |
D-1879 | cugcuucaUgCCUUucuacaA | 2979 | aUguagAaaggCaUgaagcaguu | 2980 |
D-1880 | cugcuucaUgCCUUucuacaA | 2981 | aUguagAaaggCaUgaagcaguu | 2982 |
D-1881 | ucaugccuUuCUACaguggcA | 2983 | uGccacUguagAaAggcaugauu | 2984 |
D-1882 | cugcuucaUgCCUUucuacaA | 2985 | aUguagAAaggCaUgaagcaguu | 2986 |
D-1883 | ugcuucauGcCUUUcuacagA | 2987 | uCuguaGaaagGcAugaagcauu | 2988 |
D-1884 | cugcuucaUgCCUUucuacaT | 2989 | aUguagAaaggCaUgaagcaguu | 2990 |
為提高PNPLA3 siRNA序列之效力及活體內穩定性,將化學修飾摻入PNPLA3 siRNA分子中。具體地,將核糖之2'-O-甲基及2'-氟修飾摻入PNPLA3 siRNA內之特定位置。硫代磷酸酯核苷酸間鍵亦摻入在反義及/或有義序列之末端處。下表2描述各經修飾之PNPLA3 siRNA之有義及反義序列的修飾。表2和本申請其他部分中之核苷酸序列根據以下符號列出:A、U、G及C = 相應的核糖核苷酸;dT = 去氧胸苷;dA = 去氧腺苷;dC = 去氧胞苷;dG = 去氧鳥苷;invDT = 反向去氧胸苷;invDA = 反向去氧腺苷;invDC = 反向去氧胞苷;invDG = 反向去氧鳥苷;a、u、g及c = 相應的2'-O-甲基核糖核苷酸;Af、Uf、Gf及Cf = 相應的2'-去氧-2'-氟代(「2'-氟代」)核糖核苷酸;Ab = 無鹼基;MeO-I = 2'甲氧基肌苷;GNA = 乙二醇核酸;sGNA = 帶有3'硫代磷酸酯的乙二醇核酸;LNA = 鎖核酸。在序列中***「s」 表明兩個相鄰核苷酸藉由硫代磷酸二酯基團(例如硫代磷酸酯核苷酸間鍵)連接。除非另有說明,否則所有其他核苷酸藉由3'-5'磷酸二酯基團連接。表2中之各siRNA化合物包含19個鹼基對之雙鏈體區,在兩條鏈之3'端具有2個核苷酸的突出端,或在一端或兩端具有鈍化物。GalNAc3K2AhxC6為:
[表2]. 針對具有修飾之PNPLA3之siRNA序列
實例 2 :所選擇 PNPLA3 siRNA 分子在 RNA FISH 測定中之功效
雙鏈體編號 | 有義序列 (5’-3’) | SEQ ID NO: (有義) | 反義序列 (5’-3’) | SEQ ID NO: (反義) |
D-2000 | GfsgsGfcAfaUfaAfAfGfuAfcCfuGfcUfsusUf | 167 | asGfscAfgGfuAfcuuUfaUfuGfcCfcsUfsu | 168 |
D-2001 | CfsgsGfcCfaAfuGfUfCfcAfcCfaGfcUfsusUf | 169 | asGfscUfgGfuGfgacAfuUfgGfcCfgsUfsu | 170 |
D-2002 | GfsgsUfcCfaGfcCfUfGfaAfcUfuCfuUfsusUf | 171 | asAfsgAfaGfuUfcagGfcUfgGfaCfcsUfsu | 172 |
D-2003 | GfscsUfuCfaUfcCfCfCfuUfcUfaCfaGfsusUf | 173 | csUfsgUfaGfaAfgggGfaUfgAfaGfcsUfsu | 174 |
D-2004 | GfscsGfgCfuUfcCfUfGfgGfcUfuCfuAfsusUf | 175 | usAfsgAfaGfcCfcagGfaAfgCfcGfcsUfsu | 176 |
D-2005 | GfscsCfuCfuGfaGfCfUfgAfgUfuGfgUfsusUf | 177 | asCfscAfaCfuCfagcUfcAfgAfgGfcsUfsu | 178 |
D-2006 | GfsusGfaCfaAfcGfUfAfcCfcUfuCfaUfsusUf | 179 | asUfsgAfaGfgGfuacGfuUfgUfcAfcsUfsu | 180 |
D-2007 | CfscsCfgCfcUfcCfAfGfgUfcCfcAfaAfsusUf | 181 | usUfsuGfgGfaCfcugGfaGfgCfgGfgsUfsu | 182 |
D-2008 | CfsusUfcAfuCfcCfCfUfuCfuAfcAfgUfsusUf | 183 | asCfsuGfuAfgAfaggGfgAfuGfaAfgsUfsu | 184 |
D-2009 | GfsgsUfaUfgUfuCfCfUfgCfuUfcAfuGfsusUf | 185 | csAfsuGfaAfgCfaggAfaCfaUfaCfcsUfsu | 186 |
D-2010 | GfsusAfuGfuUfcCfUfGfcUfuCfaUfgCfsusUf | 187 | gsCfsaUfgAfaGfcagGfaAfcAfuAfcsUfsu | 188 |
D-2011 | UfsasUfgUfuCfcUfGfCfuUfcAfuGfcCfsusUf | 189 | gsGfscAfuGfaAfgcaGfgAfaCfaUfasUfsu | 190 |
D-2012 | AfsusGfuUfcCfuGfCfUfuCfaUfgCfcCfsusUf | 191 | gsGfsgCfaUfgAfagcAfgGfaAfcAfusUfsu | 192 |
D-2013 | UfsgsUfuCfcUfgCfUfUfcAfuGfcCfcUfsusUf | 193 | asGfsgGfcAfuGfaagCfaGfgAfaCfasUfsu | 194 |
D-2014 | GfsusUfcCfuGfcUfUfCfaUfgCfcCfuUfsusUf | 195 | asAfsgGfgCfaUfgaaGfcAfgGfaAfcsUfsu | 196 |
D-2015 | UfsusCfcUfgCfuUfCfAfuGfcCfcUfuCfsusUf | 197 | gsAfsaGfgGfcAfugaAfgCfaGfgAfasUfsu | 198 |
D-2016 | UfscsCfuGfcUfuCfAfUfgCfcCfuUfcUfsusUf | 199 | asGfsaAfgGfgCfaugAfaGfcAfgGfasUfsu | 200 |
D-2017 | CfscsUfgCfuUfcAfUfGfcCfcUfuCfuAfsusUf | 201 | usAfsgAfaGfgGfcauGfaAfgCfaGfgsUfsu | 202 |
D-2018 | CfsusGfcUfuCfaUfGfCfcCfuUfcUfaCfsusUf | 203 | gsUfsaGfaAfgGfgcaUfgAfaGfcAfgsUfsu | 204 |
D-2019 | UfsgsCfuUfcAfuGfCfCfcUfuCfuAfcAfsusUf | 205 | usGfsuAfgAfaGfggcAfuGfaAfgCfasUfsu | 206 |
D-2020 | GfscsUfuCfaUfgCfCfCfuUfcUfaCfaGfsusUf | 207 | csUfsgUfaGfaAfgggCfaUfgAfaGfcsUfsu | 208 |
D-2021 | CfsusUfcAfuGfcCfCfUfuCfuAfcAfgUfsusUf | 209 | asCfsuGfuAfgAfaggGfcAfuGfaAfgsUfsu | 210 |
D-2022 | UfsusCfaUfgCfcCfUfUfcUfaCfaGfuGfsusUf | 211 | csAfscUfgUfaGfaagGfgCfaUfgAfasUfsu | 212 |
D-2023 | UfscsAfuGfcCfcUfUfCfuAfcAfgUfgGfsusUf | 213 | csCfsaCfuGfuAfgaaGfgGfcAfuGfasUfsu | 214 |
D-2024 | CfsasUfgCfcCfuUfCfUfaCfaGfuGfgCfsusUf | 215 | gsCfscAfcUfgUfagaAfgGfgCfaUfgsUfsu | 216 |
D-2025 | AfsusGfcCfcUfuCfUfAfcAfgUfgGfcCfsusUf | 217 | gsGfscCfaCfuGfuagAfaGfgGfcAfusUfsu | 218 |
D-2026 | UfsgsCfcCfuUfcUfAfCfaGfuGfgCfcUfsusUf | 219 | asGfsgCfcAfcUfguaGfaAfgGfgCfasUfsu | 220 |
D-2027 | GfscsCfcUfuCfuAfCfAfgUfgGfcCfuUfsusUf | 221 | asAfsgGfcCfaCfuguAfgAfaGfgGfcsUfsu | 222 |
D-2028 | GfsgsUfaUfgUfuCfCfUfgCfuUfcAfuCfsusUf | 223 | gsAfsuGfaAfgCfaggAfaCfaUfaCfcsUfsu | 224 |
D-2029 | GfsusAfuGfuUfcCfUfGfcUfuCfaUfcCfsusUf | 225 | gsGfsaUfgAfaGfcagGfaAfcAfuAfcsUfsu | 226 |
D-2030 | UfsasUfgUfuCfcUfGfCfuUfcAfuCfcCfsusUf | 227 | gsGfsgAfuGfaAfgcaGfgAfaCfaUfasUfsu | 228 |
D-2031 | AfsusGfuUfcCfuGfCfUfuCfaUfcCfcCfsusUf | 229 | gsGfsgGfaUfgAfagcAfgGfaAfcAfusUfsu | 230 |
D-2032 | UfsgsUfuCfcUfgCfUfUfcAfuCfcCfcUfsusUf | 231 | asGfsgGfgAfuGfaagCfaGfgAfaCfasUfsu | 232 |
D-2033 | GfsusUfcCfuGfcUfUfCfaUfcCfcCfuUfsusUf | 233 | asAfsgGfgGfaUfgaaGfcAfgGfaAfcsUfsu | 234 |
D-2034 | UfsusCfcUfgCfuUfCfAfuCfcCfcUfuCfsusUf | 235 | gsAfsaGfgGfgAfugaAfgCfaGfgAfasUfsu | 236 |
D-2035 | UfscsCfuGfcUfuCfAfUfcCfcCfuUfcUfsusUf | 237 | asGfsaAfgGfgGfaugAfaGfcAfgGfasUfsu | 238 |
D-2036 | CfscsUfgCfuUfcAfUfCfcCfcUfuCfuAfsusUf | 239 | usAfsgAfaGfgGfgauGfaAfgCfaGfgsUfsu | 240 |
D-2037 | CfsusGfcUfuCfaUfCfCfcCfuUfcUfaCfsusUf | 241 | gsUfsaGfaAfgGfggaUfgAfaGfcAfgsUfsu | 242 |
D-2038 | UfsgsCfuUfcAfuCfCfCfcUfuCfuAfcAfsusUf | 243 | usGfsuAfgAfaGfgggAfuGfaAfgCfasUfsu | 244 |
D-2039 | UfsusCfaUfcCfcCfUfUfcUfaCfaGfuGfsusUf | 245 | csAfscUfgUfaGfaagGfgGfaUfgAfasUfsu | 246 |
D-2040 | UfscsAfuCfcCfcUfUfCfuAfcAfgUfgGfsusUf | 247 | csCfsaCfuGfuAfgaaGfgGfgAfuGfasUfsu | 248 |
D-2041 | CfsasUfcCfcCfuUfCfUfaCfaGfuGfgCfsusUf | 249 | gsCfscAfcUfgUfagaAfgGfgGfaUfgsUfsu | 250 |
D-2042 | UfscsCfcCfuUfcUfAfCfaGfuGfgCfcUfsusUf | 251 | asGfsgCfcAfcUfguaGfaAfgGfgGfasUfsu | 252 |
D-2043 | GfsasUfcAfgGfaCfCfCfgAfgCfcGfaUfsusUf | 253 | asUfscGfgCfuCfgggUfcCfuGfaUfcsUfsu | 254 |
D-2044 | UfsgsGfgCfuUfcUfAfCfcAfcGfuCfgUfsusUf | 255 | asCfsgAfcGfuGfguaGfaAfgCfcCfasUfsu | 256 |
D-2045 | GfsasGfcGfaGfcAfCfGfcCfcCfgCfaUfsusUf | 257 | asUfsgCfgGfgGfcguGfcUfcGfcUfcsUfsu | 258 |
D-2046 | UfsgsCfaCfuGfcGfUfCfgGfcGfuCfcUfsusUf | 259 | asGfsgAfcGfcCfgacGfcAfgUfgCfasUfsu | 260 |
D-2047 | UfsgsGfaGfcAfgAfCfUfcUfgCfaGfgUfsusUf | 261 | asCfscUfgCfaGfaguCfuGfcUfcCfasUfsu | 262 |
D-2048 | UfsgsCfaGfgUfcCfUfCfuCfaGfaUfcUfsusUf | 263 | asGfsaUfcUfgAfgagGfaCfcUfgCfasUfsu | 264 |
D-2049 | CfscsCfgGfcCfaAfUfGfuCfcAfcCfaUfsusUf | 265 | asUfsgGfuGfgAfcauUfgGfcCfgGfgsUfsu | 266 |
D-2050 | UfsusCfuAfcAfgUfGfGfcCfuUfaUfcUfsusUf | 267 | asGfsaUfaAfgGfccaCfuGfuAfgAfasUfsu | 268 |
D-2051 | UfscsUfaCfaGfuGfGfCfcUfuAfuCfcUfsusUf | 269 | asGfsgAfuAfaGfgccAfcUfgUfaGfasUfsu | 270 |
D-2052 | CfsusUfcCfuUfcAfGfAfgGfcGfuGfcUfsusUf | 271 | asGfscAfcGfcCfucuGfaAfgGfaAfgsUfsu | 272 |
D-2053 | UfsusCfcUfuCfaGfAfGfgCfgUfgCfgAfsusUf | 273 | usCfsgCfaCfgCfcucUfgAfaGfgAfasUfsu | 274 |
D-2054 | GfscsGfuGfcGfaUfAfUfgUfgGfaUfgUfsusUf | 275 | asCfsaUfcCfaCfauaUfcGfcAfcGfcsUfsu | 276 |
D-2055 | CfsgsUfgCfgAfuAfUfGfuGfgAfuGfgAfsusUf | 277 | usCfscAfuCfcAfcauAfuCfgCfaCfgsUfsu | 278 |
D-2056 | UfsgsGfaUfgGfaGfGfAfgUfgAfgUfgAfsusUf | 279 | usCfsaCfuCfaCfuccUfcCfaUfcCfasUfsu | 280 |
D-2057 | AfscsGfuAfcCfcUfUfCfaUfuGfaUfgUfsusUf | 281 | asCfsaUfcAfaUfgaaGfgGfuAfcGfusUfsu | 282 |
D-2058 | UfsgsGfaCfaUfcAfCfCfaAfgCfuCfaUfsusUf | 283 | asUfsgAfgCfuUfgguGfaUfgUfcCfasUfsu | 284 |
D-2059 | CfsasCfcUfgCfgUfCfUfcAfgCfaUfcUfsusUf | 285 | asGfsaUfgCfuGfagaCfgCfaGfgUfgsUfsu | 286 |
D-2060 | AfscsCfuGfcGfuCfUfCfaGfcAfuCfcUfsusUf | 287 | asGfsgAfuGfcUfgagAfcGfcAfgGfusUfsu | 288 |
D-2061 | CfscsAfgAfgAfcUfGfGfuGfaCfaUfgUfsusUf | 289 | asCfsaUfgUfcAfccaGfuCfuCfuGfgsUfsu | 290 |
D-2062 | AfsusGfgCfuUfcCfAfGfaUfaUfgCfcUfsusUf | 291 | asGfsgCfaUfaUfcugGfaAfgCfcAfusUfsu | 292 |
D-2063 | CfscsGfcCfuCfcAfGfGfuCfcCfaAfaUfsusUf | 293 | asUfsuUfgGfgAfccuGfgAfgGfcGfgsUfsu | 294 |
D-2064 | UfsasCfcUfgCfuGfGfUfgCfuGfaGfgUfsusUf | 295 | asCfscUfcAfgCfaccAfgCfaGfgUfasUfsu | 296 |
D-2065 | AfscsCfuGfcUfgGfUfGfcUfgAfgGfgUfsusUf | 297 | asCfscCfuCfaGfcacCfaGfcAfgGfusUfsu | 298 |
D-2066 | CfsusCfuCfcAfcCfUfUfuCfcCfaGfuUfsusUf | 299 | asAfscUfgGfgAfaagGfuGfgAfgAfgsUfsu | 300 |
D-2067 | UfsusUfuUfcAfcCfUfAfaCfuAfaAfaUfsusUf | 301 | asUfsuUfuAfgUfuagGfuGfaAfaAfasUfsu | 302 |
D-2068 | CfgGfcCfaAfuGfUfCfcAfcCfaGfcUfsusUf | 303 | asGfscUfgGfuGfgacAfuUfgGfcCfgsUfsu | 304 |
D-2069 | GfgUfcCfaGfcCfUfGfaAfcUfuCfuUfsusUf | 305 | asAfsgAfaGfuUfcagGfcUfgGfaCfcsUfsu | 306 |
D-2070 | GfcGfgCfuUfcCfUfGfgGfcUfuCfuAfsusUf | 307 | usAfsgAfaGfcCfcagGfaAfgCfcGfcsUfsu | 308 |
D-2071 | GfuGfaCfaAfcGfUfAfcCfcUfuCfaUfsusUf | 309 | asUfsgAfaGfgGfuacGfuUfgUfcAfcsUfsu | 310 |
D-2072 | GfgUfaUfgUfuCfCfUfgCfuUfcAfuGfsusUf | 311 | csAfsuGfaAfgCfaggAfaCfaUfaCfcsUfsu | 312 |
D-2073 | GfuAfuGfuUfcCfUfGfcUfuCfaUfgCfsusUf | 313 | gsCfsaUfgAfaGfcagGfaAfcAfuAfcsUfsu | 314 |
D-2074 | UfgUfuCfcUfgCfUfUfcAfuGfcCfcUfsusUf | 315 | asGfsgGfcAfuGfaagCfaGfgAfaCfasUfsu | 316 |
D-2075 | GfuUfcCfuGfcUfUfCfaUfgCfcCfuUfsusUf | 317 | asAfsgGfgCfaUfgaaGfcAfgGfaAfcsUfsu | 318 |
D-2076 | CfcUfgCfuUfcAfUfGfcCfcUfuCfuAfsusUf | 319 | usAfsgAfaGfgGfcauGfaAfgCfaGfgsUfsu | 320 |
D-2077 | GfcUfuCfaUfgCfCfCfuUfcUfaCfaGfsusUf | 321 | csUfsgUfaGfaAfgggCfaUfgAfaGfcsUfsu | 322 |
D-2078 | CfuUfcAfuGfcCfCfUfuCfuAfcAfgUfsusUf | 323 | asCfsuGfuAfgAfaggGfcAfuGfaAfgsUfsu | 324 |
D-2079 | UfuCfaUfgCfcCfUfUfcUfaCfaGfuGfsusUf | 325 | csAfscUfgUfaGfaagGfgCfaUfgAfasUfsu | 326 |
D-2080 | AfuGfgCfuUfcCfAfGfaUfaUfgCfcUfsusUf | 327 | asGfsgCfaUfaUfcugGfaAfgCfcAfusUfsu | 328 |
D-2081 | AfuGfcCfcUfuCfUfAfcAfgUfgGfcCfsusUf | 329 | gsGfscCfaCfuGfuagAfaGfgGfcAfusUfsu | 330 |
D-2082 | GfcUfuCfaUfgCfCfCfuUfcUfaCfaUfsusUf | 331 | asUfsgUfaGfaAfgggCfaUfgAfaGfcsUfsu | 332 |
D-2083 | {磷酸酯}GfsgsAfaAfgAfcUfGfUfuCfcAfaAfaAfsusUf | 1295 | {磷酸酯}usUfsuUfuGfgAfacaGfuCfuUfuCfcsUfsu | 1296 |
D-2084 | {GalNAc3K2AhxC6}ggsuaugUfuCfCfUfGfcuucaugsusu | 1297 | {磷酸酯}csAfsuGfaAfGfcaggAfaCfauaccsusu | 1298 |
D-2085 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucaugcsusu | 1299 | {磷酸酯}gsCfsaUfgAfAfgcagGfaAfcauacsusu | 1300 |
D-2086 | {GalNAc3K2AhxC6}uguuccUfgCfUfUfCfaugcccususu | 1301 | {磷酸酯}asGfsgGfcAfUfgaagCfaGfgaacasusu | 1302 |
D-2087 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacagsusu | 1303 | {磷酸酯}csUfsgUfaGfAfagggCfaUfgaagcsusu | 1304 |
D-2088 | {GalNAc3K2AhxC6}cuucauGfcCfCfUfUfcuacagususu | 1305 | {磷酸酯}asCfsuGfuAfGfaaggGfcAfugaagsusu | 1306 |
D-2089 | {GalNAc3K2AhxC6}gcggcuUfcCfUfGfGfgcuucuasusu | 1307 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgccgcsusu | 1308 |
D-2090 | {GalNAc3K2AhxC6}guuccuGfcUfUfCfAfugcccuususu | 1309 | {磷酸酯}asAfsgGfgCfAfugaaGfcAfggaacsusu | 1310 |
D-2091 | {GalNAc3K2AhxC6}AfuGfgCfuUfcCfAfGfaUfaUfgCfcUfsusUf | 1311 | {磷酸酯}asGfsgCfaUfaUfcugGfaAfgCfcAfusUfsu | 1312 |
D-2092 | {GalNAc3K2AhxC6}ccugcuUfcAfUfGfCfccuucuasusu | 1313 | {磷酸酯}usAfsgAfaGfGfgcauGfaAfgcaggsusu | 1314 |
D-2093 | {GalNAc3K2AhxC6}uucaugCfcCfUfUfCfuacaguususu | 1315 | {磷酸酯}asAfscUfgUfAfgaagGfgCfaugaasusu | 1316 |
D-2094 | {GalNAc3K2AhxC6}uucaugCfcCfUfUfCfuacagugsusu | 1317 | {磷酸酯}csAfscUfgUfAfgaagGfgCfaugaasusu | 1318 |
D-2095 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1319 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1320 |
D-2096 | {GalNAc3K2AhxC6}gguccaGfcCfUfGfAfacuucuususu | 1321 | {磷酸酯}asAfsgAfaGfUfucagGfcUfggaccsusu | 1322 |
D-2097 | {GalNAc3K2AhxC6}GfcGfgCfuUfcCfUfGfGfgcUfuCfuAfsusUf | 1323 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgCfcGfcsUfsu | 1324 |
D-2098 | {GalNAc3K2AhxC6}GfcGfgCfuUfcCfuGfGfgcUfuCfuAfsusUf | 1325 | {磷酸酯}usAfsgAfaGfCfccAfgGfaAfgCfcGfcsUfsu | 1326 |
D-2099 | {GalNAc3K2AhxC6}GfuUfcCfuGfcUfUfCfAfugCfcCfuUfsusUf | 1327 | {磷酸酯}asAfsgGfgCfAfugaaGfcAfgGfaAfcsUfsu | 1328 |
D-2100 | {GalNAc3K2AhxC6}GfuUfcCfuGfcUfuCfAfugCfcCfuUfsusUf | 1329 | {磷酸酯}asAfsgGfgCfAfugAfaGfcAfgGfaAfcsUfsu | 1330 |
D-2101 | {GalNAc3K2AhxC6}CfcUfgCfuUfcAfUfGfCfccUfuCfuAfsusUf | 1331 | {磷酸酯}usAfsgAfaGfGfgcauGfaAfgCfaGfgsUfsu | 1332 |
D-2102 | {GalNAc3K2AhxC6}CfcUfgCfuUfcAfuGfCfccUfuCfuAfsusUf | 1333 | {磷酸酯}usAfsgAfaGfGfgcAfuGfaAfgCfaGfgsUfsu | 1334 |
D-2103 | {GalNAc3K2AhxC6}augcccUfuCfUfAfCfaguggccsusu | 1335 | {磷酸酯}gsGfscCfaCfUfguagAfaGfggcaususu | 1336 |
D-2104 | {GalNAc3K2AhxC6}auggcuUfcCfAfGfAfuaugccususu | 1337 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgccaususu | 1338 |
D-2105 | {GalNAc3K2AhxC6}gugacaAfcGfUfAfCfccuucaususu | 1339 | {磷酸酯}asUfsgAfaGfGfguacGfuUfgucacsusu | 1340 |
D-2106 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucaugcsusu | 1341 | {磷酸酯}gsCfsaUfgAfAfgcAfgGfaAfcauacsusu | 1342 |
D-2107 | {GalNAc3K2AhxC6}guauguUfcCfuGfCfuucaugcsusu | 1343 | {磷酸酯}gsCfsaUfgAfAfgcagGfaAfcauacsusu | 1344 |
D-2108 | {GalNAc3K2AhxC6}guauguUfcCfuGfCfuucaugcsusu | 1345 | {磷酸酯}gsCfsaUfgAfAfgcAfgGfaAfcauacsusu | 1346 |
D-2109 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucaugcscsu | 1347 | {磷酸酯}asGfsgCfaUfgAfAfgcagGfaAfcauacsusu | 1348 |
D-2110 | {GalNAc3K2AhxC6}ugguauGfuUfCfCfUfgcuucausgsu | 1349 | {磷酸酯}gsCfsaUfgAfaGfCfaggaAfcAfuaccasusu | 1350 |
D-2111 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucausgsu | 1351 | {磷酸酯}gsCfsaUfgAfAfgcagGfaAfcauacsusu | 1352 |
D-2112 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucaugcs{invAb} | 1353 | {磷酸酯}gsCfsaUfgAfAfgcagGfaAfcauacsusu | 1354 |
D-2113 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucUfaCfaUfsusUf | 1355 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgAfaGfcsUfsu | 1356 |
D-2114 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfcCfUfucUfaCfaUfsusUf | 1357 | {磷酸酯}asUfsgUfaGfAfagGfgCfaUfgAfaGfcsUfsu | 1358 |
D-2115 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1359 | {磷酸酯}asusguaGfAfagggCfaUfgaagcsusu | 1360 |
D-2116 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucUfaCfaUfsusUf | 1361 | {磷酸酯}asusguaGfAfagggCfaUfgaagcsusu | 1362 |
D-2117 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucuacaususu | 1363 | {磷酸酯}asusguaGfAfagggCfaUfgaagcsusu | 1364 |
D-2118 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1365 | {磷酸酯}asusguaGfAfagggCfaUfgAfaGfcsUfsu | 1366 |
D-2119 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1367 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgAfaGfcsUfsu | 1368 |
D-2120 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucUfaCfaUfsusUf | 1369 | {磷酸酯}asusguaGfAfagggCfaUfgaagcsusu | 1370 |
D-2121 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucuacaususu | 1371 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1372 |
D-2122 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucUfaCfaUfsusUf | 1373 | {磷酸酯}asusguaGfAfagggCfaUfgAfaGfcsUfsu | 1374 |
D-2123 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucUfaCfaUfsusUf | 1375 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1376 |
D-2124 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucuacaususu | 1377 | {磷酸酯}asusguaGfAfagggCfaUfgAfaGfcsUfsu | 1378 |
D-2125 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucuacaususu | 1379 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgAfaGfcsUfsu | 1380 |
D-2126 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucUfaCfaUfsusUf | 1381 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgAfaGfcsUfsu | 1382 |
D-2127 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucUfaCfaUfsusUf | 1383 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1384 |
D-2128 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfCfUfucUfaCfaUfsusUf | 1385 | {磷酸酯}asusguaGfAfagggCfaUfgAfaGfcsUfsu | 1386 |
D-2129 | {GalNAc3K2AhxC6}gcuucaUfg[dC]CfCfuucuacaususu | 1387 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1388 |
D-2130 | {GalNAc3K2AhxC6}gcuucaUfgCfCf[dC]Ufucuacaususu | 1389 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1390 |
D-2131 | {GalNAc3K2AhxC6}gcuucaUfgCf[dC]CfUfucuacaususu | 1391 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1392 |
D-2132 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1393 | {磷酸酯}asUfsgUfaGfAfagGfgCfaUfgaagcsusu | 1394 |
D-2133 | {GalNAc3K2AhxC6}gcuucaUfgCfcCfUfucuacaususu | 1395 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1396 |
D-2134 | {GalNAc3K2AhxC6}gcuucaUfgCfcCfUfucuacaususu | 1397 | {磷酸酯}asUfsgUfaGfAfagGfgCfaUfgaagcsusu | 1398 |
D-2135 | {GalNAc3K2AhxC6}GfscsUfuCfaUfgCfcCfUfucUfaCfaUfsusUf | 1399 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgAfaGfcsUfsu | 1400 |
D-2136 | {GalNAc3K2AhxC6}GfscsUfuCfaUfgCfCfCfUfucUfaCfaUfsusUf | 1401 | {磷酸酯}asUfsgUfaGfAfagGfgCfaUfgAfaGfcsUfsu | 1402 |
D-2137 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacagsusu | 1403 | {磷酸酯}asAfscUfgUfaGfAfagggCfaUfgaagcsusu | 1404 |
D-2138 | {GalNAc3K2AhxC6}cugcuuCfaUfGfCfCfcuucuacsasu | 1405 | {磷酸酯}asUfsgUfaGfaAfGfggcaUfgAfagcagsusu | 1406 |
D-2139 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacsasu | 1407 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1408 |
D-2140 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaus{invAb} | 1409 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1410 |
D-2141 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacauuus{invAb} | 1411 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1412 |
D-2142 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1413 | {磷酸酯}AfsusGfuAfgAfagggCfaUfgaagcsusu | 1414 |
D-2143 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfuCfuacaususu | 1415 | {磷酸酯}AfsusGfuAfgAfagggCfaUfgaagcsusu | 1416 |
D-2144 | {GalNAc3K2AhxC6}gcuucaugCfCfCfUfucuacaususu | 1417 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1418 |
D-2145 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1419 | {磷酸酯}asUfsgUfaGfAfagggCfaugaagcsusu | 1420 |
D-2146 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1421 | {磷酸酯}asusgUfaGfAfagggCfaUfgaagcsusu | 1422 |
D-2147 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1423 | {磷酸酯}asUfsgUfagAfagggCfaUfgaagcsusu | 1424 |
D-2148 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1425 | {磷酸酯}asusgUfagAfagggCfaUfgaagcsusu | 1426 |
D-2149 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1427 | {磷酸酯}asUfsguagAfagggCfaUfgaagcsusu | 1428 |
D-2150 | {GalNAc3K2AhxC6}gcuucaUfgCfCfCfUfucuacaususu | 1429 | {磷酸酯}asusguagAfagggCfaUfgaagcsusu | 1430 |
D-2151 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucaugcuus{invAb} | 1431 | {磷酸酯}gsCfsaUfgAfAfgcagGfaAfcauacsusu | 1432 |
D-2152 | {GalNAc3K2AhxC6}gguaugUfuCfCfUfGfcuucauususu | 1433 | {磷酸酯}aAfsuGfaAfGfcaggAfaCfauaccsusu | 1434 |
D-2153 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucaugususu | 1435 | {磷酸酯}asCfsaUfgAfAfgcagGfaAfcauacsusu | 1436 |
D-2154 | {GalNAc3K2AhxC6}cggccaAfuGfUfCfCfaccagcususu | 1437 | {磷酸酯}asGfscUfgGfUfggacAfuUfggccgsusu | 1438 |
D-2155 | {GalNAc3K2AhxC6}uggagcAfgAfCfUfCfugcaggususu | 1439 | {磷酸酯}asCfscUfgCfAfgaguCfuGfcuccasusu | 1440 |
D-2156 | {GalNAc3K2AhxC6}acguacCfcUfUfCfAfuugaugususu | 1441 | {磷酸酯}aCfsaUfcAfAfugaaGfgGfuacgususu | 1442 |
D-2157 | {GalNAc3K2AhxC6}ccagagAfcUfGfGfUfgacaugususu | 1443 | {磷酸酯}asCfsaUfgUfCfaccaGfuCfucuggsusu | 1444 |
D-2158 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacaususu | 1445 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcsusu | 1446 |
D-2159 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacaususu | 1447 | {磷酸酯}asAfsaUfgUfaGfAfaaggCfaUfgaagcsusu | 1448 |
D-2160 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfcUfUfUfCfuacaususu | 1449 | {磷酸酯}asAfsaUfgUfAfgaaaGfgCfaUfgaagcsusu | 1450 |
D-2161 | {GalNAc3K2AhxC6}ugcuucAfuGfCfCfUfuucuacasusu | 1451 | {磷酸酯}usGfsuAfgAfAfaggcAfuGfaagcasusu | 1452 |
D-2162 | {GalNAc3K2AhxC6}uauguuCfcUfGfCfUfucaugcususu | 1453 | {磷酸酯}asGfscAfuGfAfagcaGfgAfacauasusu | 1454 |
D-2163 | {GalNAc3K2AhxC6}uuccugCfuUfCfAfUfgccuuuususu | 1455 | {磷酸酯}asAfsaAfgGfCfaugaAfgCfaggaasusu | 1456 |
D-2164 | {GalNAc3K2AhxC6}ucaugcCfuUfUfCfUfacagugususu | 1457 | {磷酸酯}asCfsaCfuGfUfagaaAfgGfcaugasusu | 1458 |
D-2165 | {GalNAc3K2AhxC6}caugccUfuUfCfUfAfcaguggususu | 1459 | {磷酸酯}asCfscAfcUfGfuagaAfaGfgcaugsusu | 1460 |
D-2166 | {GalNAc3K2AhxC6}augccuUfuCfUfAfCfaguggcususu | 1461 | {磷酸酯}asGfscCfaCfUfguagAfaAfggcaususu | 1462 |
D-2167 | {GalNAc3K2AhxC6}gguaugUfuCfCfUfGfcuucauasusu | 1463 | {磷酸酯}usAfsuGfaAfGfcaggAfaCfauaccsusu | 1464 |
D-2168 | {GalNAc3K2AhxC6}guauguUfcCfUfGfCfuucaugasusu | 1465 | {磷酸酯}usCfsaUfgAfAfgcagGfaAfcauacsusu | 1466 |
D-2169 | {GalNAc3K2AhxC6}uauguuCfcUfGfCfUfucaugcasusu | 1467 | {磷酸酯}usGfscAfuGfAfagcaGfgAfacauasusu | 1468 |
D-2170 | {GalNAc3K2AhxC6}uuccugCfuUfCfAfUfgccuuuasusu | 1469 | {磷酸酯}usAfsaAfgGfCfaugaAfgCfaggaasusu | 1470 |
D-2171 | {GalNAc3K2AhxC6}cugcuuCfaUfGfCfCfuuucuaasusu | 1471 | {磷酸酯}usUfsaGfaAfAfggcaUfgAfagcagsusu | 1472 |
D-2172 | {GalNAc3K2AhxC6}gcuucaUfgCfCfUfUfucuacaasusu | 1473 | {磷酸酯}usUfsgUfaGfAfaaggCfaUfgaagcsusu | 1474 |
D-2173 | {GalNAc3K2AhxC6}uucaugCfcUfUfUfCfuacaguasusu | 1475 | {磷酸酯}usAfscUfgUfAfgaaaGfgCfaugaasusu | 1476 |
D-2174 | {GalNAc3K2AhxC6}ucaugcCfuUfUfCfUfacagugasusu | 1477 | {磷酸酯}usCfsaCfuGfUfagaaAfgGfcaugasusu | 1478 |
D-2175 | {GalNAc3K2AhxC6}caugccUfuUfCfUfAfcaguggasusu | 1479 | {磷酸酯}usCfscAfcUfGfuagaAfaGfgcaugsusu | 1480 |
D-2176 | {GalNAc3K2AhxC6}augccuUfuCfUfAfCfaguggcasusu | 1481 | {磷酸酯}usGfscCfaCfUfguagAfaAfggcaususu | 1482 |
D-2177 | {GalNAc3K2AhxC6}acguacCfcUfUfCfAfuugaugasusu | 1483 | {磷酸酯}usCfsaUfcAfAfugaaGfgGfuacgususu | 1484 |
D-2178 | {GalNAc3K2AhxC6}ccagagAfcUfGfGfUfgacaugasusu | 1485 | {磷酸酯}usCfsaUfgUfCfaccaGfuCfucuggsusu | 1486 |
D-2179 | {GalNAc3K2AhxC6}auggcuUfcCfAfGfAfuaugccasusu | 1487 | {磷酸酯}usGfsgCfaUfAfucugGfaAfgccaususu | 1488 |
D-2180 | {GalNAc3K2AhxC6}guuccuGfcUfUfCfAfugccuuususu | 1489 | {磷酸酯}asAfsaGfgCfAfugaaGfcAfggaacsusu | 1490 |
D-2181 | {GalNAc3K2AhxC6}ccugcuUfcAfUfGfCfcuuucuasusu | 1491 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgcaggsusu | 1492 |
D-2182 | {GalNAc3K2AhxC6}gcuucaUfgCfCfUfUfucuacaususu | 1493 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcsusu | 1494 |
D-2183 | {GalNAc3K2AhxC6}cuucauGfcCfUfUfUfcuacagususu | 1495 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1496 |
D-2184 | {GalNAc3K2AhxC6}uucaugCfcUfUfUfCfuacaguususu | 1497 | {磷酸酯}asAfscUfgUfAfgaaaGfgCfaugaasusu | 1498 |
D-2185 | {GalNAc3K2AhxC6}gcuucaUfcCfCfUfUfucuacaususu | 1499 | {磷酸酯}asUfsgUfaGfAfaaggGfaUfgaagcsusu | 1500 |
D-2186 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfCfUfucuacaususu | 1501 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1502 |
D-2187 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfCfUfucuacaususu | 1503 | {磷酸酯}asAfsaUfgUfaGfAfagggCfaUfgaagcsusu | 1504 |
D-2188 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfcCfUfUfCfuacaususu | 1505 | {磷酸酯}asAfsaUfgUfAfgaagGfgCfaUfgaagcsusu | 1506 |
D-2189 | {GalNAc3K2AhxC6}[invAb]gcggcuUfcCfUfGfGfgcuucuasusu | 1507 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgccgcsusu | 1508 |
D-2190 | {GalNAc3K2AhxC6}[invAb]CfuGfcGfgCfuUfcCfuGfGfgcUfuCfsusAf | 1509 | {磷酸酯}usAfsgAfaGfCfccAfgGfaAfgCfcGfcAfgsUfsu | 1510 |
D-2191 | {GalNAc3K2AhxC6}cugcggCfuUfCfCfUfgggcuucus{invAb} | 1511 | {磷酸酯}usAfsgAfaGfcCfCfaggaAfgCfcgcagsusu | 1512 |
D-2192 | {GalNAc3K2AhxC6}[invAb]cugcggCfuUfCfCfUfgggcuucsusa | 1513 | {磷酸酯}usAfsgAfaGfcCfCfaggaAfgCfcgcagsusu | 1514 |
D-2193 | {GalNAc3K2AhxC6}[invAb]gcggcuUfcCfUfGfGfgcUfuCfuAfsusUf | 1515 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgccgcsusu | 1516 |
D-2194 | {GalNAc3K2AhxC6}cugcggCfuUfCfCfUfggGfcUfuCfus{invAb} | 1517 | {磷酸酯}usAfsgAfaGfcCfCfaggaAfgCfcgcagsusu | 1518 |
D-2195 | {GalNAc3K2AhxC6}[invAb]cugcggCfuUfCfCfUfggGfcUfuCfsusAf | 1519 | {磷酸酯}usAfsgAfaGfcCfCfaggaAfgCfcgcagsusu | 1520 |
D-2196 | {GalNAc3K2AhxC6}[invAb]GfcGfgCfuUfcCfuGfGfgcUfuCfuAfsusUf | 1521 | {磷酸酯}usAfsgAfaGfCfccAfgGfaAfgCfcGfcsUfsu | 1522 |
D-2197 | {GalNAc3K2AhxC6}CfuGfcGfgCfuUfcCfUfggGfcUfuCfus{invAb} | 1523 | {磷酸酯}usAfsgAfaGfcCfCfagGfaAfgCfcGfcAfgsUfsu | 1524 |
D-2198 | {GalNAc3K2AhxC6}[invAb]CfuGfcGfgCfuUfcCfUfggGfcUfuCfsusAf | 1525 | {磷酸酯}usAfsgAfaGfcCfCfagGfaAfgCfcGfcAfgsUfsu | 1526 |
D-2199 | {GalNAc3K2AhxC6}[invAb]GfcGfgCfuUfcCfUfGfGfgcuucuasusu | 1527 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgCfcGfcsUfsu | 1528 |
D-2200 | {GalNAc3K2AhxC6}[invAb]CfuGfcGfgCfuUfCfCfUfgggcuucsusa | 1529 | {磷酸酯}usAfsgAfaGfcCfCfaggaAfgCfcGfcAfgsUfsu | 1530 |
D-2201 | {GalNAc3K2AhxC6}[invAb]cugcggcuUfcCfUfGfGfgcUfuCfusAf | 1531 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgccgcagsusu | 1532 |
D-2202 | {GalNAc3K2AhxC6}[invAb]CfuGfcGfgCfuUfcCfUfGfGfgcuucsusa | 1533 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgCfcGfcAfgsUfsu | 1534 |
D-2203 | {GalNAc3K2AhxC6}[invAb]auggcuUfcCfAfGfAfuaugccususu | 1535 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgccaususu | 1536 |
D-2204 | {GalNAc3K2AhxC6}[invAb]AfcAfuGfgCfuUfcCfaGfAfuaUfgCfscsUf | 1537 | {磷酸酯}asGfsgCfaUfAfucUfgGfaAfgCfcAfuGfusUfsu | 1538 |
D-2205 | {GalNAc3K2AhxC6}acauggCfuUfCfCfAfgauaugccs{invAb} | 1539 | {磷酸酯}asGfsgCfaUfaUfCfuggaAfgCfcaugususu | 1540 |
D-2206 | {GalNAc3K2AhxC6}[invAb]acauggCfuUfCfCfAfgauaugcscsu | 1541 | {磷酸酯}asGfsgCfaUfaUfCfuggaAfgCfcaugususu | 1542 |
D-2207 | {GalNAc3K2AhxC6}[invAb]auggcuUfcCfAfGfAfuaUfgCfcUfsusUf | 1543 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgccaususu | 1544 |
D-2208 | {GalNAc3K2AhxC6}acauggCfuUfCfCfAfgaUfaUfgCfcs{invAb} | 1545 | {磷酸酯}asGfsgCfaUfaUfCfuggaAfgCfcaugususu | 1546 |
D-2209 | {GalNAc3K2AhxC6}[invAb]acauggCfuUfCfCfAfgaUfaUfgCfscsUf | 1547 | {磷酸酯}asGfsgCfaUfaUfCfuggaAfgCfcaugususu | 1548 |
D-2210 | {GalNAc3K2AhxC6}[invAb]AfuGfgCfuUfcCfaGfAfuaUfgCfcUfsusUf | 1549 | {磷酸酯}asGfsgCfaUfAfucUfgGfaAfgCfcAfusUfsu | 1550 |
D-2211 | {GalNAc3K2AhxC6}AfcAfuGfgCfuUfcCfAfgaUfaUfgCfcs{invAb} | 1551 | {磷酸酯}asGfsgCfaUfaUfCfugGfaAfgCfcAfuGfusUfsu | 1552 |
D-2212 | {GalNAc3K2AhxC6}[invAb]AfcAfuGfgCfuUfcCfAfgaUfaUfgCfscsUf | 1553 | {磷酸酯}asGfsgCfaUfaUfCfugGfaAfgCfcAfuGfusUfsu | 1554 |
D-2213 | {GalNAc3K2AhxC6}[invAb]AfuGfgCfuUfcCfAfGfAfuaugccususu | 1555 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgCfcAfusUfsu | 1556 |
D-2214 | {GalNAc3K2AhxC6}AfcAfuGfgCfuUfCfCfAfgauaugccs{invAb} | 1557 | {磷酸酯}asGfsgCfaUfaUfCfuggaAfgCfcAfuGfusUfsu | 1558 |
D-2215 | {GalNAc3K2AhxC6}[invAb]AfcAfuGfgCfuUfCfCfAfgauaugcscsu | 1559 | {磷酸酯}asGfsgCfaUfaUfCfuggaAfgCfcAfuGfusUfsu | 1560 |
D-2216 | {GalNAc3K2AhxC6}[invAb]acauggcuUfcCfAfGfAfuaugcscsu | 1561 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgccaugususu | 1562 |
D-2217 | {GalNAc3K2AhxC6}[invAb]acauggcuUfcCfAfGfAfuaUfgCfcsUf | 1563 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgccaugususu | 1564 |
D-2218 | {GalNAc3K2AhxC6}[invAb]AfcAfuGfgCfuUfcCfAfGfAfuaugcscsu | 1565 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgCfcAfuGfusUfsu | 1566 |
D-2219 | {GalNAc3K2AhxC6}[invAb]acguacCfcUfUfCfAfuugaugususu | 1567 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuacgususu | 1568 |
D-2220 | {GalNAc3K2AhxC6}[invAb]CfaAfcGfuAfcCfcUfuCfAfuuGfaUfsgsUf | 1569 | {磷酸酯}asCfsaUfcAfAfugAfaGfgGfuAfcGfuUfgsUfsu | 1570 |
D-2221 | {GalNAc3K2AhxC6}[invAb]caacguAfcCfCfUfUfcauugausgsu | 1571 | {磷酸酯}asCfsaUfcAfaUfGfaaggGfuAfcguugsusu | 1572 |
D-2222 | {GalNAc3K2AhxC6}[invAb]acguacCfcUfUfCfAfuuGfaUfgUfsusUf | 1573 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuacgususu | 1574 |
D-2223 | {GalNAc3K2AhxC6}caacguAfcCfCfUfUfcaUfuGfaUfgs{invAb} | 1575 | {磷酸酯}asCfsaUfcAfaUfGfaaggGfuAfcguugsusu | 1576 |
D-2224 | {GalNAc3K2AhxC6}[invAb]AfcGfuAfcCfcUfuCfAfuuGfaUfgUfsusUf | 1577 | {磷酸酯}asCfsaUfcAfAfugAfaGfgGfuAfcGfusUfsu | 1578 |
D-2225 | {GalNAc3K2AhxC6}CfaAfcGfuAfcCfcUfUfcaUfuGfaUfgs{invAb} | 1579 | {磷酸酯}asCfsaUfcAfaUfGfaaGfgGfuAfcGfuUfgsUfsu | 1580 |
D-2226 | {GalNAc3K2AhxC6}[invAb]CfaAfcGfuAfcCfcUfUfcaUfuGfaUfsgsUf | 1581 | {磷酸酯}asCfsaUfcAfaUfGfaaGfgGfuAfcGfuUfgsUfsu | 1582 |
D-2227 | {GalNAc3K2AhxC6}[invAb]AfcGfuAfcCfcUfUfCfAfuugaugususu | 1583 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuAfcGfusUfsu | 1584 |
D-2228 | {GalNAc3K2AhxC6}CfaAfcGfuAfcCfCfUfUfcauugaugs{invAb} | 1585 | {磷酸酯}asCfsaUfcAfaUfGfaaggGfuAfcGfuUfgsUfsu | 1586 |
D-2229 | {GalNAc3K2AhxC6}[invAb]CfaAfcGfuAfcCfCfUfUfcauugausgsu | 1587 | {磷酸酯}asCfsaUfcAfaUfGfaaggGfuAfcGfuUfgsUfsu | 1588 |
D-2230 | {GalNAc3K2AhxC6}[invAb]caacguacCfcUfUfCfAfuugausgsu | 1589 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuacguugsusu | 1590 |
D-2231 | {GalNAc3K2AhxC6}[invAb]caacguacCfcUfUfCfAfuuGfaUfgsUf | 1591 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuacguugsusu | 1592 |
D-2232 | {GalNAc3K2AhxC6}[invAb]CfaAfcGfuAfcCfcUfUfCfAfuugausgsu | 1593 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuAfcGfuUfgsUfsu | 1594 |
D-2233 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacsasu | 1595 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1596 |
D-2234 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucUfaCfsasUf | 1597 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1598 |
D-2235 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfcUfUfucUfaCfaUfsusUf | 1599 | {磷酸酯}asUfsgUfaGfAfaaGfgCfaUfgAfaGfcsUfsu | 1600 |
D-2236 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfCfUfUfucuacaususu | 1601 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgAfaGfcsUfsu | 1602 |
D-2237 | {GalNAc3K2AhxC6}CfuGfcUfuCfaUfgCfcUfUfucUfaCfsasUf | 1603 | {磷酸酯}asUfsgUfaGfAfaaGfgCfaUfgAfaGfcAfgsUfsu | 1604 |
D-2238 | {GalNAc3K2AhxC6}CfuGfcUfuCfaUfgCfCfUfUfucuacsasu | 1605 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgAfaGfcAfgsUfsu | 1606 |
D-2239 | {GalNAc3K2AhxC6}[invAb]CfuGfcUfuCfaUfgCfCfUfUfucuacsasu | 1607 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgAfaGfcAfgsUfsu | 1608 |
D-2240 | {GalNAc3K2AhxC6}CfuGfcUfuCfaUfgCfCfUfUfucuacas{invAb} | 1609 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1610 |
D-2241 | {GalNAc3K2AhxC6}[invAb]CfuGfcUfuCfaUfgCfCfUfUfucuacsasu | 1611 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1612 |
D-2242 | {GalNAc3K2AhxC6}[invAb]cugcuucaUfgCf[dC]UfUfucuacsasu | 1613 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1614 |
D-2243 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacsasu | 1615 | {磷酸酯}asUfsgUfaGfAfaaGfgCfaUfgaagcagsusu | 1616 |
D-2244 | {GalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasu | 1617 | {磷酸酯}asUfsgUfaGfAfaaGfgCfaUfgaagcagsusu | 1618 |
D-2245 | {GalNAc3K2AhxC6}cugcuucaUfgCfcUfUfucuacsasu | 1619 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1620 |
D-2246 | {GalNAc3K2AhxC6}cugcuucaUfgCfcUfUfucuacas{invAb} | 1621 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1622 |
D-2247 | {GalNAc3K2AhxC6}[invAb]cugcuucaUfgCfcUfUfucuacsasu | 1623 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1624 |
D-2248 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacsasu | 1625 | {磷酸酯}asUfsgUfagAfaaggCfaUfgaagcagsusu | 1626 |
D-2249 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1627 | {磷酸酯}asUfsgUfagAfaaggCfaUfgaagcagsusu | 1628 |
D-2250 | {GalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasu | 1629 | {磷酸酯}asUfsgUfagAfaaggCfaUfgaagcagsusu | 1630 |
D-2251 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1631 | {磷酸酯}asUfsguagAfaaggCfaUfgaagcagsusu | 1632 |
D-2252 | {GalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasu | 1633 | {磷酸酯}asUfsguagAfaaggCfaUfgaagcagsusu | 1634 |
D-2253 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfcUfUfucUfaCfaUfsusUf | 1635 | {磷酸酯}asUfsgUfagAfaaGfgCfaUfgAfaGfcsUfsu | 1636 |
D-2254 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfCfUfUfucuacaususu | 1637 | {磷酸酯}asUfsgUfagAfaaggCfaUfgAfaGfcsUfsu | 1638 |
D-2255 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfCfUfUfucuacaususu | 1639 | {磷酸酯}asUfsgUfagAfaaggCfaUfgaagcsusu | 1640 |
D-2256 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacaususu | 1641 | {磷酸酯}asUfsgUfagAfaaGfgCfaUfgaagcsusu | 1642 |
D-2257 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfcUfUfucuacaususu | 1643 | {磷酸酯}asUfsgUfagAfaaGfgCfaUfgaagcsusu | 1644 |
D-2258 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfCfUfucuacsasu | 1645 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcagsusu | 1646 |
D-2259 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfCfUfucUfaCfaUfsusUf | 1647 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1648 |
D-2260 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfcCfUfucUfaCfaUfsusUf | 1649 | {磷酸酯}asUfsgUfaGfAfagGfgCfaUfgAfaGfcsUfsu | 1650 |
D-2261 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfCfCfUfucuacaususu | 1651 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgAfaGfcsUfsu | 1652 |
D-2262 | {GalNAc3K2AhxC6}cugcuucaUfgCfCfCfUfucuacas{invAb} | 1653 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcagsusu | 1654 |
D-2263 | {GalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfCfUfucuacsasu | 1655 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcagsusu | 1656 |
D-2264 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfCfCfUfucuacaususu | 1657 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1658 |
D-2265 | {GalNAc3K2AhxC6}auguucCfuGfCfUfUfcaugccususu | 1659 | {磷酸酯}asGfsgCfaUfGfaagcAfgGfaacaususu | 1660 |
D-2266 | {GalNAc3K2AhxC6}uguuccUfgCfUfUfCfaugccuususu | 1661 | {磷酸酯}asAfsgGfcAfUfgaagCfaGfgaacasusu | 1662 |
D-2267 | {GalNAc3K2AhxC6}ugccuuUfcUfAfCfAfguggccususu | 1663 | {磷酸酯}asGfsgCfcAfCfuguaGfaAfaggcasusu | 1664 |
D-2268 | cguacuUfcGfUfCfCfuuguaugsusu | 1665 | {磷酸酯}csAfsuAfcAfAfggacGfaAfguacgsusu | 1666 |
D-2269 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCf[dC]CfUfucuacaususu | 1667 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1668 |
D-2270 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfCfUfucuacaususu | 1669 | {磷酸酯}asUfsgUfaGfAfagGfgCfaUfgaagcsusu | 1670 |
D-2271 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfcCfUfucuacaususu | 1671 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1672 |
D-2272 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfCfUfucuacaususu | 1673 | {磷酸酯}asUfsgUfagAfagggCfaUfgaagcsusu | 1674 |
D-2273 | {GalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfCfUfucuacaususu | 1675 | {磷酸酯}asUfsguagAfagggCfaUfgaagcsusu | 1676 |
D-2274 | {GalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfcCfUfucuacaususu | 1677 | {磷酸酯}asUfsgUfaGfAfagggCfaUfgaagcsusu | 1678 |
D-2275 | {sGalNAc3K2AhxC6}[invAb]ccugcuUfcAfUfGfCfcuUfuCfuAfsusUf | 1679 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgcaggsusu | 1680 |
D-2276 | {sGalNAc3K2AhxC6}[invAb]CfcUfgCfuUfcAfuGfCfcuUfuCfuAfsusUf | 1681 | {磷酸酯}usAfsgAfaAfGfgcAfuGfaAfgCfaGfgsUfsu | 1682 |
D-2277 | {sGalNAc3K2AhxC6}UfuCfcUfgCfuUfcAfUfgcCfuUfuCfus{invAb} | 1683 | {磷酸酯}usAfsgAfaAfgGfCfauGfaAfgCfaGfgAfasUfsu | 1684 |
D-2278 | {sGalNAc3K2AhxC6}[invAb]CfcUfgCfuUfcAfUfGfCfcuuucuasusu | 1685 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgCfaGfgsUfsu | 1686 |
D-2279 | {sGalNAc3K2AhxC6}UfuCfcUfgCfuUfCfAfUfgccuuucus{invAb} | 1687 | {磷酸酯}usAfsgAfaAfgGfCfaugaAfgCfaGfgAfasUfsu | 1688 |
D-2280 | {sGalNAc3K2AhxC6}[invAb]augccuUfuCfUfAfCfaguggcususu | 1689 | {磷酸酯}asGfscCfaCfUfguagAfaAfggcaususu | 1690 |
D-2281 | {sGalNAc3K2AhxC6}[invAb]augccuUfuCfUfAfCfagUfgGfcUfsusUf | 1691 | {磷酸酯}asGfscCfaCfUfguagAfaAfggcaususu | 1692 |
D-2282 | {sGalNAc3K2AhxC6}[invAb]AfuGfcCfuUfuCfuAfCfagUfgGfcUfsusUf | 1693 | {磷酸酯}asGfscCfaCfUfguAfgAfaAfgGfcAfusUfsu | 1694 |
D-2283 | {sGalNAc3K2AhxC6}UfcAfuGfcCfuUfuCfUfacAfgUfgGfcs{invAb} | 1695 | {磷酸酯}asGfscCfaCfuGfUfagAfaAfgGfcAfuGfasUfsu | 1696 |
D-2284 | {sGalNAc3K2AhxC6}[invAb]AfuGfcCfuUfuCfUfAfCfaguggcususu | 1697 | {磷酸酯}asGfscCfaCfUfguagAfaAfgGfcAfusUfsu | 1698 |
D-2285 | {sGalNAc3K2AhxC6}[invAb]UfcAfuGfcCfuUfUfCfUfacaguggscsu | 1699 | {磷酸酯}asGfscCfaCfuGfUfagaaAfgGfcAfuGfasUfsu | 1700 |
D-2286 | {sGalNAc3K2AhxC6}[invAb]ugcuucAfuGfCfCfUfuucuacasgsu | 1701 | {磷酸酯}asCfsuGfuAfgAfAfaggcAfuGfaagcasusu | 1702 |
D-2287 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuAfcAfgUfsusUf | 1703 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1704 |
D-2288 | {sGalNAc3K2AhxC6}UfgCfuUfcAfuGfcCfUfuuCfuAfcAfgs{invAb} | 1705 | {磷酸酯}asCfsuGfuAfgAfAfagGfcAfuGfaAfgCfasUfsu | 1706 |
D-2289 | {sGalNAc3K2AhxC6}[invAb]CfuUfcAfuGfcCfUfUfUfcuacagususu | 1707 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfuGfaAfgsUfsu | 1708 |
D-2290 | {sGalNAc3K2AhxC6}UfgCfuUfcAfuGfCfCfUfuucuacags{invAb} | 1709 | {磷酸酯}asCfsuGfuAfgAfAfaggcAfuGfaAfgCfasUfsu | 1710 |
D-2291 | {sGalNAc3K2AhxC6}[invAb]UfgCfuUfcAfuGfCfCfUfuucuacasgsu | 1711 | {磷酸酯}asCfsuGfuAfgAfAfaggcAfuGfaAfgCfasUfsu | 1712 |
D-2292 | {sGalNAc3K2AhxC6}[invAb]ugcuucauGfcCfUfUfUfcuacasgsu | 1713 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagcasusu | 1714 |
D-2293 | {sGalNAc3K2AhxC6}[invAb]ugcuucauGfcCfUfUfUfcuAfcAfgsUf | 1715 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagcasusu | 1716 |
D-2294 | {sGalNAc3K2AhxC6}[invAb]UfgCfuUfcAfuGfcCfUfUfUfcuacasgsu | 1717 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfuGfaAfgCfasUfsu | 1718 |
D-2295 | {sGalNAc3K2AhxC6}[invAb]guuccuGfcUfUfCfAfugccuuususu | 1719 | {磷酸酯}asAfsaGfgCfAfugaaGfcAfggaacsusu | 1720 |
D-2296 | {sGalNAc3K2AhxC6}[invAb]ccugcuUfcAfUfGfCfcuuucuasusu | 1721 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgcaggsusu | 1722 |
D-2297 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuacagususu | 1723 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1724 |
D-2298 | {sGalNAc3K2AhxC6}[invAb]UfuCfcUfgCfuUfcAfuGfCfcuUfuCfsusAf | 1725 | {磷酸酯}usAfsgAfaAfGfgcAfuGfaAfgCfaGfgAfasUfsu | 1726 |
D-2299 | {sGalNAc3K2AhxC6}uuccugCfuUfCfAfUfgccuuucus{invAb} | 1727 | {磷酸酯}usAfsgAfaAfgGfCfaugaAfgCfaggaasusu | 1728 |
D-2300 | {sGalNAc3K2AhxC6}[invAb]uuccugCfuUfCfAfUfgccuuucsusa | 1729 | {磷酸酯}usAfsgAfaAfgGfCfaugaAfgCfaggaasusu | 1730 |
D-2301 | {sGalNAc3K2AhxC6}uuccugCfuUfCfAfUfgcCfuUfuCfus{invAb} | 1731 | {磷酸酯}usAfsgAfaAfgGfCfaugaAfgCfaggaasusu | 1732 |
D-2302 | {sGalNAc3K2AhxC6}[invAb]uuccugCfuUfCfAfUfgcCfuUfuCfsusAf | 1733 | {磷酸酯}usAfsgAfaAfgGfCfaugaAfgCfaggaasusu | 1734 |
D-2303 | {sGalNAc3K2AhxC6}[invAb]UfuCfcUfgCfuUfcAfUfgcCfuUfuCfsusAf | 1735 | {磷酸酯}usAfsgAfaAfgGfCfauGfaAfgCfaGfgAfasUfsu | 1736 |
D-2304 | {sGalNAc3K2AhxC6}[invAb]UfuCfcUfgCfuUfCfAfUfgccuuucsusa | 1737 | {磷酸酯}usAfsgAfaAfgGfCfaugaAfgCfaGfgAfasUfsu | 1738 |
D-2305 | {sGalNAc3K2AhxC6}[invAb]uuccugcuUfcAfUfGfCfcuuucsusa | 1739 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgcaggaasusu | 1740 |
D-2306 | {sGalNAc3K2AhxC6}[invAb]uuccugcuUfcAfUfGfCfcuUfuCfusAf | 1741 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgcaggaasusu | 1742 |
D-2307 | {sGalNAc3K2AhxC6}[invAb]UfuCfcUfgCfuUfcAfUfGfCfcuuucsusa | 1743 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgCfaGfgAfasUfsu | 1744 |
D-2308 | {sGalNAc3K2AhxC6}[invAb]UfcAfuGfcCfuUfuCfuAfCfagUfgGfscsUf | 1745 | {磷酸酯}asGfscCfaCfUfguAfgAfaAfgGfcAfuGfasUfsu | 1746 |
D-2309 | {sGalNAc3K2AhxC6}ucaugcCfuUfUfCfUfacaguggcs{invAb} | 1747 | {磷酸酯}asGfscCfaCfuGfUfagaaAfgGfcaugasusu | 1748 |
D-2310 | {sGalNAc3K2AhxC6}[invAb]ucaugcCfuUfUfCfUfacaguggscsu | 1749 | {磷酸酯}asGfscCfaCfuGfUfagaaAfgGfcaugasusu | 1750 |
D-2311 | {sGalNAc3K2AhxC6}ucaugcCfuUfUfCfUfacAfgUfgGfcs{invAb} | 1751 | {磷酸酯}asGfscCfaCfuGfUfagaaAfgGfcaugasusu | 1752 |
D-2312 | {sGalNAc3K2AhxC6}[invAb]ucaugcCfuUfUfCfUfacAfgUfgGfscsUf | 1753 | {磷酸酯}asGfscCfaCfuGfUfagaaAfgGfcaugasusu | 1754 |
D-2313 | {sGalNAc3K2AhxC6}[invAb]UfcAfuGfcCfuUfuCfUfacAfgUfgGfscsUf | 1755 | {磷酸酯}asGfscCfaCfuGfUfagAfaAfgGfcAfuGfasUfsu | 1756 |
D-2314 | {sGalNAc3K2AhxC6}UfcAfuGfcCfuUfUfCfUfacaguggcs{invAb} | 1757 | {磷酸酯}asGfscCfaCfuGfUfagaaAfgGfcAfuGfasUfsu | 1758 |
D-2315 | {sGalNAc3K2AhxC6}[invAb]ucaugccuUfuCfUfAfCfaguggscsu | 1759 | {磷酸酯}asGfscCfaCfUfguagAfaAfggcaugasusu | 1760 |
D-2316 | {sGalNAc3K2AhxC6}[invAb]ucaugccuUfuCfUfAfCfagUfgGfcsUf | 1761 | {磷酸酯}asGfscCfaCfUfguagAfaAfggcaugasusu | 1762 |
D-2317 | {sGalNAc3K2AhxC6}[invAb]UfcAfuGfcCfuUfuCfUfAfCfaguggscsu | 1763 | {磷酸酯}asGfscCfaCfUfguagAfaAfgGfcAfuGfasUfsu | 1764 |
D-2318 | {sGalNAc3K2AhxC6}[invAb]UfgCfuUfcAfuGfcCfuUfUfcuAfcAfsgsUf | 1765 | {磷酸酯}asCfsuGfuAfGfaaAfgGfcAfuGfaAfgCfasUfsu | 1766 |
D-2319 | {sGalNAc3K2AhxC6}ugcuucAfuGfCfCfUfuucuacags{invAb} | 1767 | {磷酸酯}asCfsuGfuAfgAfAfaggcAfuGfaagcasusu | 1768 |
D-2320 | {sGalNAc3K2AhxC6}ugcuucAfuGfCfCfUfuuCfuAfcAfgs{invAb} | 1769 | {磷酸酯}asCfsuGfuAfgAfAfaggcAfuGfaagcasusu | 1770 |
D-2321 | {sGalNAc3K2AhxC6}[invAb]ugcuucAfuGfCfCfUfuuCfuAfcAfsgsUf | 1771 | {磷酸酯}asCfsuGfuAfgAfAfaggcAfuGfaagcasusu | 1772 |
D-2322 | {sGalNAc3K2AhxC6}[invAb]CfuUfcAfuGfcCfuUfUfcuAfcAfgUfsusUf | 1773 | {磷酸酯}asCfsuGfuAfGfaaAfgGfcAfuGfaAfgsUfsu | 1774 |
D-2323 | {sGalNAc3K2AhxC6}[invAb]UfgCfuUfcAfuGfcCfUfuuCfuAfcAfsgsUf | 1775 | {磷酸酯}asCfsuGfuAfgAfAfagGfcAfuGfaAfgCfasUfsu | 1776 |
D-2324 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacaususu | 1777 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcsusu | 1778 |
D-2325 | {sGalNAc3K2AhxC6}[invAb]caugccUfuUfCfUfAfcaguggususu | 1779 | {磷酸酯}asCfscAfcUfGfuagaAfaGfgcaugsusu | 1780 |
D-2326 | {sGalNAc3K2AhxC6}[invAb]cugcuuCfaUfGfCfCfuuucuaasusu | 1781 | {磷酸酯}usUfsaGfaAfAfggcaUfgAfagcagsusu | 1782 |
D-2327 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacaasusu | 1783 | {磷酸酯}usUfsgUfaGfAfaaggCfaUfgaagcsusu | 1784 |
D-2328 | {sGalNAc3K2AhxC6}[invAb]uucaugCfcUfUfUfCfuacaguasusu | 1785 | {磷酸酯}usAfscUfgUfAfgaaaGfgCfaugaasusu | 1786 |
D-2329 | {sGalNAc3K2AhxC6}[invAb]guuccuGfcUfUfCfAfugCfcUfuUfsusUf | 1787 | {磷酸酯}asAfsaGfgCfAfugaaGfcAfggaacsusu | 1788 |
D-2330 | {sGalNAc3K2AhxC6}auguucCfuGfCfUfUfcaUfgCfcUfus{invAb} | 1789 | {磷酸酯}asAfsaGfgCfaUfGfaagcAfgGfaacaususu | 1790 |
D-2331 | {sGalNAc3K2AhxC6}[invAb]auguucCfuGfCfUfUfcaUfgCfcUfsusUf | 1791 | {磷酸酯}asAfsaGfgCfaUfGfaagcAfgGfaacaususu | 1792 |
D-2332 | {sGalNAc3K2AhxC6}[invAb]GfuUfcCfuGfcUfuCfAfugCfcUfuUfsusUf | 1793 | {磷酸酯}asAfsaGfgCfAfugAfaGfcAfgGfaAfcsUfsu | 1794 |
D-2333 | {sGalNAc3K2AhxC6}AfuGfuUfcCfuGfcUfUfcaUfgCfcUfus{invAb} | 1795 | {磷酸酯}asAfsaGfgCfaUfGfaaGfcAfgGfaAfcAfusUfsu | 1796 |
D-2334 | {sGalNAc3K2AhxC6}[invAb]AfuGfuUfcCfuGfcUfUfcaUfgCfcUfsusUf | 1797 | {磷酸酯}asAfsaGfgCfaUfGfaaGfcAfgGfaAfcAfusUfsu | 1798 |
D-2335 | {sGalNAc3K2AhxC6}[invAb]GfuUfcCfuGfcUfUfCfAfugccuuususu | 1799 | {磷酸酯}asAfsaGfgCfAfugaaGfcAfgGfaAfcsUfsu | 1800 |
D-2336 | {sGalNAc3K2AhxC6}AfuGfuUfcCfuGfCfUfUfcaugccuus{invAb} | 1801 | {磷酸酯}asAfsaGfgCfaUfGfaagcAfgGfaAfcAfusUfsu | 1802 |
D-2337 | {sGalNAc3K2AhxC6}[invAb]AfuGfuUfcCfuGfCfUfUfcaugccususu | 1803 | {磷酸酯}asAfsaGfgCfaUfGfaagcAfgGfaAfcAfusUfsu | 1804 |
D-2338 | {sGalNAc3K2AhxC6}[invAb]auguuccuGfcUfUfCfAfugccususu | 1805 | {磷酸酯}asAfsaGfgCfAfugaaGfcAfggaacaususu | 1806 |
D-2339 | {sGalNAc3K2AhxC6}[invAb]auguuccuGfcUfUfCfAfugCfcUfusUf | 1807 | {磷酸酯}asAfsaGfgCfAfugaaGfcAfggaacaususu | 1808 |
D-2340 | {sGalNAc3K2AhxC6}[invAb]GfcUfuCfaUfgCfcUfuUfCfuaCfaGfsusAf | 1809 | {磷酸酯}usAfscUfgUfAfgaAfaGfgCfaUfgAfaGfcsUfsu | 1810 |
D-2341 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacagsusa | 1811 | {磷酸酯}usAfscUfgUfaGfAfaaggCfaUfgaagcsusu | 1812 |
D-2342 | {sGalNAc3K2AhxC6}[invAb]uucaugCfcUfUfUfCfuaCfaGfuAfsusUf | 1813 | {磷酸酯}usAfscUfgUfAfgaaaGfgCfaugaasusu | 1814 |
D-2343 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucUfaCfaGfsusAf | 1815 | {磷酸酯}usAfscUfgUfaGfAfaaggCfaUfgaagcsusu | 1816 |
D-2344 | {sGalNAc3K2AhxC6}GfcUfuCfaUfgCfCfUfUfucuacagus{invAb} | 1817 | {磷酸酯}usAfscUfgUfaGfAfaaggCfaUfgAfaGfcsUfsu | 1818 |
D-2345 | {sGalNAc3K2AhxC6}[invAb]GfcGfgCfuUfcCfUfGfGfgcuucuasusu | 1819 | {磷酸酯}usAfsgAfaGfCfccagGfaAfgCfcGfcsUfsu | 1820 |
D-2346 | {sGalNAc3K2AhxC6}[invAb]auggcuUfcCfAfGfAfuaugccususu | 1821 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgccaususu | 1822 |
D-2347 | {sGalNAc3K2AhxC6}[invAb]acauggCfuUfCfCfAfgaUfaUfgCfscsUf | 1823 | {磷酸酯}asGfsgCfaUfaUfCfuggaAfgCfcaugususu | 1824 |
D-2348 | {sGalNAc3K2AhxC6}[invAb]AfcAfuGfgCfuUfcCfAfGfAfuaugcscsu | 1825 | {磷酸酯}asGfsgCfaUfAfucugGfaAfgCfcAfuGfusUfsu | 1826 |
D-2349 | {sGalNAc3K2AhxC6}[invAb]caacguAfcCfCfUfUfcauugausgsu | 1827 | {磷酸酯}asCfsaUfcAfaUfGfaaggGfuAfcguugsusu | 1828 |
D-2350 | {sGalNAc3K2AhxC6}[invAb]caacguacCfcUfUfCfAfuuGfaUfgsUf | 1829 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuacguugsusu | 1830 |
D-2351 | {sGalNAc3K2AhxC6}[invAb]acguacCfcUfUfCfAfuugaugususu | 1831 | {磷酸酯}asCfsaUfcAfAfugaaGfgGfuacgususu | 1832 |
D-2352 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1833 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1834 |
D-2353 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasu | 1835 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1836 |
D-2354 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucUfaCfaUfsusUf | 1837 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcsusu | 1838 |
D-2355 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucUfaCfas{invAb} | 1839 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1840 |
D-2356 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucUfaCfasUf | 1841 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1842 |
D-2357 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1843 | {磷酸酯}asUfsguagAfaaggCfaUfgaagcagsusu | 1844 |
D-2358 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacas{invAb} | 1845 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1846 |
D-2359 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacaususu | 1847 | {磷酸酯}asUfsgUfaGfAfaaGfgCfaUfgaagcsusu | 1848 |
D-2360 | {sGalNAc3K2AhxC6}[invAb]CfuUfcAfuGfcCfUfUfUfcuacagususu | 1849 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1850 |
D-2361 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 1851 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagcasusu | 1852 |
D-2362 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasu | 1853 | {磷酸酯}asUfsgUfaGfAfaaGfgCfaUfgaagcagsusu | 1854 |
D-2363 | {sGalNAc3K2AhxC6}cugcuucaUfgCfcUfUfucuacas{invAb} | 1855 | {磷酸酯}asUfsgUfaGfAfaaggcaUfgaagcagsusu | 1856 |
D-2364 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1857 | {磷酸酯}asUfsgUfaGfAfaaGfgCfaUfgaagcagsusu | 1858 |
D-2365 | {sGalNAc3K2AhxC6}[invAb]cuucauCfcCfUfUfUfcuacagususu | 1859 | {磷酸酯}asCfsuGfuAfGfaaagGfgAfugaagsusu | 1860 |
D-2366 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfcCfCfUfUfucuacaususu | 1861 | {磷酸酯}asUfsgUfaGfAfaaggGfaUfgaagcsusu | 1862 |
D-2367 | {sGalNAc3K2AhxC6}ugcuucauCfcCfUfUfUfcuacags{invAb} | 1863 | {磷酸酯}asCfsuGfuAfGfaaagGfgAfugaagcasusu | 1864 |
D-2368 | {sGalNAc3K2AhxC6}cugcuucaUfcCfCfUfUfucuacas{invAb} | 1865 | {磷酸酯}asUfsgUfaGfAfaaggGfaUfgaagcagsusu | 1866 |
D-2369 | {sGalNAc3K2AhxC6}[invAb]acauugCfuCfUfUfUfcaccugasusu | 1867 | {磷酸酯}usCfsaGfgUfGfaaagAfgCfaaugususu | 1868 |
D-2370 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1869 | asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1870 |
D-2371 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1871 | asUfsgUfaGfAfaaggCfaUfgaagcasgsusu | 1872 |
D-2372 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1873 | asUfsgsUfaGfAfaaggCfaUfgaagcagsusu | 1874 |
D-2373 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1875 | asUfsgUfaGfAfaaggCfaUfgaagcagusu | 1876 |
D-2374 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1877 | asUfsgsUfaGfAfaaggCfaUfgaagcagusu | 1878 |
D-2375 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1879 | asUfgUfaGfAfaaggCfaUfgaagcagsusu | 1880 |
D-2376 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1881 | asUfgUfaGfAfaaggCfaUfgaagcasgsusu | 1882 |
D-2377 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacsas{invAb} | 1883 | asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1884 |
D-2378 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuascsas{invAb} | 1885 | asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1886 |
D-2379 | {sGalNAc3K2AhxC6}csugcuucaUfgCfCfUfUfucuacas{invAb} | 1887 | asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1888 |
D-2380 | {sGalNAc3K2AhxC6}csusgcuucaUfgCfCfUfUfucuacas{invAb} | 1889 | asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1890 |
D-2381 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCf[dT]UfUfcuacagususu | 1891 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1892 |
D-2382 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuacagususu | 1893 | {磷酸酯}asCfsuGfuAfGfaaAfgGfcAfugaagsusu | 1894 |
D-2383 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfuUfUfcuacagususu | 1895 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1896 |
D-2384 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuacagususu | 1897 | {磷酸酯}asCfsuGfuaGfaaagGfcAfugaagsusu | 1898 |
D-2385 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuacagususu | 1899 | {磷酸酯}asCfsuguaGfaaagGfcAfugaagsusu | 1900 |
D-2386 | {sGalNAc3K2AhxC6}[invAb]cuucauGfc[dC]UfUfUfcuacagususu | 1901 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1902 |
D-2387 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUf[dT]Ufcuacagususu | 1903 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1904 |
D-2388 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgGfGfAfUfucuacaususu | 1905 | {磷酸酯}asUfsgUfaGfAfauccCfaUfgaagcsusu | 1906 |
D-2389 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcGfAfAfUfcuacagususu | 1907 | {磷酸酯}asCfsuGfuAfGfauucGfcAfugaagsusu | 1908 |
D-2390 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1909 | {磷酸酯}usUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1910 |
D-2391 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasa | 1911 | {磷酸酯}usUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1912 |
D-2392 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invDA} | 1913 | {磷酸酯}usUfsgUfaGfAfaaggCfaUfgaagcagsusu | 1914 |
D-2393 | {sGalNAc3K2AhxC6}cugcuucaUfgGfGfAfUfucuacas{invAb} | 1915 | {磷酸酯}asUfsgUfaGfAfauccCfaUfgaagcagsusu | 1916 |
D-2394 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgGfGfAfUfucuacsasu | 1917 | {磷酸酯}asUfsgUfaGfAfauccCfaUfgaagcagsusu | 1918 |
D-2395 | {sGalNAc3K2AhxC6}[invAb]CfuUfcAfuGfcCfUfUfUfcuAfcAfgUfsusUf | 1919 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfugaagsusu | 1920 |
D-2396 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuAfcAfgUfsusUf | 1921 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfuGfaAfgsUfsu | 1922 |
D-2397 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgCfCfUfUfucuacaususu | 1923 | asUfsgUfaGfAfaaggCfaUfgaagcsusu | 1924 |
D-2398 | {sGalNAc3K2AhxC6}[invAb]ugcuucauGfcCfUfUfUfcuacags{invAb} | 1925 | asCfsuGfuAfGfaaagGfcAfugaagcasusu | 1926 |
D-2399 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuacagususu | 1927 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfuGfaAfgsUfsu | 1928 |
D-2400 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuAfcAfgs{invAb} | 1929 | asCfsuGfuAfGfaaagGfcAfugaagcasusu | 1930 |
D-2401 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 1931 | asCfsuGfuAfGfaaAfgGfcAfugaagcasusu | 1932 |
D-2402 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 1933 | asCfsuguaGfaaagGfcAfugaagcasusu | 1934 |
D-2403 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfUfUfUfcuacagususu | 1935 | asCfsuGfuAfGfaaagGfcAfugaagsusu | 1936 |
D-2404 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 1937 | asCfsuGfuAfGfaaagGfcAfugaagcasusu | 1938 |
D-2405 | {GalNAc3K2AhxC6}augccuuuCfuAfCfAfGfuggcusus{invAb} | 1939 | {磷酸酯}asGfscCfaCfUfguAfgAfaAfgGfcAfuGfasUfsu | 1940 |
D-2406 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1941 | asGfscCfaCfUfguagAfaAfggcaugasusu | 1942 |
D-2407 | {sGalNAc3K2AhxC6}[invAb]augccuUfuCfUfAfCfaguggcususu | 1943 | asGfscCfaCfUfguagAfaAfggcaususu | 1944 |
D-2408 | {sGalNAc3K2AhxC6}[invAb]augccuUfuCfUfAfCfagUfgGfcUfsusUf | 1945 | asGfscCfaCfUfguagAfaAfggcaususu | 1946 |
D-2409 | {sGalNAc3K2AhxC6}[invAb]ucaugccuUfuCfUfAfCfaguggscsu | 1947 | asGfscCfaCfUfguagAfaAfggcaugasusu | 1948 |
D-2410 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfagUfgGfcs{invAb} | 1949 | asGfscCfaCfUfguagAfaAfggcaugasusu | 1950 |
D-2411 | {sGalNAc3K2AhxC6}[invAb]augccuUfuCfUfAfCfaguggcususu | 1951 | asGfscCfaCfUfguAfgAfaAfggcaususu | 1952 |
D-2412 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1953 | asGfscCfaCfUfguAfgAfaAfggcaugasusu | 1954 |
D-2413 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1955 | asGfsccacUfguagAfaAfggcaugasusu | 1956 |
D-2414 | {sGalNAc3K2AhxC6}[invAb]ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1957 | asGfscCfaCfUfguagAfaAfggcaugasusu | 1958 |
D-2415 | {sGalNAc3K2AhxC6}[invAb]augccuUfuCfUfAfCfaguggcasusu | 1959 | usGfscCfaCfUfguagAfaAfggcaususu | 1960 |
D-2416 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1961 | usGfscCfaCfUfguagAfaAfggcaugasusu | 1962 |
D-2417 | {sGalNAc3K2AhxC6}[invAb]ucaugccuUfuCfUfAfCfaguggscsa | 1963 | usGfscCfaCfUfguagAfaAfggcaugasusu | 1964 |
D-2418 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invDA} | 1965 | usGfscCfaCfUfguagAfaAfggcaugasusu | 1966 |
D-2419 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1967 | asUfsguagAfaaggCfaUfgaagcagsusu | 1968 |
D-2420 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1969 | usUfsguagAfaaggCfaUfgaagcagsusu | 1970 |
D-2421 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invDA} | 1971 | usUfsguagAfaaggCfaUfgaagcagsusu | 1972 |
D-2422 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1973 | usGfsccacUfguagAfaAfggcaugasusu | 1974 |
D-2423 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invDA} | 1975 | usGfsccacUfguagAfaAfggcaugasusu | 1976 |
D-2424 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcCfuUfUfcuacagususu | 1977 | {磷酸酯}asCfsuGfuAfGfaaAfgGfcAfugaagsusu | 1978 |
D-2425 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1979 | asUfsgUfa[Ab]AfaaggCfaUfgaagcagsusu | 1980 |
D-2426 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1981 | asUfsgua[Ab]AfaaggCfaUfgaagcagsusu | 1982 |
D-2427 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 1983 | asCfsuGfu[Ab]GfaaagGfcAfugaagcasusu | 1984 |
D-2428 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 1985 | asCfsugu[Ab]GfaaagGfcAfugaagcasusu | 1986 |
D-2429 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1987 | asGfscCfa[Ab]UfguagAfaAfggcaugasusu | 1988 |
D-2430 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 1989 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 1990 |
D-2431 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1991 | asUfs[GNA-G]uagAfaaggCfaUfgaagcagsusu | 1992 |
D-2432 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1993 | asUfsg[GNA-U]agAfaaggCfaUfgaagcagsusu | 1994 |
D-2433 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1995 | asUfsgu[GNA-A]gAfaaggCfaUfgaagcagsusu | 1996 |
D-2434 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1997 | asUfsgua[GNA-G]AfaaggCfaUfgaagcagsusu | 1998 |
D-2435 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 1999 | asUfsguag[GNA-A]aaggCfaUfgaagcagsusu | 2000 |
D-2436 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2001 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2002 |
D-2437 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2003 | asUfsgUfagAfaaggCfaUfgaagcagsusu | 2004 |
D-2438 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasu | 2005 | asUfsguagAfaaggCfaUfgaagcagsusu | 2006 |
D-2439 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacas{invAb} | 2007 | asUfsguagAfaaggCfaUfgaagcagsusu | 2008 |
D-2440 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2009 | asUfsguagAfaaGfgCfaUfgaagcagsusu | 2010 |
D-2441 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucUfaCfas{invAb} | 2011 | asUfsguagAfaaggCfaUfgaagcagsusu | 2012 |
D-2442 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucUfaCfasUf | 2013 | asUfsguagAfaaggCfaUfgaagcagsusu | 2014 |
D-2443 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucUfaCfas{invAb} | 2015 | asUfsguagAfaaGfgCfaUfgaagcagsusu | 2016 |
D-2444 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2017 | asUfsgUfaGfAfaaGfgCfaUfgaagcagsusu | 2018 |
D-2445 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacsasu | 2019 | asUfsgUfaGfAfaaGfgCfaUfgaagcagsusu | 2020 |
D-2446 | {sGalNAc3K2AhxC6}[invAb]cugcuucaUfgCfCfUfUfucuacas{invAb} | 2021 | asUfsgUfaGfAfaaggCfaUfgaagcagsusu | 2022 |
D-2447 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2023 | as[Ab]guagAfaaggCfaUfgaagcagsusu | 2024 |
D-2448 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2025 | asUfs[Ab]uagAfaaggCfaUfgaagcagsusu | 2026 |
D-2449 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2027 | asUfsg[Ab]agAfaaggCfaUfgaagcagsusu | 2028 |
D-2450 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2029 | asUfsgu[Ab]gAfaaggCfaUfgaagcagsusu | 2030 |
D-2451 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2031 | asUfsguag[Ab]aaggCfaUfgaagcagsusu | 2032 |
D-2452 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2033 | asUfsguagAf[Ab]aggCfaUfgaagcagsusu | 2034 |
D-2453 | {sGalNAc3K2AhxC6}caacguacCfcUfUfCfAfuugaugs{invAb} | 2035 | asCfsaucaAfugaaGfgGfuacguugsusu | 2036 |
D-2454 | {sGalNAc3K2AhxC6}caacguacCfcUfUfCfAfuugaugs{invAb} | 2037 | asCfsaUfcAfAfugaaGfgGfuacguugsusu | 2038 |
D-2455 | {sGalNAc3K2AhxC6}acauggcuUfcCfAfGfAfuaugccs{invAb} | 2039 | asGfsgcauAfucugGfaAfgccaugususu | 2040 |
D-2456 | {sGalNAc3K2AhxC6}acauggcuUfcCfAfGfAfuaugccs{invAb} | 2041 | asGfsgCfaUfAfucugGfaAfgccaugususu | 2042 |
D-2457 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2043 | asUfsguaGfAfaaggCfaUfgaagcagsusu | 2044 |
D-2458 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucUfacas{invAb} | 2045 | asUfsguagAfaaggCfaUfgaagcagsusu | 2046 |
D-2459 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuaCfas{invAb} | 2047 | asUfsguagAfaaggCfaUfgaagcagsusu | 2048 |
D-2460 | {sGalNAc3K2AhxC6}cugcggcuUfcCfUfGfGfgcuucus{invAb} | 2049 | usAfsgAfaGfCfccagGfaAfgccgcagsusu | 2050 |
D-2461 | {sGalNAc3K2AhxC6}cugcggcuUfcCfUfGfGfgcuucus{invAb} | 2051 | usAfsgaagCfccagGfaAfgccgcagsusu | 2052 |
D-2462 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 2053 | asCfsuGfuaGfaaagGfcAfugaagcasusu | 2054 |
D-2463 | {sGalNAc3K2AhxC6}[invAb]ugcuucauGfcCfUfUfUfcuacasgsu | 2055 | asCfsuGfuaGfaaagGfcAfugaagcasusu | 2056 |
D-2464 | {sGalNAc3K2AhxC6}[invAb]ugcuucauGfcCfUfUfUfcuacags{invAb} | 2057 | asCfsuGfuaGfaaagGfcAfugaagcasusu | 2058 |
D-2465 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 2059 | asCfsuGfuaGfaaAfgGfcAfugaagcasusu | 2060 |
D-2466 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuAfcAfgs{invAb} | 2061 | asCfsuGfuaGfaaagGfcAfugaagcasusu | 2062 |
D-2467 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuAfcAfgs{invAb} | 2063 | asCfsuGfuAfGfaaagGfcAfuGfaAfgCfasUfsu | 2064 |
D-2468 | {sGalNAc3K2AhxC6}cugcuuCfaUfGfCfcuuucuacsasu | 2065 | asUfsguaGfaaAfggcaUfgAfagcagsusu | 2066 |
D-2469 | {sGalNAc3K2AhxC6}cugcuuCfaUfGfCfcuuucuacsasu | 2067 | asUfsguaGfaaaggcaUfgAfagcagsusu | 2068 |
D-2470 | {sGalNAc3K2AhxC6}cugcuuCfaUfGfCfcuuucuacsasu | 2069 | asUfsguaGfaAfAfggcaUfgAfagcagsusu | 2070 |
D-2471 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 2071 | usCfsuguaGfaaagGfcAfugaagcasusu | 2072 |
D-2472 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invDA} | 2073 | usCfsuguaGfaaagGfcAfugaagcasusu | 2074 |
D-2473 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invDT} | 2075 | asUfsguagAfaaggCfaUfgaagcagsusu | 2076 |
D-2474 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2077 | asUfsguagAf[sGNA-A]aggCfaUfgaagcagsusu | 2078 |
D-2475 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2079 | asUfsguagAfs[GNA-A]aggCfaUfgaagcagsusu | 2080 |
D-2476 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2081 | asUfsguagAfs[sGNA-A]aggCfaUfgaagcagsusu | 2082 |
D-2477 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2083 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2084 |
D-2478 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2085 | asUfsguagAf[GNA-A]AfggCfaUfgaagcagsusu | 2086 |
D-2479 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2087 | asUfsguaga[GNA-A]aggCfaUfgaagcagsusu | 2088 |
D-2480 | csusgcuucaUfgCfCfUfuucuacas{invAb} | 2089 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2090 |
D-2481 | csusgcuucaUfgCfCfUfuucuacas{invAb} | 2091 | asUfsguaga[GNA-A]aggCfaUfgaagcagsusu | 2092 |
D-2482 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2093 | asUfsguaga[GNA-A]AfggCfaUfgaagcagsusu | 2094 |
D-2483 | csusgcuucaUfgCfCfUfuucuacas{invAb} | 2095 | asUfsguagAf[GNA-A]AfggCfaUfgaagcagsusu | 2096 |
D-2484 | csusgcuucaUfgCfCfUfuucuacas{invAb} | 2097 | asUfsguaga[GNA-A]AfggCfaUfgaagcagsusu | 2098 |
D-2485 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2099 | asUfsgUfaGfAf[GNA-A]aggCfaUfgaagcagsusu | 2100 |
D-2486 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2101 | asUfsgUfagAf[GNA-A]aGfgCfaUfgaagcagsusu | 2102 |
D-2487 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2103 | asUfsgUfagAf[GNA-A]aggCfaUfgaagcagsusu | 2104 |
D-2488 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2105 | asUfsgUfaGfAf[GNA-A]aGfgCfaUfgaagcagsusu | 2106 |
D-2489 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2107 | asUfsguagAf[GNA-A]aGfgCfaUfgaagcagsusu | 2108 |
D-2490 | csusgcuucaUfgCfCfUfUfucUfaCfas{invAb} | 2109 | asUfsguagAf[GNA-A]aGfgCfaUfgaagcagsusu | 2110 |
D-2491 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2111 | asUfsguagAf[GNA-A]a[dG]gCfaUfgaagcagsusu | 2112 |
D-2492 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2113 | asUfsguagAf[GNA-A][dA]ggCfaUfgaagcagsusu | 2114 |
D-2493 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2115 | asUfsguagAf[GNA-A]ag[dG]CfaUfgaagcagsusu | 2116 |
D-2494 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2117 | asUfsguaGfAf[GNA-A]aggCfaUfgaagcagsusu | 2118 |
D-2495 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2119 | asUfsguaGfa[GNA-A]aggCfaUfgaagcagsusu | 2120 |
D-2496 | csusgcuucaUfgCfCfUfUfucuacas{invAb} | 2121 | asUfsguaGfa[GNA-A]AfggCfaUfgaagcagsusu | 2122 |
D-2497 | csusgcuuCfaUfGfCfcuuucuacas{invAb} | 2123 | asUfsguaGfa[GNA-A]aggcaUfgAfagcagsusu | 2124 |
D-2498 | csusgcuuCfaUfGfCfcuuucuacas{invAb} | 2125 | asUfsguaGfa[GNA-A]AfggcaUfgAfagcagsusu | 2126 |
D-2499 | csusgcuuCfaUfgCfcuuucuacas{invAb} | 2127 | asUfsguaga[GNA-A]aggCfaUfgAfagcagsusu | 2128 |
D-2500 | csusgcuuCfaUfgCfcuuucuacas{invAb} | 2129 | asUfsguagaaaggCfaUfgAfagcagsusu | 2130 |
D-2501 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2131 | us[sGNA-C]uguaGfaaagGfcAfugaagcasusu | 2132 |
D-2502 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2133 | usCfs[GNA-U]guaGfaaagGfcAfugaagcasusu | 2134 |
D-2503 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2135 | usCfsug[GNA-U]aGfaaagGfcAfugaagcasusu | 2136 |
D-2504 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2137 | usCfsugu[GNA-A]GfaaagGfcAfugaagcasusu | 2138 |
D-2505 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2139 | usCfsuguaGf[GNA-A]aagGfcAfugaagcasusu | 2140 |
D-2506 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2141 | us[Ab]uguaGfaaagGfcAfugaagcasusu | 2142 |
D-2507 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2143 | usCfs[Ab]guaGfaaagGfcAfugaagcasusu | 2144 |
D-2508 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2145 | usCfsu[Ab]uaGfaaagGfcAfugaagcasusu | 2146 |
D-2509 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2147 | usCfsug[Ab]aGfaaagGfcAfugaagcasusu | 2148 |
D-2510 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2149 | usCfsugu[Ab]GfaaagGfcAfugaagcasusu | 2150 |
D-2511 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2151 | usCfsugua[Ab]aaagGfcAfugaagcasusu | 2152 |
D-2512 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2153 | usCfsuguaGf[Ab]aagGfcAfugaagcasusu | 2154 |
D-2513 | uscsaugccuUfuCfUfAfCfaguggcs{invDT} | 2155 | asGfsccacUfguagAfaAfggcaugasusu | 2156 |
D-2514 | uscsaugccuUfuCfUfAfCfaguggcs{invAb} | 2157 | usGfsccacUfguagAfaAfggcaugasusu | 2158 |
D-2515 | uscsaugccuUfuCfUfAfCfaguggcs{invDA} | 2159 | usGfsccacUfguagAfaAfggcaugasusu | 2160 |
D-2516 | uscscugcuuCfaUfGfCfCfuuucuas{invDT} | 2161 | asUfsagaaAfggcaUfgAfagcaggasusu | 2162 |
D-2517 | uscscugcuuCfaUfGfCfCfuuucuas{invAb} | 2163 | usUfsagaaAfggcaUfgAfagcaggasusu | 2164 |
D-2518 | uscscugcuuCfaUfGfCfCfuuucuas{invDA} | 2165 | usUfsagaaAfggcaUfgAfagcaggasusu | 2166 |
D-2519 | usasuguuccUfgCfUfUfCfaugccus{invDT} | 2167 | asAfsggcaUfgaagCfaGfgaacauasusu | 2168 |
D-2520 | usasuguuccUfgCfUfUfCfaugccus{invAb} | 2169 | usAfsggcaUfgaagCfaGfgaacauasusu | 2170 |
D-2521 | usasuguuccUfgCfUfUfCfaugccus{invDA} | 2171 | usAfsggcaUfgaagCfaGfgaacauasusu | 2172 |
D-2522 | {sGalNAc3K2AhxC6}uccugcuuCfaUfGfCfCfuuucuas{invAb} | 2173 | asUfsagaaAfggcaUfgAfagcaggasusu | 2174 |
D-2523 | {sGalNAc3K2AhxC6}uauguuccUfgCfUfUfCfaugccus{invAb} | 2175 | asAfsggcaUfgaagCfaGfgaacauasusu | 2176 |
D-2524 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invDT} | 2177 | asGfsccacUfguagAfaAfggcaugasusu | 2178 |
D-2525 | {sGalNAc3K2AhxC6}uccugcuuCfaUfGfCfCfuuucuas{invDT} | 2179 | asUfsagaaAfggcaUfgAfagcaggasusu | 2180 |
D-2526 | {sGalNAc3K2AhxC6}uccugcuuCfaUfGfCfCfuuucuas{invDA} | 2181 | usUfsagaaAfggcaUfgAfagcaggasusu | 2182 |
D-2527 | {sGalNAc3K2AhxC6}uauguuccUfgCfUfUfCfaugccus{invDA} | 2183 | usAfsggcaUfgaagCfaGfgaacauasusu | 2184 |
D-2528 | usgscuucauGfcCfUfUfUfcuacags{invDA} | 2185 | usCfsugua[GNA-G]aaagGfcAfugaagcasusu | 2186 |
D-2529 | cugcuucaUfgCfCfUfUfsucuacas{invAb} | 2187 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2188 |
D-2530 | cugcuucaUfgCfCfUfsUfucuacas{invAb} | 2189 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2190 |
D-2531 | cugcuucaUfgCfCfUfsUfsucuacas{invAb} | 2191 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2192 |
D-2532 | cugcuucaUfgCfCfUfsUfucuacas{invAb} | 2193 | asUfsguagAfs[GNA-A]aggCfaUfgaagcagsusu | 2194 |
D-2533 | cugcuucaUfgCfCfUfsUfsucuacas{invAb} | 2195 | asUfsguagAfs[GNA-A]aggCfaUfgaagcagsusu | 2196 |
D-2534 | cugcuucaUfgCfCfUfUfsucuacas{invAb} | 2197 | asUfsguagAf[sGNA-A]aggCfaUfgaagcagsusu | 2198 |
D-2535 | cugcuucaUfgCfCfUfsUfsucuacas{invAb} | 2199 | asUfsguagAf[sGNA-A]aggCfaUfgaagcagsusu | 2200 |
D-2536 | cugcuucaUfgCfCfUfUfsucuacas{invAb} | 2201 | asUfsguagAfs[sGNA-A]aggCfaUfgaagcagsusu | 2202 |
D-2537 | cugcuucaUfgCfCfUfsUfucuacas{invAb} | 2203 | asUfsguagAfs[sGNA-A]aggCfaUfgaagcagsusu | 2204 |
D-2538 | cugcuucaUfgCfCfUf[LNA-T]ucuacas{invAb} | 2205 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2206 |
D-2539 | cugcuucaUfgCfCf[LNA-T]Ufucuacas{invAb} | 2207 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2208 |
D-2540 | cugcuucaUfgCfCfUfUf[LNA-T]cuacas{invAb} | 2209 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2210 |
D-2541 | cugcuucaUfgCfCfUfUfuc[LNA-T]acas{invAb} | 2211 | asUfsgu[GNA-A]gAfaaggCfaUfgaagcagsusu | 2212 |
D-2542 | cugcuucaUfgCfCfUfUfucu[LNA-A]cas{invAb} | 2213 | asUfsg[GNA-U]agAfaaggCfaUfgaagcagsusu | 2214 |
D-2543 | cugcuucaUfgCfCfUfUfucuac[sLNA-A]{invAb} | 2215 | as[sGNA-U]guagAfaaggCfaUfgaagcagsusu | 2216 |
D-2544 | cugcuucaUfgCfCfUfUf[LNA-T]cuacas{invAb} | 2217 | asUfsguag[Ab]aaggCfaUfgaagcagsusu | 2218 |
D-2545 | cugcuucaUfgCfCfUfUfuc[LNA-T]acas{invAb} | 2219 | asUfsgu[Ab]gAfaaggCfaUfgaagcagsusu | 2220 |
D-2546 | cugcuucaUfgCfCfUfUfucu[LNA-A]cas{invAb} | 2221 | asUfsg[Ab]agAfaaggCfaUfgaagcagsusu | 2222 |
D-2547 | cugcuucaUfgCfCfUfUfucUfaCfas{invAb} | 2223 | asUfsgUfagAf[GNA-A]aggCfaUfgaagcagsusu | 2224 |
D-2548 | cugcuucaUfgCfCfUfUfucUfaCfas{invAb} | 2225 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2226 |
D-2549 | cugcuucaUfgCfCfUfs[LNA-T]ucuacas{invAb} | 2227 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2228 |
D-2550 | cugcuucaUfgCfCfUfs[sLNA-T]ucuacas{invAb} | 2229 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2230 |
D-2551 | cugcuucaUfgCfCfUf[LNA-T]ucuacas{invAb} | 2231 | asUfsguagAf[sGNA-A]aggCfaUfgaagcagsusu | 2232 |
D-2552 | cugcuucaUfgCfCfUf[LNA-T]ucuacas{invAb} | 2233 | asUfsguagAfs[GNA-A]aggCfaUfgaagcagsusu | 2234 |
D-2553 | usgscuucauGfcCfUfUfUfcu[LNA-A]cags{invDA} | 2235 | usCfsug[Ab]aGfaaagGfcAfugaagcasusu | 2236 |
D-2554 | usgscuucauGfcCfUfUfUfc[LNA-T]acags{invDA} | 2237 | usCfsugu[Ab]GfaaagGfcAfugaagcasusu | 2238 |
D-2555 | usgscuucauGfcCfUfUf[LNA-T]cuacags{invDA} | 2239 | usCfsuguaGf[Ab]aagGfcAfugaagcasusu | 2240 |
D-2556 | usgscuucauGfcCfUfUfUfcuac[LNA-A]gs{invDA} | 2241 | usCfs[GNA-U]guaGfaaagGfcAfugaagcasusu | 2242 |
D-2557 | usgscuucauGfcCfUfUfUfcu[LNA-A]cags{invDA} | 2243 | usCfsug[GNA-U]aGfaaagGfcAfugaagcasusu | 2244 |
D-2558 | usgscuucauGfcCfUfUfUfc[LNA-T]acags{invDA} | 2245 | usCfsugu[GNA-A]GfaaagGfcAfugaagcasusu | 2246 |
D-2559 | usgscuucauGfcCfUfUfUfcuaca[sLNA-G]{invDA} | 2247 | us[Ab]uguaGfaaagGfcAfugaagcasusu | 2248 |
D-2560 | usgscuucauGfcCfUfUfUfcuac[LNA-A]gs{invDA} | 2249 | usCfs[Ab]guaGfaaagGfcAfugaagcasusu | 2250 |
D-2561 | ucaugccuUfuCfUfAfCfa[LNA-G]uggcs{invAb} | 2251 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2252 |
D-2562 | ucaugccuUfuCfUfAfCfag[LNA-T]ggcs{invAb} | 2253 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2254 |
D-2563 | ucaugccuUfuCfUfAfCfasgsuggcs{invAb} | 2255 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2256 |
D-2564 | {GalNAc3K2AhxC6}GfgUfaUfgUfuCfCfUfGfcuUfcAfuUfsusUf | 2349 | {磷酸酯}asAfsuGfaAfGfcaggAfaCfaUfaCfcsUfsu | 2350 |
D-2565 | {GalNAc3K2AhxC6}GfgUfaUfgUfuCfCfUfGfcuUfcAfuAfsusUf | 2351 | {磷酸酯}usAfsuGfaAfGfcaggAfaCfaUfaCfcsUfsu | 2352 |
D-2566 | {GalNAc3K2AhxC6}GfuAfuGfuUfcCfUfGfCfuuCfaUfgUfsusUf | 2353 | {磷酸酯}asCfsaUfgAfAfgcagGfaAfcAfuAfcsUfsu | 2354 |
D-2567 | {GalNAc3K2AhxC6}UfaUfgUfuCfcUfGfCfUfucAfuGfcAfsusUf | 2355 | {磷酸酯}usGfscAfuGfAfagcaGfgAfaCfaUfasUfsu | 2356 |
D-2568 | {GalNAc3K2AhxC6}AfuGfuUfcCfuGfCfUfUfcaUfgCfcUfsusUf | 2357 | {磷酸酯}asGfsgCfaUfGfaagcAfgGfaAfcAfusUfsu | 2358 |
D-2569 | {GalNAc3K2AhxC6}UfgUfuCfcUfgCfUfUfCfauGfcCfuUfsusUf | 2359 | {磷酸酯}asAfsgGfcAfUfgaagCfaGfgAfaCfasUfsu | 2360 |
D-2570 | {GalNAc3K2AhxC6}GfuUfcCfuGfcUfUfCfAfugCfcUfuUfsusUf | 2361 | {磷酸酯}asAfsaGfgCfAfugaaGfcAfgGfaAfcsUfsu | 2362 |
D-2571 | {GalNAc3K2AhxC6}UfuCfcUfgCfuUfCfAfUfgcCfuUfuUfsusUf | 2363 | {磷酸酯}asAfsaAfgGfCfaugaAfgCfaGfgAfasUfsu | 2364 |
D-2572 | {GalNAc3K2AhxC6}UfuCfcUfgCfuUfCfAfUfgcCfuUfuAfsusUf | 2365 | {磷酸酯}usAfsaAfgGfCfaugaAfgCfaGfgAfasUfsu | 2366 |
D-2573 | {GalNAc3K2AhxC6}UfcCfuGfcUfuCfAfUfGfccUfuUfcUfsusUf | 2367 | {磷酸酯}asGfsaAfaGfGfcaugAfaGfcAfgGfasUfsu | 2368 |
D-2574 | {GalNAc3K2AhxC6}CfcUfgCfuUfcAfUfGfCfcuUfuCfuAfsusUf | 2369 | {磷酸酯}usAfsgAfaAfGfgcauGfaAfgCfaGfgsUfsu | 2370 |
D-2575 | {GalNAc3K2AhxC6}CfuGfcUfuCfaUfGfCfCfuuUfcUfaUfsusUf | 2371 | {磷酸酯}asUfsaGfaAfAfggcaUfgAfaGfcAfgsUfsu | 2372 |
D-2576 | {GalNAc3K2AhxC6}CfuGfcUfuCfaUfGfCfCfuuUfcUfaAfsusUf | 2373 | {磷酸酯}usUfsaGfaAfAfggcaUfgAfaGfcAfgsUfsu | 2374 |
D-2577 | {GalNAc3K2AhxC6}UfgCfuUfcAfuGfCfCfUfuuCfuAfcAfsusUf | 2375 | {磷酸酯}usGfsuAfgAfAfaggcAfuGfaAfgCfasUfsu | 2376 |
D-2578 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfUfUfucUfaCfaUfsusUf | 2377 | {磷酸酯}asUfsgUfaGfAfaaggCfaUfgAfaGfcsUfsu | 2378 |
D-2579 | {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCfUfUfucUfaCfaAfsusUf | 2379 | {磷酸酯}usUfsgUfaGfAfaaggCfaUfgAfaGfcsUfsu | 2380 |
D-2580 | {GalNAc3K2AhxC6}CfuUfcAfuGfcCfUfUfUfcuAfcAfgUfsusUf | 2381 | {磷酸酯}asCfsuGfuAfGfaaagGfcAfuGfaAfgsUfsu | 2382 |
D-2581 | {GalNAc3K2AhxC6}UfuCfaUfgCfcUfUfUfCfuaCfaGfuUfsusUf | 2383 | {磷酸酯}asAfscUfgUfAfgaaaGfgCfaUfgAfasUfsu | 2384 |
D-2582 | {GalNAc3K2AhxC6}UfuCfaUfgCfcUfUfUfCfuaCfaGfuAfsusUf | 2385 | {磷酸酯}usAfscUfgUfAfgaaaGfgCfaUfgAfasUfsu | 2386 |
D-2583 | {GalNAc3K2AhxC6}UfcAfuGfcCfuUfUfCfUfacAfgUfgUfsusUf | 2387 | {磷酸酯}asCfsaCfuGfUfagaaAfgGfcAfuGfasUfsu | 2388 |
D-2584 | {GalNAc3K2AhxC6}UfcAfuGfcCfuUfUfCfUfacAfgUfgAfsusUf | 2389 | {磷酸酯}usCfsaCfuGfUfagaaAfgGfcAfuGfasUfsu | 2390 |
D-2585 | {GalNAc3K2AhxC6}CfaUfgCfcUfuUfCfUfAfcaGfuGfgUfsusUf | 2391 | {磷酸酯}asCfscAfcUfGfuagaAfaGfgCfaUfgsUfsu | 2392 |
D-2586 | {GalNAc3K2AhxC6}CfaUfgCfcUfuUfCfUfAfcaGfuGfgAfsusUf | 2393 | {磷酸酯}usCfscAfcUfGfuagaAfaGfgCfaUfgsUfsu | 2394 |
D-2587 | {GalNAc3K2AhxC6}AfuGfcCfuUfuCfUfAfCfagUfgGfcUfsusUf | 2395 | {磷酸酯}asGfscCfaCfUfguagAfaAfgGfcAfusUfsu | 2396 |
D-2588 | {GalNAc3K2AhxC6}AfuGfcCfuUfuCfUfAfCfagUfgGfcAfsusUf | 2397 | {磷酸酯}usGfscCfaCfUfguagAfaAfgGfcAfusUfsu | 2398 |
D-2589 | {GalNAc3K2AhxC6}UfgCfcUfuUfcUfAfCfAfguGfgCfcUfsusUf | 2399 | {磷酸酯}asGfsgCfcAfCfuguaGfaAfaGfgCfasUfsu | 2400 |
D-2590 | {GalNAc3K2AhxC6}GfcCfuUfuCfuAfCfAfGfugGfcCfuUfsusUf | 2401 | {磷酸酯}asAfsgGfcCfAfcuguAfgAfaAfgGfcsUfsu | 2402 |
D-2591 | {GalNAc3K2AhxC6}GfgUfaUfgUfuCfCfUfGfcuUfcAfuCfsusUf | 2403 | {磷酸酯}gsAfsuGfaAfGfcaggAfaCfaUfaCfcsUfsu | 2404 |
D-2592 | {GalNAc3K2AhxC6}GfuAfuGfuUfcCfUfGfCfuuCfaUfcCfsusUf | 2405 | {磷酸酯}gsGfsaUfgAfAfgcagGfaAfcAfuAfcsUfsu | 2406 |
D-2593 | {GalNAc3K2AhxC6}UfaUfgUfuCfcUfGfCfUfucAfuCfcCfsusUf | 2407 | {磷酸酯}gsGfsgAfuGfAfagcaGfgAfaCfaUfasUfsu | 2408 |
D-2594 | {GalNAc3K2AhxC6}AfuGfuUfcCfuGfCfUfUfcaUfcCfcCfsusUf | 2409 | {磷酸酯}gsGfsgGfaUfGfaagcAfgGfaAfcAfusUfsu | 2410 |
D-2595 | {GalNAc3K2AhxC6}UfgUfuCfcUfgCfUfUfCfauCfcCfcUfsusUf | 2411 | {磷酸酯}asGfsgGfgAfUfgaagCfaGfgAfaCfasUfsu | 2412 |
D-2596 | {GalNAc3K2AhxC6}GfuUfcCfuGfcUfUfCfAfucCfcCfuUfsusUf | 2413 | {磷酸酯}asAfsgGfgGfAfugaaGfcAfgGfaAfcsUfsu | 2414 |
D-2597 | {GalNAc3K2AhxC6}UfuCfcUfgCfuUfCfAfUfccCfcUfuCfsusUf | 2415 | {磷酸酯}gsAfsaGfgGfGfaugaAfgCfaGfgAfasUfsu | 2416 |
D-2598 | {GalNAc3K2AhxC6}UfcCfuGfcUfuCfAfUfCfccCfuUfcUfsusUf | 2417 | {磷酸酯}asGfsaAfgGfGfgaugAfaGfcAfgGfasUfsu | 2418 |
D-2599 | {GalNAc3K2AhxC6}CfcUfgCfuUfcAfUfCfCfccUfuCfuAfsusUf | 2419 | {磷酸酯}usAfsgAfaGfGfggauGfaAfgCfaGfgsUfsu | 2420 |
D-2600 | {GalNAc3K2AhxC6}CfuGfcUfuCfaUfCfCfCfcuUfcUfaCfsusUf | 2421 | {磷酸酯}gsUfsaGfaAfGfgggaUfgAfaGfcAfgsUfsu | 2422 |
D-2601 | {GalNAc3K2AhxC6}UfgCfuUfcAfuCfCfCfCfuuCfuAfcAfsusUf | 2423 | {磷酸酯}usGfsuAfgAfAfggggAfuGfaAfgCfasUfsu | 2424 |
D-2602 | {GalNAc3K2AhxC6}GfcUfuCfaUfcCfCfCfUfucUfaCfaGfsusUf | 2425 | {磷酸酯}csUfsgUfaGfAfagggGfaUfgAfaGfcsUfsu | 2426 |
D-2603 | {GalNAc3K2AhxC6}CfuUfcAfuCfcCfCfUfUfcuAfcAfgUfsusUf | 2427 | {磷酸酯}asCfsuGfuAfGfaaggGfgAfuGfaAfgsUfsu | 2428 |
D-2604 | {GalNAc3K2AhxC6}UfuCfaUfcCfcCfUfUfCfuaCfaGfuGfsusUf | 2429 | {磷酸酯}csAfscUfgUfAfgaagGfgGfaUfgAfasUfsu | 2430 |
D-2605 | {GalNAc3K2AhxC6}UfcAfuCfcCfcUfUfCfUfacAfgUfgGfsusUf | 2431 | {磷酸酯}csCfsaCfuGfUfagaaGfgGfgAfuGfasUfsu | 2432 |
D-2606 | {GalNAc3K2AhxC6}CfaUfcCfcCfuUfCfUfAfcaGfuGfgCfsusUf | 2433 | {磷酸酯}gsCfscAfcUfGfuagaAfgGfgGfaUfgsUfsu | 2434 |
D-2607 | {GalNAc3K2AhxC6}AfuCfcCfcUfuCfUfAfCfagUfgGfcCfsusUf | 2435 | {磷酸酯}gsGfscCfaCfUfguagAfaGfgGfgAfusUfsu | 2436 |
D-2608 | {GalNAc3K2AhxC6}UfcCfcCfuUfcUfAfCfAfguGfgCfcUfsusUf | 2437 | {磷酸酯}asGfsgCfcAfCfuguaGfaAfgGfgGfasUfsu | 2438 |
D-2609 | {GalNAc3K2AhxC6}CfcCfcUfuCfuAfCfAfGfugGfcCfuUfsusUf | 2439 | {磷酸酯}asAfsgGfcCfAfcuguAfgAfaGfgGfgsUfsu | 2440 |
D-2610 | cugcuucaUfgCfCfUfUfucua[LNA-mC]as{invAb} | 2805 | asUfs[GNA-G]uagAfaaggCfaUfgaagcagsusu | 2806 |
D-2611 | cugcuucaUfgCfCfUfUfucua[LNA-mC]as{invAb} | 2807 | asUfs[Ab]uagAfaaggCfaUfgaagcagsusu | 2808 |
D-2612 | usgscuucauGfcCfUfUfUfcua[LNA-mC]ags{invDA} | 2809 | usCfsu[Ab]uaGfaaagGfcAfugaagcasusu | 2810 |
D-2613 | usgscuucauGfcCfUfUfUfcua[LNA-mC]ags{invDA} | 2811 | usCfsu[GNA-G]uaGfaaagGfcAfugaagcasusu | 2812 |
D-2614 | ucaugccuUfuCfUfAfCfasguggcs{invAb} | 2813 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2814 |
D-2615 | ucaugccuUfuCfUfAfCfagsuggcs{invAb} | 2815 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2816 |
D-2616 | ucaugccuUfuCfUfAfCfas[LNA-G]uggcs{invAb} | 2817 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2818 |
D-2617 | ucaugccuUfuCfUfAfCfas[sLNA-G]uggcs{invAb} | 2819 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2820 |
D-2618 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2821 | as[sMeO-I]guagAfaaggCfaUfgaagcagsusu | 2822 |
D-2619 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2823 | asUfsg[MeO-I]agAfaaggCfaUfgaagcagsusu | 2824 |
D-2620 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2825 | asUfsgu[MeO-I]gAfaaggCfaUfgaagcagsusu | 2826 |
D-2621 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2827 | asUfsgua[MeO-I]AfaaggCfaUfgaagcagsusu | 2828 |
D-2622 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2829 | asUfsguagAf[MeO-I]aggCfaUfgaagcagsusu | 2830 |
D-2623 | cugcuucaUfgCfCfUfUfucuaccs{invAb} | 2831 | as[sMeO-I]guagAfaaggCfaUfgaagcagsusu | 2832 |
D-2624 | cugcuucaUfgCfCfUfUfucuccas{invAb} | 2833 | asUfsg[MeO-I]agAfaaggCfaUfgaagcagsusu | 2834 |
D-2625 | cugcuucaUfgCfCfUfUfccuacas{invAb} | 2835 | asUfsguag[MeO-I]aaggCfaUfgaagcagsusu | 2836 |
D-2626 | cugcuucaUfgCfCfUfCfucuacas{invAb} | 2837 | asUfsguagAf[MeO-I]aggCfaUfgaagcagsusu | 2838 |
D-2627 | cugcuucaUfgCfCfUfUfucuacus{invAb} | 2839 | asAfsguagAfaaggCfaUfgaagcagsusu | 2840 |
D-2628 | cugcuucaUfgCfCfUfUfucuucas{invAb} | 2841 | asUfsgaagAfaaggCfaUfgaagcagsusu | 2842 |
D-2629 | cugcuucaUfgCfCfUfUfucaacas{invAb} | 2843 | asUfsguugAfaaggCfaUfgaagcagsusu | 2844 |
D-2630 | cugcuucaUfgCfCfUfAfucuacas{invAb} | 2845 | asUfsguagAfuaggCfaUfgaagcagsusu | 2846 |
D-2631 | ugcuucauGfcCfUfUfUfcuacacs{invAb} | 2847 | as[sMeO-I]uguaGfaaagGfcAfugaagcasusu | 2848 |
D-2632 | ugcuucauGfcCfUfUfUfcuacags{invAb} | 2849 | asCfs[MeO-I]guaGfaaagGfcAfugaagcasusu | 2850 |
D-2633 | ugcuucauGfcCfUfUfUfcuacags{invAb} | 2851 | asCfsug[MeO-I]aGfaaagGfcAfugaagcasusu | 2852 |
D-2634 | ugcuucauGfcCfUfUfUfcuacags{invAb} | 2853 | asCfsugu[MeO-I]GfaaagGfcAfugaagcasusu | 2854 |
D-2635 | ugcuucauGfcCfUfUfUfcuacags{invAb} | 2855 | asCfsugua[MeO-I]aaagGfcAfugaagcasusu | 2856 |
D-2636 | ugcuucauGfcCfUfUfUfcuaccgs{invAb} | 2857 | asCfs[MeO-I]guaGfaaagGfcAfugaagcasusu | 2858 |
D-2637 | ugcuucauGfcCfUfUfUfcuccags{invAb} | 2859 | asCfsug[MeO-I]aGfaaagGfcAfugaagcasusu | 2860 |
D-2638 | ugcuucauGfcCfUfUfUfccacags{invAb} | 2861 | asCfsugu[MeO-I]GfaaagGfcAfugaagcasusu | 2862 |
D-2639 | cugcuucaUfgCfCfUfUfucuaaas{invAb} | 2863 | asUfsuuagAfaaggCfaUfgaagcagsusu | 2864 |
D-2640 | cugcuucaUfgCfCfUfUfuauacas{invAb} | 2865 | asUfsguauAfaaggCfaUfgaagcagsusu | 2866 |
D-2641 | cugcuucaUfgCfCfUfsUfucuacas{invAb} | 2867 | asUfsguagAf[sGNA-A]aggCfaUfgaagcagsusu | 2868 |
D-2642 | cugcuucaUfgCfCfUf[LNA-T]ucuacas{invAb} | 2869 | asUfsguagAfs[sGNA-A]aggCfaUfgaagcagsusu | 2870 |
D-2643 | usgscuucauGfcCfUfUfUfcuaca[sLNA-G]{invDA} | 2871 | us[sGNA-C]uguaGfaaagGfcAfugaagcasusu | 2872 |
D-2644 | usgscuucauGfcCfUfUf[LNA-T]cuacags{invDA} | 2873 | usCfsuguaGf[GNA-A]aagGfcAfugaagcasusu | 2874 |
D-2645 | ucaugccuUfuCfUfAfCf[LNA-A]guggcs{invAb} | 2875 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2876 |
D-2646 | cugcuucaUfgCfCfUfUfu[LNA-mC]uacas{invAb} | 2877 | asUfsgua[Ab]AfaaggCfaUfgaagcagsusu | 2878 |
D-2647 | ucaugccuUfuCfUfAfCfa[sLNA-G]uggcs{invAb} | 2879 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2880 |
D-2648 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2881 | asUfs[MeO-I]uagAfaaggCfaUfgaagcagsusu | 2882 |
D-2649 | cugcuucaUfgCfCfUfUfucuagas{invAb} | 2883 | asUfscuagAfaaggCfaUfgaagcagsusu | 2884 |
D-2650 | cugcuucaUfgCfCfUfUfacuacas{invAb} | 2885 | asUfsguagUfaaggCfaUfgaagcagsusu | 2886 |
D-2651 | ugcuucauGfcCfUfUfUfcuacags{invAb} | 2887 | asCfsu[MeO-I]uaGfaaagGfcAfugaagcasusu | 2888 |
D-2652 | ugcuucauGfcCfUfUfUfcuacags{invAb} | 2889 | asCfsuguaGf[MeO-I]aagGfcAfugaagcasusu | 2890 |
D-2653 | ugcuucauGfcCfUfUfCfcuacags{invAb} | 2891 | asCfsuguaGf[MeO-I]aagGfcAfugaagcasusu | 2892 |
D-2654 | cugcuucaUfgCfCfUfUfucuauas{invAb} | 2893 | asUfsauagAfaaggCfaUfgaagcagsusu | 2894 |
D-2655 | cugcuucaUfgCfCfUfUfuuuacas{invAb} | 2895 | asUfsguaaAfaaggCfaUfgaagcagsusu | 2896 |
D-2656 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2897 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2898 |
D-2657 | cugcuucaUfgCfCfUfUfsucuacas{invAb} | 2899 | asUfsguagAfs[GNA-A]aggCfaUfgaagcagsusu | 2900 |
D-2658 | cugcuucaUfgCfCfUfUf[LNA-T]cuacas{invAb} | 2901 | asUfsguag[GNA-A]aaggCfaUfgaagcagsusu | 2902 |
D-2659 | cugcuucaUfgCfCfUf[LNA-T]ucuacas{invAb} | 2903 | asUfsguagAf[Ab]aggCfaUfgaagcagsusu | 2904 |
D-2660 | cugcuucaUfgCfCfUfUfucuac[sLNA-A]{invAb} | 2905 | as[sAb]guagAfaaggCfaUfgaagcagsusu | 2906 |
D-2661 | cugcuucaUfgCfCfUfUfu[LNA-mC]uacas{invAb} | 2907 | asUfsgua[GNA-G]AfaaggCfaUfgaagcagsusu | 2908 |
D-2662 | usgscuucauGfcCfUfUfUf[LNA-mC]uacags{invDA} | 2909 | usCfsugua[Ab]aaagGfcAfugaagcasusu | 2910 |
D-2663 | cugcuucaUfgCfCfUfUfucuacas{invAb} | 2911 | asUfsguag[MeO-I]aaggCfaUfgaagcagsusu | 2912 |
D-2664 | cugcuucaUfgCfCfUfsUfsucuacas{invAb} | 2913 | asUfsguagAfs[sGNA-A]aggCfaUfgaagcagsusu | 2914 |
D-2665 | cugcuucaUfgCfCfUf[sLNA-T]ucuacas{invAb} | 2915 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2916 |
D-2666 | {sGalNAc3K2AhxC6}cusgcuucaUfgCfCfUfUfucUfaCfas{invAb} | 2917 | asUfsguagAf[GNA-A]aGfgCfaUfgaagcagsusu | 2918 |
D-2667 | {sGalNAc3K2AhxC6}cugcuuCfaUfGfCfCfuuucuacas{invAb} | 2919 | asUfsgUfaGfa[GNA-A]aggcaUfgAfagcagsusu | 2920 |
D-2668 | {sGalNAc3K2AhxC6}cugcuuCfaUfGfCfCfuuucuacas{invAb} | 2921 | asUfsgUfaGfa[GNA-A]AfggcaUfgAfagcagsusu | 2922 |
D-2669 | {sGalNAc3K2AhxC6}cugcuuCfaUfgCfcuuucuacas{invAb} | 2923 | asUfsguaga[GNA-A]aggCfaUfgAfagcagsusu | 2924 |
D-2670 | {sGalNAc3K2AhxC6}cugcuucaUfgCfcUfuucuacas{invAb} | 2925 | asUfsguagAf[GNA-A]AfggCfaUfgaagcagsusu | 2926 |
D-2671 | {sGalNAc3K2AhxC6}cugcuucaUfgCfcUfuucuacas{invAb} | 2927 | asUfsguaga[GNA-A]AfggCfaUfgaagcagsusu | 2928 |
D-2672 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUf[LNA-T]ucuacas{invAb} | 2929 | asUfsguagAf[GNA-A]aggCfaUfgaagcagsusu | 2930 |
D-2673 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucua[LNA-mC]as{invAb} | 2931 | asUfs[GNA-G]uagAfaaggCfaUfgaagcagsusu | 2932 |
D-2674 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfa[LNA-G]uggcs{invAb} | 2933 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2934 |
D-2675 | {sGalNAc3K2AhxC6}cugcuucaUfgGfGfAfUfucuacas{invAb} | 2935 | asUfsguagAfauccCfaUfgaagcagsusu | 2936 |
D-2676 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2937 | asUfsguaGfa[GNA-A]AfggCfaUfgaagcagsusu | 2938 |
D-2677 | {sGalNAc3K2AhxC6}guauguucCfuGfCfUfUfcaugccs{invAb} | 2939 | asGfsgcauGfaagcAfgGfaacauacsusu | 2940 |
D-2678 | {sGalNAc3K2AhxC6}uguccugcUfuCfAfUfGfccuuucs{invAb} | 2941 | asGfsaaagGfcaugAfaGfcaggacasusu | 2942 |
D-2679 | {sGalNAc3K2AhxC6}gcuucaugCfcUfUfUfCfuacagus{invAb} | 2943 | asAfscuguAfgaaaGfgCfaugaagcsusu | 2944 |
D-2680 | {sGalNAc3K2AhxC6}cuucaugcCfuUfUfCfUfacagugs{invAb} | 2945 | asCfsacugUfagaaAfgGfcaugaagsusu | 2946 |
D-2681 | {sGalNAc3K2AhxC6}uucaugccUfuUfCfUfAfcaguggs{invAb} | 2947 | asCfscacuGfuagaAfaGfgcaugaasusu | 2948 |
D-2682 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucaacas{invAb} | 2949 | asUfsguugAfaaggCfaUfgaagcagsusu | 2950 |
D-2683 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuccas{invAb} | 2951 | asUfsg[MeO-I]agAfaaggCfaUfgaagcagsusu | 2952 |
D-2684 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuccags{invAb} | 2953 | asCfsug[MeO-I]aGfaaagGfcAfugaagcasusu | 2954 |
D-2685 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2955 | asUfsgu[MeO-I]gAfaaggCfaUfgaagcagsusu | 2956 |
D-2686 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 2957 | asCfsugu[MeO-I]GfaaagGfcAfugaagcasusu | 2958 |
D-2687 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2959 | asUfsgua[MeO-I]AfaaggCfaUfgaagcagsusu | 2960 |
D-2688 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 2961 | asCfsugua[MeO-I]aaagGfcAfugaagcasusu | 2962 |
D-2689 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfccuacas{invAb} | 2963 | asUfsguag[MeO-I]aaggCfaUfgaagcagsusu | 2964 |
D-2690 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfCfucuacas{invAb} | 2965 | asUfsguagAf[MeO-I]aggCfaUfgaagcagsusu | 2966 |
D-2691 | {sGalNAc3K2AhxC6}usgscuucauGfcCfUfUfUfcu[LNA-A]cags{invDA} | 2967 | usCfsug[Ab]aGfaaagGfcAfugaagcasusu | 2968 |
D-2692 | {sGalNAc3K2AhxC6}usgscuucauGfcCfUfUfUfcuac[LNA-A]gs{invDA} | 2969 | usCfs[GNA-U]guaGfaaagGfcAfugaagcasusu | 2970 |
D-2693 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfasgsuggcs{invAb} | 2971 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2972 |
D-2694 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfuc[LNA-T]acas{invAb} | 2973 | asUfsgu[GNA-A]gAfaaggCfaUfgaagcagsusu | 2974 |
D-2695 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfuc[LNA-T]acas{invAb} | 2975 | asUfsgu[Ab]gAfaaggCfaUfgaagcagsusu | 2976 |
D-2696 | {sGalNAc3K2AhxC6}usgscuucauGfcCfUfUfUfc[LNA-T]acags{invDA} | 2977 | usCfsugu[GNA-A]GfaaagGfcAfugaagcasusu | 2978 |
D-2697 | {sGalNAc3K2AhxC6}usgscuucauGfcCfUfUfUfcua[LNA-mC]ags{invDA} | 2979 | usCfsu[GNA-G]uaGfaaagGfcAfugaagcasusu | 2980 |
D-2698 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfas[sLNA-G]uggcs{invAb} | 2981 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 2982 |
D-2699 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2983 | asUfs[MeO-I]uagAfaaggCfaUfgaagcagsusu | 2984 |
D-2700 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacas{invAb} | 2985 | asUfsguag[MeO-I]aaggCfaUfgaagcagsusu | 2986 |
D-2701 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfacuacas{invAb} | 2987 | asUfsguagUfaaggCfaUfgaagcagsusu | 2988 |
D-2702 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 2989 | asCfsuguaGf[MeO-I]aagGfcAfugaagcasusu | 2990 |
D-2703 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfCfcuacags{invAb} | 2991 | asCfsuguaGf[MeO-I]aagGfcAfugaagcasusu | 2992 |
D-2704 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUfcuacags{invAb} | 2993 | asCfsu[MeO-I]uaGfaaagGfcAfugaagcasusu | 2994 |
D-2705 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucusacas{invAb} | 2995 | asUfsgu[MeO-I]gAfaaggCfaUfgaagcagsusu | 2996 |
D-2706 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucsuacas{invAb} | 2997 | asUfsgu[MeO-I]gAfaaggCfaUfgaagcagsusu | 2998 |
D-2707 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucsusacas{invAb} | 2999 | asUfsgu[MeO-I]gAfaaggCfaUfgaagcagsusu | 3000 |
D-2708 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfuscuacas{invAb} | 3001 | asUfsguag[MeO-I]aaggCfaUfgaagcagsusu | 3002 |
D-2709 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfa[sLNA-G]uggcs{invAb} | 3003 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 3004 |
D-2710 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCf[LNA-A]guggcs{invAb} | 3005 | asGfscca[Ab]UfguagAfaAfggcaugasusu | 3006 |
D-2711 | {sGalNAc3K2AhxC6}cugcuucaUfgGfGfAfUfuguacas{invAb} | 3007 | asUfsguacAfauccCfaUfgaagcagsusu | 3008 |
D-2712 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfuc[LNA-T]acas{invAb} | 3009 | asUfsgu[MeO-I]gAfaaggCfaUfgaagcagsusu | 3010 |
D-2713 | {sGalNAc3K2AhxC6}ugcuucauGfcCfUfUfUf[LNA-mC]uacags{invAb} | 3011 | asCfsugua[MeO-I]aaagGfcAfugaagcasusu | 3012 |
D-2714 | {sGalNAc3K2AhxC6}cugcuuCfaUfGfCfCfu[LNA-T]ucuacas{invAb} | 3013 | asUfsgUfaGfa[GNA-A]aggcaUfgAfagcagsusu | 3014 |
D-2715 | {sGalNAc3K2AhxC6}cugcuuCfaUfGfCfCfu[LNA-T]ucuacas{invAb} | 3015 | asUfsgUfaGfa[GNA-A]AfggcaUfgAfagcagsusu | 3016 |
D-2716 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUf[LNA-T]cuacas{invAb} | 3017 | asUfsguag[MeO-I]aaggCfaUfgaagcagsusu | 3018 |
D-2717 | {sGalNAc3K2AhxC6}ucaugcCfuUfuCfuacaguggcs{invAb} | 3019 | asGfsccacuguagAfaAfgGfcaugasusu | 3020 |
D-2718 | {sGalNAc3K2AhxC6}uauguuCfcUfgCfuucaugccus{invAb} | 3021 | asAfsggcaugaagCfaGfgAfacauasusu | 3022 |
D-2719 | {sGalNAc3K2AhxC6}ugcuucAfuGfcCfuuucuacags{invAb} | 3023 | asCfsuguagaaagGfcAfuGfaagcasusu | 3024 |
D-2720 | {sGalNAc3K2AhxC6}uguuccUfgCfUfUfCfaugccuuus{invAb} | 3025 | asAfsggcaUfgaagCfaGfgaacasusu | 3026 |
D-2721 | {sGalNAc3K2AhxC6}cugcuuCfaUfgCfcuuucuacas{invAb} | 3027 | asUfsguagaaaggCfaUfgAfagcagsusu | 3028 |
D-2722 | {sGalNAc3K2AhxC6}uauguuccUfgCfuUfCfaugccus{invAb} | 3029 | asAfsggcaUfgaagCfaGfgaacauasusu | 3030 |
D-2723 | {sGalNAc3K2AhxC6}uguuccUfgCfUfUfCfaugccus{invAb} | 3031 | asAfsggcaUfgaagCfaGfgaacasusu | 3032 |
D-2724 | {sGalNAc3K2AhxC6}uauguuCfcUfgCfuucaugccuus{invAb} | 3033 | asAfsggcaugaagCfaGfgAfacauasusu | 3034 |
D-2725 | {sGalNAc3K2AhxC6}uauguuCfcUfgCfuucaugccus{invAb} | 3035 | asAfsggcaugaagCfaGfgAfacauascsc | 3036 |
D-2726 | {sGalNAc3K2AhxC6}uauguuCfcUfgCfuucaugccuus{invAb} | 3037 | asAfsggcaugaagCfaGfgAfacauascsc | 3038 |
D-2727 | uauguuccUfgCfUfUfCfaugccus{invAb} | 3039 | asAfsggcaUfgaagCfaGfgaacauasusu | 3040 |
D-2728 | uauguuccUfgCfUfUfCfaugcgus{invAb} | 3041 | asAfscgcaUfgaagCfaGfgaacauasusu | 3042 |
D-2729 | uauguuccUfgCfUfUfCfauggcus{invAb} | 3043 | asAfsgccaUfgaagCfaGfgaacauasusu | 3044 |
D-2730 | uauguuccUfgCfUfUfCfaagccus{invAb} | 3045 | asAfsggcuUfgaagCfaGfgaacauasusu | 3046 |
D-2731 | uauguuccUfgCfUfUfCfuugccus{invAb} | 3047 | asAfsggcaAfgaagCfaGfgaacauasusu | 3048 |
D-2732 | uauguuccUfgCfUfUfGfaugccus{invAb} | 3049 | asAfsggcaUfcaagCfaGfgaacauasusu | 3050 |
D-2733 | uauguuccUfgCfUfAfCfaugccus{invAb} | 3051 | asAfsggcaUfguagCfaGfgaacauasusu | 3052 |
D-2734 | uauguuccUfgCfAfUfCfaugccus{invAb} | 3053 | asAfsggcaUfgaugCfaGfgaacauasusu | 3054 |
D-2735 | uauguuccUfgGfUfUfCfaugccus{invAb} | 3055 | asAfsggcaUfgaacCfaGfgaacauasusu | 3056 |
D-2736 | uauguuccUfcCfUfUfCfaugccus{invAb} | 3057 | asAfsggcaUfgaagGfaGfgaacauasusu | 3058 |
D-2737 | uauguuccAfgCfUfUfCfaugccus{invAb} | 3059 | asAfsggcaUfgaagCfuGfgaacauasusu | 3060 |
D-2738 | uauguucgUfgCfUfUfCfaugccus{invAb} | 3061 | asAfsggcaUfgaagCfaCfgaacauasusu | 3062 |
D-2739 | uauguugcUfgCfUfUfCfaugccus{invAb} | 3063 | asAfsggcaUfgaagCfaGfcaacauasusu | 3064 |
D-2740 | uauguaccUfgCfUfUfCfaugccus{invAb} | 3065 | asAfsggcaUfgaagCfaGfguacauasusu | 3066 |
D-2741 | uaugauccUfgCfUfUfCfaugccus{invAb} | 3067 | asAfsggcaUfgaagCfaGfgaucauasusu | 3068 |
D-2742 | uaucuuccUfgCfUfUfCfaugccus{invAb} | 3069 | asAfsggcaUfgaagCfaGfgaagauasusu | 3070 |
D-2743 | uaaguuccUfgCfUfUfCfaugccus{invAb} | 3071 | asAfsggcaUfgaagCfaGfgaacuuasusu | 3072 |
D-2744 | uuuguuccUfgCfUfUfCfaugccus{invAb} | 3073 | asAfsggcaUfgaagCfaGfgaacaaasusu | 3074 |
D-2745 | aauguuccUfgCfUfUfCfaugccus{invAb} | 3075 | asAfsggcaUfgaagCfaGfgaacauususu | 3076 |
D-2746 | ucaugccuUfuCfUfAfCfaguggcs{invAb} | 3077 | asGfsccacUfguagAfaAfggcaugasusu | 3078 |
D-2747 | ucaugccuUfuCfUfAfCfagugggs{invAb} | 3079 | asCfsccacUfguagAfaAfggcaugasusu | 3080 |
D-2748 | ucaugccuUfuCfUfAfCfagugccs{invAb} | 3081 | asGfsgcacUfguagAfaAfggcaugasusu | 3082 |
D-2749 | ucaugccuUfuCfUfAfCfagucgcs{invAb} | 3083 | asGfscgacUfguagAfaAfggcaugasusu | 3084 |
D-2750 | ucaugccuUfuCfUfAfCfagaggcs{invAb} | 3085 | asGfsccucUfguagAfaAfggcaugasusu | 3086 |
D-2751 | ucaugccuUfuCfUfAfCfacuggcs{invAb} | 3087 | asGfsccagUfguagAfaAfggcaugasusu | 3088 |
D-2752 | ucaugccuUfuCfUfAfCfuguggcs{invAb} | 3089 | asGfsccacAfguagAfaAfggcaugasusu | 3090 |
D-2753 | ucaugccuUfuCfUfAfGfaguggcs{invAb} | 3091 | asGfsccacUfcuagAfaAfggcaugasusu | 3092 |
D-2754 | ucaugccuUfuCfUfUfCfaguggcs{invAb} | 3093 | asGfsccacUfgaagAfaAfggcaugasusu | 3094 |
D-2755 | ucaugccuUfuCfAfAfCfaguggcs{invAb} | 3095 | asGfsccacUfguugAfaAfggcaugasusu | 3096 |
D-2756 | ucaugccuUfuGfUfAfCfaguggcs{invAb} | 3097 | asGfsccacUfguacAfaAfggcaugasusu | 3098 |
D-2757 | ucaugccuAfuCfUfAfCfaguggcs{invAb} | 3099 | asGfsccacUfguagAfuAfggcaugasusu | 3100 |
D-2758 | ucaugcguUfuCfUfAfCfaguggcs{invAb} | 3101 | asGfsccacUfguagAfaAfcgcaugasusu | 3102 |
D-2759 | ucauggcuUfuCfUfAfCfaguggcs{invAb} | 3103 | asGfsccacUfguagAfaAfgccaugasusu | 3104 |
D-2760 | ucaagccuUfuCfUfAfCfaguggcs{invAb} | 3105 | asGfsccacUfguagAfaAfggcuugasusu | 3106 |
D-2761 | ucuugccuUfuCfUfAfCfaguggcs{invAb} | 3107 | asGfsccacUfguagAfaAfggcaagasusu | 3108 |
D-2762 | ugaugccuUfuCfUfAfCfaguggcs{invAb} | 3109 | asGfsccacUfguagAfaAfggcaucasusu | 3110 |
D-2763 | acaugccuUfuCfUfAfCfaguggcs{invAb} | 3111 | asGfsccacUfguagAfaAfggcaugususu | 3112 |
D-2764 | ucaugccuUfaCfUfAfCfaguggcs{invAb} | 3113 | asGfsccacUfguagUfaAfggcaugasusu | 3114 |
D-2765 | {sGalNAc3K2AhxC6}augccuUfuCfUfAfCfaguggcusus{invAb} | 3115 | asGfsccacUfguagAfaAfggcaususu | 3116 |
D-2766 | {sGalNAc3K2AhxC6}augccuUfuCfUfAfCfaguggcs{invAb} | 3117 | asGfsccacUfguagAfaAfggcaususu | 3118 |
D-2767 | {sGalNAc3K2AhxC6}ucaugccuUfuCfuAfCfaguggcs{invAb} | 3119 | asGfsccacUfguagAfaAfggcaugasusu | 3120 |
D-2768 | {sGalNAc3K2AhxC6}[invAb]augccuUfUfCfUfacaguggscsu | 3121 | asGfsccacUfguagaaAfgGfcaususu | 3122 |
D-2769 | {sGalNAc3K2AhxC6}ucaugcCfuUfuCfuacaguggcus{invAb} | 3123 | asGfsccacuguagAfaAfgGfcaugasusu | 3124 |
D-2770 | {sGalNAc3K2AhxC6}ucaugcCfuUfuCfuacaguggcs{invAb} | 3125 | asGfsccacuguagAfaAfgGfcaugasasc | 3126 |
D-2771 | {sGalNAc3K2AhxC6}ucaugcCfuUfuCfuacaguggcus{invAb} | 3127 | asGfsccacuguagAfaAfgGfcaugasasc | 3128 |
D-2772 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcus{invAb} | 3129 | asGfsccacUfguagAfaAfggcaugasusu | 3130 |
D-2773 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcs{invAb} | 3131 | asGfsccacUfguagAfaAfggcaugasasc | 3132 |
D-2774 | {sGalNAc3K2AhxC6}ucaugccuUfuCfUfAfCfaguggcus{invAb} | 3133 | asGfsccacUfguagAfaAfggcaugasasc | 3134 |
D-2775 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfu[LNA-mC]uacas{invAb} | 3135 | asUfsgua[MeO-I]AfaaggCfaUfgaagcagsusu | 3136 |
D-2776 | {GalNAc3K2AhxC6}gcggcuUfcGfAfCfGfgcuucuasusu | 3137 | {磷酸酯}usAfsgAfaGfCfcgucGfaAfgccgcsusu | 3138 |
D-2777 | {GalNAc3K2AhxC6}gcggcuUfcGfAfCfGfgguucuasusu | 3139 | {磷酸酯}usAfsgAfaCfCfcgucGfaAfgccgcsusu | 3140 |
D-2778 | {GalNAc3K2AhxC6}gcggcuUfcGfAfCfGfccuucuasusu | 3141 | {磷酸酯}usAfsgAfaGfGfcgucGfaAfgccgcsusu | 3142 |
D-2779 | {GalNAc3K2AhxC6}gcggcuUfcGfAfCfCfgcuucuasusu | 3143 | {磷酸酯}usAfsgAfaGfCfggucGfaAfgccgcsusu | 3144 |
D-2780 | {GalNAc3K2AhxC6}gcggcuUfcCfUfGfGfgcuucuasusu | 3145 | [sInvAb]usAfgAfaGfCfccagGfaAfgccgcsusu | 3146 |
D-2781 | {sGalNAc3K2AhxC6}[invAb]gcuucaUfgGfGfAfUfuguacaususu | 3147 | {磷酸酯}asUfsgUfaCfAfauccCfaUfgaagcsusu | 3148 |
D-2782 | {sGalNAc3K2AhxC6}ucaugccuUfuGfAfUfCfacuggcs{invAb} | 3149 | asGfsccagUfgaucAfaAfggcaugasusu | 3150 |
D-2783 | {sGalNAc3K2AhxC6}cugcuucaUfgCfCfUfUfucuacasc | 3151 | csUfsguagAfaaggCfaUfgaagcagsusu | 3152 |
D-2784 | {sGalNAc3K2AhxC6}[invAb]cuucauGfcGfAfAfUfcaAfcAfgUfsusUf | 3153 | {磷酸酯}asCfsuGfuUfGfauucGfcAfugaagsusu | 3154 |
D-2785 | {sGalNAc3K2AhxC6}ggccuuAfuCfCfCfuccuuccususa | 3155 | usAfsaggAfaGfGfagggAfuAfaggccsasc | 3156 |
D-2786 | {sGalNAc3K2AhxC6}gugucuGfaGfUfUfccauuccasasa | 3157 | usUfsuggAfaUfGfgaacUfcAfgacacscsa | 3158 |
製備一組經完全化學修飾之siRNA,包括跨越PNPLA3中之rs738409及/或rs738408 SNP的siRNA,且測試活體外mRNA敲低之效力及選擇性。各siRNA雙鏈體由兩條鏈組成,即有義鏈或「隨從」鏈及反義或「引導」鏈。鏈長21或23個核苷酸,具有19個互補鹼基對。在某些情況下,存在兩個鹼基對3'突出端。製備siRNA,其中用2'-OMe或2'-F基團取代各核苷酸之核糖中的天然2'-OH。視需要,用硫代磷酸酯替換一或兩條鏈上之磷酸二酯核苷酸間鍵以減少核酸外切酶之降解。
使用384孔格式的活體外siRNA轉染測定,然後進行螢光原位雜交靶向核糖核酸分子(RNA FISH)測定來評估各siRNA分子在降低PNPLA3表現中之功效,以判定IC50及最大活性值。該測定在人肝細胞癌細胞株Hep3B細胞(ATCC HB-8064)及表現人PNPLA3 I148I之中國倉鼠卵巢(CHO)細胞上進行。在37°C及5% CO2下,將人肝細胞癌HepB3維持在EMEM培養基(ATCC 30-2003)中,該培養基補充有10%胎牛血清及1%抗生素/抗真菌劑。在37°C及5% CO2下,將表現人PNPLA3 I148I之CHO細胞維持在含有50% CD-CHO(生命技術公司(Life Technologies))、50% Ex-Cell CHO 5培養基(西格瑪公司(Sigma))、8 mM L-穀胺醯胺、1 x HT、1%抗生素/抗真菌劑、及10 μg/mL嘌呤黴素之培養基中。
對於Hep3B細胞測定,根據製造商之建議,在384孔板(珀金埃爾默公司(PerkinElmer))中以10 μg/孔製備siRNA分子及Lipofectamine RNAiMAX轉染試劑(生命技術公司(Life Technologies))在EMEM培養基(ATCC 30-2003)中的轉染複合物。對於CHO人細胞測定,根據製造商之建議,在384孔板中以10μg/孔製備siRNA分子及Lipofectamine RNAiMAX轉染試劑在F12K培養基(聯發科技(Mediatech))中的轉染複合物。將細胞在無抗生素/抗真菌劑之培養基中稀釋至67,000個細胞/ml,且向各孔中加入30 μl,在40 μl培養基中最終密度為2000個細胞/孔。在室溫下溫育20分鐘後,將板轉移至37°C及5% CO2培養箱中。將Hep3B細胞轉染測定溫育72小時,且將CHO人PNPLA3 I148I轉染測定溫育48小時。
收穫時,將細胞在8%甲醛固定溶液(賽默科技公司(Thermo Scientific))中在室溫下固定15分鐘。然後將板藉由連續50%、70%及100%乙醇洗滌進行脫水。然後將板密封且儲存在-20°C。
使用Affymetrix QuantiGene® View RNA HC篩選測定套組(QVP0011)、Affymetrix View HC信號擴增套組3-plex(QVP0213)、及以下Affymetrix基因特異性探針進行RNA FISH測定:PNPLA3人0.33 mL View RNA Type 6(650標記)VA6-20279-01及PPIB人0.44 mL View RNA Type 1(488標記)VA1-10148-01。
首先將板藉由連續100%、70%及50%乙醇洗滌再水化。然後用PBS洗滌細胞,且隨後根據套組說明書進行透化及蛋白酶消化。根據製造商之方案製備目標工作探針組,將其加入孔中,且在40°C下溫育3小時。遵循製造商之方案進行使用工作探針組、工作前置放大器、工作放大器及工作LP之連續雜交。最後,應用細胞核複染色(Hoechst 33342及Cell Mask Blue; 分子探針公司(Molecular Probes))。將板在室溫下溫育30分鐘,用PBS洗滌,用80 μl之PBS覆蓋,且然後將板密封用於成像。
在Opera Phenix高含量篩選系統(珀金埃爾默公司(PerkinElmer))上,使用針對Hoechst 33342及Cell Mask Blue之UV通道、針對類型1探針之488通道、及針對類型6探針之647通道,對所有板成像。
使用Columbus軟體分析RNA FISH數據,且使用Genedata Screener生成影像。經CHO轉染之PNPLA3 I148I之測定結果如表3所示。經CHO轉染之PNPLA3 I148M之測定結果如表4所示。PNPLA3敲低提供與對照相比之敲低百分比。負值表示PNPLA3水平下降。
[表3]. 經CHO轉染之PNPLA3 I148I之RNA FISH測定
[表4]. 經CHO轉染之PNPLA3 I148M之RNA FISH測定
RNA FISH亦在含有雙突變體PNPLA3-rs738408-rs738409之肝細胞株以及對照野生型細胞株Hep3B上運行。將Hep3B及HepG2細胞(購自ATCC)在最小必需培養基(對於Hep3B,來自康寧公司(Corning)之MEM,且對於HepG2,來自ATCC之EMEM)中培養,該培養基補充有10%胎牛血清(FBS,西格瑪公司(Sigma))及1%青黴素 - 鏈黴素(P-S,康寧公司(Corning))。siRNA轉染如下進行:取決於細胞株,將1 μL測試siRNA及4 μL普通MEM或EMEM藉由BioMek FX(貝克曼庫爾特公司(Beckman Coulter))加入到經PDL塗佈之CellCarrier-384 Ultra測定板(珀金埃爾默公司(PerkinElmer))中。將5 μL之Lipofectamine RNAiMAX(賽默飛世爾科技公司(Thermo Fisher Scientific))在普通MEM或EMEM中預稀釋(具體而言,對於Hep3B,為在5μLMEM中之0.035 μL RNAiMAX,且對於HepG2,為在5 μL EMEM中之0.06 μL之RNAiMAX),然後藉由Multidrop Combi試劑分配器(賽默飛世爾科技公司(Thermo Fisher Scientific))分配到測定板中。在室溫(RT)下溫育siRNA / RNAiMAX混合物20分鐘後,使用Multidrop Combi試劑分配器,將補充有10% FBS及1% P-S之MEM或EMEM中的30μL Hep3B或HepG2細胞(每孔2000個細胞)添加至轉染複合物中。將測定板在室溫下溫育20分鐘,然後置於培養箱中。然後將細胞在37°C及5% CO2下溫育72小時。遵循製造方案(賽默飛世爾科技公司(Thermo Fisher Scientific))使用用於液體處理的內部組裝的自動FISH測定平台進行ViewRNA ISH細胞測定。簡言之,將細胞在4%甲醛(賽默飛世爾科技公司(Thermo Fisher Scientific))中在室溫下固定15分鐘,在室溫下用去污劑透化3分鐘,且然後在室溫下用蛋白酶溶液處理10分鐘。將靶特異性探針對(賽默飛世爾科技公司(Thermo Fisher Scientific))之溫育進行3小時,而對於前置放大器、放大器及標記探針(賽默飛世爾科技公司(Thermo Fisher Scientific))各1小時。所有雜交步驟均在40°C下在Cytomat 2 C-LIN自動培養箱(賽默飛世爾科技公司(Thermo Fisher Scientific))中進行。在雜交反應後,將細胞用Hoechst及CellMask Blue(賽默飛世爾科技公司(Thermo Fisher Scientific))染色30分鐘,且然後在Opera Phenix(珀金埃爾默公司(PerkinElmer))上成像。使用Columbus影像數據存儲及分析系統(珀金埃爾默公司(PerkinElmer))分析影像以獲得每個細胞之平均斑點計數。使用高(含有磷酸鹽緩衝鹽水,康寧公司(Corning))及低(無靶探針對)對照孔來標準化斑點計數。繪製相對於總siRNA濃度之標準化值,且將數據擬合到Genedata Screener(基因數據公司(Genedata))中之四參數S形模型以獲得IC50及最大活性。HepG2細胞之測定結果如表5所示,且Hep3B細胞之測定結果如表6所示。PNPLA3敲低提供與對照相比之敲低百分比。負值表示PNPLA3水平下降。在雙鏈體運行超過一次的情況下,顯示平均IC50,以及標準偏差。
[表5]. 肝臟HepG2細胞之RNA FISH測定
[表6]. 對肝Hep3B細胞之RNA FISH測定
實例 3 :用於 PNPLA3-rs738409 及 PNPLA3-rs738409-rs738408 之 siRNA 的微滴式數位 PCR 測定
雙鏈體編號 | IC50 (μM) | PNPLA3敲低 (%) |
D-2001 | .0589 | -33.9 |
D-2002 | .0158 | -67.2 |
D-2003 | .0427 | -43.4 |
D-2004 | .00835 | -63.5 |
D-2006 | .0177 | -77.8 |
D-2008 | .125 | -10.7 |
D-2009 | .00769 | -45.5 |
D-2010 | .00558 | -80 |
D-2011 | .035 | -3 |
D-2012 | > 0.5 | -3.7 |
D-2013 | .036 | -6.4 |
D-2014 | .0122 | -58.2 |
D-2015 | > 0.5 | 8 |
D-2016 | > 0.5 | 8 |
D-2017 | .0153 | -73.2 |
D-2018 | .00386 | -31.5 |
D-2019 | .125 | |
D-2020 | > 0.5 | |
D-2021 | > 0.5 | 8 |
D-2022 | > 0.167 | -6.8 |
D-2023 | .0257 | -36.9 |
D-2024 | > 0.5 | 4 |
D-2025 | > 0.5 | -2.5 |
D-2026 | .022 | -35.1 |
D-2027 | .00172 | -16.5 |
D-2028 | > 0.5 | 10 |
D-2029 | .0106 | -56.2 |
D-2032 | .00205 | -52.9 |
D-2033 | .0107 | -61.7 |
D-2034 | > 0.5 | 6 |
D-2035 | > 0.5 | -3.8 |
D-2036 | .00665 | -55.9 |
D-2037 | > 0.5 | 4 |
D-2038 | > 0.5 | 10 |
D-2039 | .0116 | -23.9 |
D-2040 | > 0.5 | -25.4 |
D-2041 | > 0.5 | 9 |
D-2042 | .00959 | -25.5 |
D-2043 | > 0.5 | 9 |
D-2044 | .00552 | -29 |
D-2045 | > 0.5 | 9 |
D-2046 | > 0.5 | 9 |
D-2047 | > 0.5 | -5.9 |
D-2048 | .00618 | -56.3 |
D-2049 | > 0.5 | 12 |
D-2050 | > 0.5 | 10 |
D-2051 | > 0.5 | -17.2 |
D-2052 | > 0.5 | -8.3 |
D-2053 | > 0.5 | 11 |
D-2054 | > 0.5 | -14.9 |
D-2055 | > 0.5 | -10.6 |
D-2056 | > 0.5 | 10 |
D-2057 | .00485 | -59.2 |
D-2058 | .014 | -53 |
D-2059 | > 0.5 | -4.9 |
D-2060 | > 0.5 | 18 |
D-2061 | .00795 | -44.8 |
D-2062 | .000668 | -74.6 |
D-2063 | > 0.5 | -21.8 |
D-2064 | > 0.5 | 9 |
D-2065 | > 0.5 | -10.5 |
D-2066 | .0412 | -42.2 |
D-2067 | > 0.5 | 10 |
雙鏈體編號 | IC50 (μM) | PNPLA3敲低 (%) |
D-2000 | .0316 | -29.2 |
D-2001 | .0131 | -81.8 |
D-2002 | .00216 | -90.5 |
D-2003 | .022 | -50.4 |
D-2004 | .00429 | -88 |
D-2005 | > 0.5 | 15 |
D-2006 | .00301 | -89.2 |
D-2007 | > 0.5 | 6 |
D-2009 | .00274 | -86.9 |
D-2010 | .00203 | -93.3 |
D-2011 | .000694 | -11.9 |
D-2012 | > 0.5 | -18 |
D-2013 | .011 | -66.4 |
D-2014 | .0057 | -84.3 |
D-2015 | > 0.5 | -13.5 |
D-2016 | > 0.5 | -12.4 |
D-2017 | .00448 | -89.4 |
D-2018 | .0104 | -36.2 |
D-2019 | .0302 | -7.7 |
D-2020 | .01 | -78.9 |
D-2021 | .00435 | -83.5 |
D-2022 | .00628 | -88.6 |
D-2023 | .0143 | -44.3 |
D-2024 | > 0.5 | 11 |
D-2025 | .00355 | -58.2 |
D-2026 | .000867 | -39.4 |
D-2027 | > 0.5 | 32 |
D-2028 | .00205 | -89.9 |
D-2029 | .0019 | -94 |
D-2030 | > 0.5 | -9.4 |
D-2031 | > 0.5 | 4 |
雙鏈體編號 | IC50 (μM) | PNPLA3敲低 (%) |
D-2001 | .0132 | -70.5 |
D-2003 | .0458 | -46.9 |
D-2004 | .0049 | -74.2 |
D-2006 | .00283 | -69.6 |
D-2009 | .00448 | -62.2 |
D-2010 | .00206 | -52 |
D-2013 | .00319 | -71.7 |
D-2014 | .00164 | -67.4 |
D-2017 | .00222 | -60.9 |
D-2018 | .00717 | -52.7 |
D-2020 | .00664 | -68.3 |
D-2021 | .00559 | -61.5 |
D-2022 | .004 | -30.5 |
D-2023 | > 0.5 | -18 |
D-2025 | .0041 | -61.9 |
D-2026 | .00937 | -34.6 |
D-2043 | > 0.1 | -5.394 |
D-2045 | > 0.1 | -37.195 |
D-2046 | > 0.1 | 0.94 |
D-2047 | > 0.1 | -11.446 |
D-2048 | 0.000355 | -56.824 |
D-2049 | 未定義 | -30.881 |
D-2050 | > 0.1 | -11.814 |
D-2051 | 0.000903 | -73.749 |
D-2052 | 0.000235 | -38.467 |
D-2053 | 0.000142 | -19.405 |
D-2054 | > 0.1 | -29.069 |
D-2055 | > 0.1 | -31.986 |
D-2056 | 未定義 | -38.596 |
D-2057 | 0.000197 | -87.199 |
D-2058 | 0.00126 | -40.177 |
D-2059 | > 0.1 | -11.162 |
D-2060 | > 0.1 | -8.147 |
D-2069 | 0.0329 | -62.804 |
D-2072 | 0.00377 | -47.552 |
D-2078 | > 0.1 | -25.077 |
D-2073 | 0.00602 | -54.092 |
D-2084 | 0.000549 | -45.854 |
D-2084 | 0.0018 | -60.106 |
D-2085 | 0.000122 | -38.539 |
D-2085 | 0.0076 | -68.83 |
D-2086 | 0.00182 | -25.524 |
D-2087 | 0.00103 | -53.242 |
D-2088 | 0.00111 | -26.614 |
D-2089 | 0.000776 | -55.436 |
D-2089 | 0.000457 +/- 0.000174 | -62.57 +/- 1.46 |
D-2090 | 未定義 | -56.094 |
D-2092 | 0.000979 | -52.168 |
D-2099 | 0.000727 | -43.233 |
D-2100 | 0.000152 | -40.651 |
D-2104 | 0.000712 | -72.734 |
D-2105 | 0.000129 | -75.994 |
D-2128 | 0.00154 | -60.927 |
D-2138 | 0.0142 | -63.864 |
D-2149 | 0.000518 | -74.288 |
D-2152 | 0.00524 | -64.758 |
D-2153 | 0.00149 | -72.861 |
D-2155 | > 0.1 | -32.937 |
D-2156 | 0.0183 | -75.145 |
D-2157 | > 0.0333 | -37.881 |
D-2158 | 0.00368 | -79.231 |
D-2159 | 0.00167 | -85.848 |
D-2160 | 0.00442 | -37.827 |
D-2172 | 0.0000615 | -74.462 |
D-2182 | 0.000211 | -79.605 |
D-2183 | 0.000159 | -80.865 |
D-2187 | 0.00109 | -67.777 |
D-2188 | 0.000728 | -71.503 |
D-2189 | 0.000224 | -58.772 |
D-2190 | 0.000803 | -72.508 |
D-2192 | 0.00125 | -33.021 |
D-2193 | 0.000152 | -74.239 |
D-2194 | 0.00385 | -50.848 |
D-2196 | 0.000362 | -70.158 |
D-2197 | 0.000180 | -54.44 |
D-2198 | 0.000269 | -45.556 |
D-2199 | 0.000198 | -81.082 |
D-2201 | 0.000356 | -82.2 |
D-2202 | 0.000123 | -62.466 |
D-2203 | 0.00476 | -68.112 |
D-2204 | 0.000300 | -63.383 |
D-2205 | 0.000442 | -70.712 |
D-2206 | 0.000969 | -74.194 |
D-2208 | 0.000202 | -64.046 |
D-2209 | 0.000752 | -63.083 |
D-2210 | 0.0000991 | -60.426 |
D-2211 | 0.0000948 | -71.098 |
D-2213 | 0.00119 | -65.57 |
D-2214 | 0.000293 | -72.959 |
D-2215 | 0.000307 | -64.561 |
D-2217 | 0.00198 | -87.273 |
D-2218 | 0.000186 | -64.522 |
D-2219 | 0.000577 | -74.879 |
D-2220 | 0.000189 | -74.278 |
D-2221 | 0.000173 | -54.639 |
D-2223 | 0.00131 | -69.834 |
D-2224 | 0.000527 | -84.911 |
D-2225 | 0.000561 | -68.969 |
D-2226 | 0.00109 | -75.536 |
D-2227 | 0.000227 | -85.915 |
D-2228 | 0.00202 | -50.227 |
D-2229 | 未定義 | -59.847 |
D-2230 | 0.000156 | -53.194 |
D-2231 | 0.000351 | -65.054 |
D-2232 | 0.0000619 | -63.991 |
D-2233 | 0.0000982 | -74.304 |
D-2234 | 0.000290 | -81.992 |
D-2235 | 0.000127 | -83.593 |
D-2236 | 0.000243 | -80.826 |
D-2237 | 0.0000711 | -62.814 |
D-2238 | 0.0000690 | -76.476 |
D-2239 | 0.000123 | -74.747 |
D-2240 | 0.000120 | -79.301 |
D-2241 | 0.000201 | -88.769 |
D-2242 | 0.000179 | -84.503 |
D-2243 | 0.000264 | -88.712 |
D-2244 | 0.0000605 | -82.556 |
D-2245 | 0.000125 | -83.93 |
D-2246 | 0.000122 | -84.41 |
D-2247 | 0.000100 | -81.777 |
D-2248 | 0.000141 | -79.18 |
D-2249 | 0.0000710 | -85.093 |
D-2250 | 0.000494 | -81.724 |
D-2251 | 0.000196 | -87.601 |
D-2252 | 0.0000796 | -62.87 |
D-2253 | 0.000501 | -78.699 |
D-2254 | 0.0000979 | -67.617 |
D-2255 | 0.000359 | -81.18 |
D-2256 | 0.000610 | -98.206 |
D-2257 | 0.000151 | -76.718 |
D-2258 | 0.000325 | -87.548 |
D-2259 | 0.000124 | -82.545 |
D-2260 | 0.000109 | -85.47 |
D-2261 | 0.000231 | -75.386 |
D-2262 | 0.000162 | -72.407 |
D-2263 | 0.000271 | -74.968 |
D-2264 | 0.000132 | -76.418 |
D-2265 | > 0.1 | -17.144 |
D-2266 | 未定義 | -58.475 |
D-2267 | > 0.1 | -3.991 |
D-2280 | 0.0000390 | -85.368 |
D-2286 | 0.00304 | -89.536 |
D-2287 | 0.00142 | -85.435 |
D-2287 | 0.000261 | -66.501 |
D-2288 | 0.000839 | -81.988 |
D-2289 | 0.0012 | -83.437 |
D-2289 | 0.00168 | -69.471 |
D-2290 | 0.00157 | -73.089 |
D-2291 | 0.0112 | -64.108 |
D-2292 | 0.00411 | -66.681 |
D-2292 | 0.00138 | -56.253 |
D-2293 | 0.00112 | -61.118 |
D-2294 | 0.000408 | -70.684 |
D-2295 | 0.000195 | -65.493 |
D-2297 | 0.00165 | -61.821 |
D-2297 | 0.000684 | -69.444 |
D-2318 | 0.000986 | -69.28 |
D-2319 | 0.00136 | -67.654 |
D-2320 | 0.00129 | -83.723 |
D-2323 | 0.00194 | -74.525 |
D-2324 | 0.00209 | -63.745 |
D-2325 | 0.000301 | -81.302 |
D-2327 | 0.000306 | -77.959 |
D-2327 | 0.000610 | -56.293 |
D-2328 | 0.000131 | -72.631 |
D-2345 | 0.000131 | -72.677 |
D-2346 | 0.0000274 | -71.459 |
D-2347 | 0.000640 | -78.414 |
D-2348 | 0.000101 | -64.627 |
D-2349 | 0.000487 | -52.131 |
D-2350 | 0.000102 | -68.668 |
D-2351 | 未定義 | -70.229 |
D-2352 | 0.000332 | -81.083 |
D-2352 | 0.0027713 +/- 0.00426 | -68.138 +/- 9.28 |
D-2353 | 0.000410 | -83.001 |
D-2353 | 0.00215 | -64.343 |
D-2354 | 0.003405 +/- 0.00185 | -72.56 +/- 7.42 |
D-2354 | 0.000362 | -72.776 |
D-2355 | 0.00215 | -69.321 |
D-2358 | 0.0011 | -61.081 |
D-2359 | 0.000654 | -76.258 |
D-2360 | 未定義 | -59.027 |
D-2361 | 0.00414 | -57.88 |
D-2362 | 0.000772 | -87.512 |
D-2364 | 0.00104 | -53.895 |
D-2370 | 0.0024 | -83.423 |
D-2371 | 0.00418 | -72.262 |
D-2372 | 0.00492 | -67.648 |
D-2373 | 0.000906 | -64.099 |
D-2374 | 0.00282 | -64.049 |
D-2375 | 0.00125 | -59.813 |
D-2376 | 0.00171 | -63.025 |
D-2377 | 0.00168 | -61.597 |
D-2378 | 0.00505 | -72.446 |
D-2379 | 0.00511 | -80.576 |
D-2380 | 0.0497 | -82.835 |
D-2381 | 0.000944 | -71.788 |
D-2382 | 0.000830 | -65.087 |
D-2383 | 0.00118 | -71.393 |
D-2386 | 0.0000367 | -44.073 |
D-2387 | 0.00224 | -74.24 |
D-2390 | 0.00188 | -69.313 |
D-2391 | 0.000493 | -72.314 |
D-2393 | 0.008875 +/- 0.0112 | -48.855 +/- 15.6 |
D-2401 | 0.000125 | -69.217 |
D-2409 | 0.00479 | -68.703 |
D-2402 | > 0.5 | 21.01 +/- 6.08 |
D-2410 | 0.00533 | -64.812 |
D-2411 | 0.00283 | -64.889 |
D-2412 | 0.00211 | -67.781 |
D-2413 | 0.006575 +/- 0.0000212 | -64.021 +/- 15.8 |
D-2417 | 0.00558 | -50.293 |
D-2418 | 0.00566 | -59.533 |
D-2419 | > 0.5 | -15.364 +/- 7.56 |
D-2423 | 0.000498 +/- 0.000414 | -65.15 +/- 17.5 |
D-2426 | > 0.1 | -15.207 |
D-2430 | 0.0000511 | -63.602 |
D-2444 | 0.000262 +/- 0.000192 | -67.206 +/- 9.13 |
D-2454 | 0.001795 +/- 0.000106 | -51.619 +/- 0.163 |
D-2456 | 0.000274 | -61.291 |
D-2472 | 0.000314 | -70.624 |
D-2473 | > 0.1 | -19.624 +/- 7.27 |
D-2474 | 0.000339 | -37.846 |
D-2475 | 0.002249 +/- 0.00231 | -41.661 +/- 8.64 |
D-2476 | 0.000594 | -26.48 |
D-2477 | 0.000547 | -61.592 |
D-2478 | 0.000168 | -65.904 |
D-2479 | 0.0005565 | -65.14 |
D-2480 | 0.000907 | -53.271 |
D-2481 | 0.0004005 | -59.5 |
D-2482 | 0.000493 | -61.712 |
D-2483 | 0.0004015 +/- 0.000218 | -66.803 +/- .197 |
D-2484 | 0.0003375 | -67.4 |
D-2485 | 0.0002865 | -44.529 |
D-2486 | 0.0012575 | -62.847 |
D-2487 | 0.000454 | -54.396 |
D-2488 | 0.0002415 | -66.315 |
D-2489 | > 0.5 | -2.973 +/- 1.98 |
D-2490 | 0.0011555 | -62.566 |
D-2491 | 0.0006005 | -60.304 |
D-2492 | 0.0007775 | -47.748 |
D-2493 | 0.001465 | -48.8 |
D-2494 | 0.0008985 | -50.707 |
D-2495 | 0.001317 | -55.5 |
D-2496 | 0.0001374 | -68.949 |
D-2497 | 0.00013615 +/- 0.0000691 | -72.763 +/- 4.02 |
D-2498 | 0.000332 +/- 0.000259 | -82.507 +/- 6.15 |
D-2499 | 0.0004155 | -75.4 |
D-2500 | 0.0000742 +/- 0.0000407 | -74.968 +/- 5.41 |
D-2501 | > 0.5 | -1.648 +/- 2.2 |
D-2502 | 0.0002705 +/- 0.000252 | -68.406 +/- 5.36 |
D-2503 | 0.000313 +/- 0.000198 | -67.858 +/- 8.2 |
D-2504 | 0.0006445 +/- 0.000385 | -59.733 +/- 7.96 |
D-2505 | 0.0004985 | -53.3 |
D-2506 | > 0.1 | -16.588 +/- 14.9 |
D-2507 | 0.002136 | -64.978 |
D-2508 | > 0.1 | -20.61 +/- 11.1 |
D-2509 | 0.001242 | -65.112 |
D-2510 | 0.0015895 | -56.416 |
D-2511 | 0.00166 | -19.901 |
D-2512 | 0.000593 | -0.374 |
D-2513 | 0.0001497 +/- 0.000123 | -69.663 +/- 11.6 |
D-2514 | 0.000204 +/- 0.0000594 | -69.924 +/- 13.7 |
D-2515 | 0.000331 +/- 0.000129 | -71.29 +/- 8.74 |
D-2516 | 0.000348 +/- 0.000249 | -65.905 +/- 14.8 |
D-2517 | 0.000452 +/- 0.000322 | -68.212 +/- 11.2 |
D-2518 | 0.0006965 | -73.226 |
D-2519 | > 0.5 | -1.486 +/- 4.67 |
D-2520 | 0.000098 | -75.127 |
D-2521 | 0.000717 +/- 0.000378 | -61.216 +/- 12.8 |
D-2522 | 0.000661 +/- 0.000748 | -56.771 +/- 16.9 |
D-2523 | 0.0003685 | -73.312 |
D-2524 | 0.0002745 +/- 0.0000686 | -65.341 +/- 12.7 |
D-2525 | 0.003855 +/- 0.0033 | -44.288 +/- 19.0 |
D-2526 | 0.0006115 +/- 0.000204 | -57.77 +/- 13.2 |
D-2527 | 0.000333 | -77.405 |
D-2528 | 0.0002665 +/- 0.000105 | -62.407 +/- 8.65 |
D-2529 | 0.0076967 +/- 0.00555 | -44.194 +/- 6.73 |
D-2530 | 0.0132 | -42.657 +/- 0.222 |
D-2531 | 0.00312 | -32.789 +/- 12.0 |
D-2532 | 0.005795 +/- 0.00268 | -44.051 +/- 2.82 |
D-2533 | 0.005515 +/- 0.00356 | -43.145 |
D-2534 | 0.0129 | -28.734 +/- 16.4 |
D-2535 | 0.003195 +/- 0.000233 | -29.79 |
D-2536 | 0.005865 +/- 0.00456 | -25.918 |
D-2537 | 0.0133 | -25.755 +/- 3.32 |
D-2538 | > 0.1 | -40.955 +/- 5.3 |
D-2539 | 0.004785 +/- 0.00183 | -34.334 +/- 11.0 |
D-2540 | 0.0044 +/- 0.00075 | -39.221 |
D-2541 | 0.002735 +/- 0.00141 | -62.39 +/- 3.03 |
D-2542 | 0.01323 +/- 0.0108 | -30.917 +/- 1.57 |
D-2543 | > 0.1 | -8.54 +/- 8.37 |
D-2544 | 0.017 | -23.757 +/- 2.2 |
D-2545 | 0.01642 +/- 0.0164 | -52.394 +/- 7.21 |
D-2546 | > 0.1 | -25.351 +/- 7.84 |
D-2547 | 0.00412 | -25.042 +/- 5.28 |
D-2548 | 0.00411 +/- 0.00184 | -40.406 +/- 1.26 |
D-2549 | 0.02285 +/- 0.00318 | -53.713 +/- 14.1 |
D-2550 | 0.015935 +/- 0.0115 | -41.907 |
D-2551 | 0.00795 | -26.39 |
D-2552 | 0.01165 +/- 0.00841 | -41.306 |
D-2553 | 0.00361 | -70.457 |
D-2554 | 0.00656 | -42.866 |
D-2555 | > 0.1 | -11.425 |
D-2556 | 0.00336 | -63.996 |
D-2557 | > 0.1 | -2.734 |
D-2558 | 0.00251 | -66.775 |
D-2559 | > 0.1 | -7.034 |
D-2560 | 0.0234 | -44.657 |
D-2561 | 0.00154 | -74.225 |
D-2562 | 0.00454 | -66.578 |
D-2563 | 0.00182 | -77.025 |
D-2564 | 0.009 | -64.021 |
D-2565 | 0.00609 | -70.965 |
D-2566 | 0.00255 | -52.65 |
D-2567 | 0.00584 | -56.603 |
D-2568 | 0.0157 | -63.002 |
D-2569 | 0.00327 | -64.898 |
D-2570 | 0.00144 | -55.424 |
D-2571 | > 0.1 | -18.661 |
D-2572 | 0.00557 | -25.668 |
D-2573 | 0.0115 | -55.329 |
D-2574 | 0.00289 | -69.84 |
D-2575 | 0.00378 | -69.491 |
D-2576 | 0.00527 | -64.841 |
D-2577 | 0.00511 | -46.449 |
D-2578 | 0.0026 | -67.821 |
D-2579 | 0.00402 | -67.057 |
D-2580 | 0.00119 | -64.422 |
D-2581 | 0.00915 | -73.008 |
D-2582 | 0.000823 | -77.053 |
D-2583 | 0.00851 | -66.555 |
D-2584 | 0.00513 | -54.442 |
D-2585 | 0.0154 | -67.707 |
D-2586 | 0.00701 | -69.624 |
D-2587 | 0.00732 | -66.627 |
D-2588 | 0.00226 | -70.854 |
D-2589 | 0.00837 | -31.221 |
D-2590 | 0.0249 | -41.857 |
D-2591 | 0.009 | -64.021 |
D-2592 | 0.00609 | -70.965 |
D-2593 | 0.00255 | -52.65 |
D-2594 | 0.00584 | -56.603 |
D-2595 | 0.0157 | -63.002 |
D-2596 | 0.00327 | -64.898 |
D-2597 | 0.00144 | -55.424 |
D-2598 | > 0.1 | -18.661 |
D-2599 | 0.00557 | -25.668 |
D-2600 | 0.0115 | -55.329 |
D-2601 | 0.00289 | -69.84 |
D-2602 | 0.00378 | -69.491 |
D-2603 | 0.00527 | -64.841 |
D-2604 | 0.00511 | -46.449 |
D-2605 | 0.0026 | -67.821 |
D-2606 | 0.00402 | -67.057 |
D-2607 | 0.00119 | -64.422 |
D-2608 | 0.00915 | -73.008 |
D-2609 | 0.000823 | -77.053 |
D-2610 | 0.00141 | -37.663 +/- 39.4 |
D-2611 | > 0.1 | -2.829 +/- 0.006 |
D-2612 | > 0.1 | -15.894 |
D-2613 | 0.00809 | -58.833 |
D-2614 | 0.00204 | -73.042 |
D-2615 | 0.00306 | -70.087 |
D-2616 | 7.95E-04 | -71.014 |
D-2617 | 0.00218 | -75.769 |
D-2618 | 0.0429 | -31.19 +/- 2.61 |
D-2619 | 0.02495 +/- 0.0192 | -59.645 +/- 13.4 |
D-2620 | 0.0055 +/- 0.00329 | -73.308 +/- 5.33 |
D-2621 | 0.004705 +/- 0.00176 | -63.053 +/- 7.66 |
D-2622 | 0.004525 +/- 0.00231 | -66.895 +/- 7.78 |
D-2623 | 0.00435 | -31.366 +/- 14.5 |
D-2624 | 0.0056 +/- 0.0016 | -65.16 +/- 10.9 |
D-2625 | 0.00764 +/- 0.00115 | -73.065 +/- 9.97 |
D-2626 | 0.021645 +/- 0.0267 | -71.117 +/- 3.63 |
D-2627 | 0.00651 +/- 0.00489 | -63.009 +/- 8.83 |
D-2628 | 0.01334 +/- 0.00588 | -48.771 +/- 2.35 |
D-2629 | 0.005504 +/- 0.00434 | -74.111 +/- 4.61 |
D-2630 | 0.007965 +/- 0.00684 | -70.17+/- 5.32 |
D-2631 | > 0.1 | -8.645 |
D-2632 | > 0.1 | -37.841 |
D-2633 | 0.0111 | -53.354 |
D-2634 | 0.0187 | -63.686 |
D-2635 | 0.00797 | -58.605 |
D-2636 | > 0.1 | -34.727 |
D-2637 | 0.00541 | -61.225 |
D-2638 | 0.00397 | -62.221 |
D-2639 | 0.0295 +/- 0.0187 | -51.328 +/- 12.8 |
D-2640 | 0.0212 | -37.799 +/- 16.1 |
D-2641 | 0.00164 | -21.913 +/- 14.1 |
D-2642 | > 0.1 | -11.733 +/- 23.5 |
D-2643 | > 0.1 | -23.009 |
D-2644 | 0.00165 | -46.514 |
D-2645 | 0.00242 | -75.956 |
D-2646 | > 0.1 | -15.539 +/- 1.03 |
D-2647 | 0.00243 | -73.363 |
D-2648 | 0.00117 +/- 0.0000849 | -74.63 +/- 1.55 |
D-2649 | 0.007455 +/- 0.00756 | -46.662 +/- 2.86 |
D-2650 | 0.003845 +/- 0.00122 | -67.914 +/- 1.87 |
D-2651 | 0.000449 | -70.274 |
D-2652 | 0.0014 | -66.6 |
D-2653 | 0.00299 | -59.695 |
D-2654 | 0.00431 | -26.338 +/- 4.54 |
D-2655 | 0.00254 +/- 0.0012 | -52.632 +/- 0.938 |
D-2656 | 0.003275 +/- 0.00112 | -37.351 +/- 0.79 |
D-2657 | 0.007435 +/- 0.00357 | -37.313 +/- 3.03 |
D-2658 | > 0.1 | -22.486 +/- 10.6 |
D-2659 | > 0.1 | -10.905 +/- 3.7 |
D-2660 | > 0.1 | -4.489 +/- 8.45 |
D-2661 | 0.00875 +/- 0.00728 | -56.877 +/- 9.41 |
D-2662 | > 0.1 | -8.133 |
D-2663 | 0.00268 +/- 0.000778 | -73.024 +/- 1.16 |
D-2664 | 0.008 | -30.908 +/- 1.19 |
D-2665 | 0.004675 +/- 0.000516 | -39.914 +/- 3.72 |
D-2666 | 0.004085 +/- 0.000106 | -46.423 +/- 3.66 |
D-2667 | 0.017107 +/- 0.0145 | -72.967 +/- 2.96 |
D-2668 | 0.00589 +/- 0.000919 | -73.779 +/- 2.9 |
D-2669 | 0.0035033 +/- 0.00167 | -67.153 +/- 0.761 |
D-2670 | 0.0129 | -59.149 +/- 0.371 |
D-2671 | 0.0157 +/- 0.00156 | -57.5 +/- 3.88 |
D-2672 | > 0.1 | -3.262 +/- 6.36 |
D-2673 | 0.00461 +/- 0.00173 | -50.295 +/- 14.1 |
D-2674 | 0.00279 | -54.886 +/- 32.2 |
D-2675 | > 0.1 | -13.5 +/- 21.4 |
D-2676 | 0.015075 +/- 0.0156 | -50.255 +/- 8.86 |
D-2677 | > 0.1 | -22.123 |
D-2678 | > 0.1 | -43.995 |
D-2679 | 0.00268 | -83.804 |
D-2680 | > 0.1 | -47.702 |
D-2681 | > 0.1 | -38.246 |
D-2682 | 0.00305 +/- 0.000255 | -70.849 +/- 11.3 |
D-2683 | 0.00363 | -59.037 +/- 15.6 |
D-2684 | 0.00763 | -74.35 |
D-2685 | 0.00585 +/- 0.00379 | -72.725 +/- 18.8 |
D-2686 | > 0.1 | -6.429 |
D-2687 | 0.0031933 +/- 0.000225 | -70.122 +/- 5.47 |
D-2688 | 0.0023063 +/- 0.00152 | -65.267 +/- 8.73 |
D-2689 | 0.0025767 +/- 0.000721 | -66.016 +/- 11.4 |
D-2690 | 0.0032367 +/- 0.00272 | -63.04+/- 13.4 |
D-2691 | > 0.1 | -63.208 |
D-2692 | 0.00376 | -62.833 |
D-2693 | 0.002915 +/- 0.00131 | -71.229 +/- 10.9 |
D-2694 | 0.003245 +/- 0.000573 | -69.264 |
D-2695 | 0.01652 +/- 0.0179 | -51.139 +/- 17.8 |
D-2696 | 0.0024 | 12.88 |
D-2697 | 0.013 | -63.845 |
D-2698 | 0.003285 +/- 0.000304 | -79.24 |
D-2699 | 0.002205 +/- 0.00117 | -67.306 +/- 0.216 |
D-2700 | 0.0022297 +/- 0.00129 | -67.949 +/- 7.66 |
D-2701 | 0.0026037 +/- 0.00196 | -54.669 +/- 7.49 |
D-2702 | 0.00448 | -64.496 |
D-2703 | 0.00254 | -65.869 |
D-2704 | 0.00257 | -71.285 |
D-2705 | 0.002505 +/- 0.000346 | -64.722 +/- 4.62 |
D-2706 | 0.003865 +/- 0.00264 | -63.247 +/- 10.0 |
D-2707 | 0.003425 +/- 0.000318 | -65.82+/- 2.07 |
D-2708 | 0.0055112 +/- 0.00582 | -66.395 +/- 10.4 |
D-2709 | 0.00243 | -76.778 |
D-2710 | 0.000912 | -78.289 |
D-2711 | > 0.1 | 4.37 +/- 9.89 |
D-2712 | 0.0046104 +/- 0.00608 | -65.876 +/- 9.59 |
D-2713 | 0.0010115 +/- 0.00102 | -67.741 +/- 2.46 |
D-2714 | 0.0063585 +/- 0.00869 | -66.904 +/- 0.044 |
D-2715 | 0.0061022 +/- 0.00862 | -74.054 |
D-2716 | 0.00338 +/- 0.00308 | -70.054 +/- 9.47 |
D-2717 | 0.0033 +/- 0.00155 | -72.932 +/- 7.6 |
D-2718 | 0.0155 | -58.784 |
D-2719 | 0.00527 | -57.281 |
D-2720 | 0.0215 | -41.76 |
D-2721 | 0.01829 +/- 0.0132 | -64.649 +/- 3.84 |
D-2722 | 0.0164 | -53.261 |
D-2723 | 0.00733 | -47.56 |
D-2724 | 0.00564 | -37.19 |
D-2725 | 0.00776 | -42.77 |
D-2726 | 0.00675 | -42.811 |
D-2727 | 0.00667 | -57.414 |
D-2728 | 0.0438 | -37.169 |
D-2729 | 0.00454 | -25.538 |
D-2730 | 0.0241 | -47.501 |
D-2731 | 0.0088 | -44.866 |
D-2732 | 0.0326 | -44.68 |
D-2733 | 0.0217 | -25.146 |
D-2734 | > 0.1 | -14.582 |
D-2735 | 0.0202 | -28.294 |
D-2736 | 0.014 | -38.251 |
D-2737 | > 0.1 | -17.833 |
D-2738 | 0.00514 | -62.424 |
D-2739 | 0.0317 | -41.409 |
D-2740 | 0.0111 | -59.253 |
D-2741 | 0.0111 | -59.687 |
D-2742 | 0.0229 | -82.351 |
D-2743 | 0.00933 | -59.271 |
D-2744 | 0.0215 | -54.24 |
D-2745 | 0.00619 | -57.406 |
D-2746 | 0.00654 | -69.738 |
D-2747 | 0.0307 | -63.431 |
D-2748 | 0.0152 | -59.477 |
D-2749 | 0.0329 | -45.543 |
D-2750 | 0.0149 | -47.589 |
D-2751 | 0.0261 | -54.747 |
D-2752 | 0.0106 | -65.912 |
D-2753 | 0.0113 | -66.051 |
D-2754 | 0.0201 | -65.582 |
D-2755 | > 0.1 | -25.144 |
D-2756 | 0.0179 | -65.965 |
D-2757 | 0.0251 | -40.795 |
D-2758 | 0.0312 | -40.55 |
D-2759 | 0.00936 | -63.545 |
D-2760 | 0.00859 | -74.99 |
D-2761 | 0.0115 | -71.716 |
D-2762 | 0.00777 | -68.169 |
D-2763 | 0.0106 | -72.426 |
D-2764 | 0.0327 | -58.009 |
D-2766 | 0.003215 +/- 0.000516 | -77.976 |
D-2767 | 0.00376 +/- 0.000552 | -74.346 |
D-2768 | 0.00396 +/- 0.0000566 | -76.319 |
D-2769 | 0.005655 +/- 0.00165 | -72.844 +/- 3.26 |
D-2770 | 0.0041 +/- 0.000778 | -79.301 +/- 1.36 |
D-2771 | 0.00891 +/- 0.00137 | -70.223 |
D-2772 | 0.004855 +/- 0.00000707 | -72.651 |
D-2773 | 0.004115 +/- 0.000544 | -77.716 +/- 5.2 |
D-2774 | 0.00467 +/- 0.000354 | -76.397 +/- 3.67 |
雙鏈體編號 | IC50 (μM) | PNPLA3敲低 (%) |
D-2001 | .00842 | -37 |
D-2003 | .0158 | -32.1 |
D-2004 | .00266 | -32.4 |
D-2006 | .00948 | -54.1 |
D-2009 | .00228 | -29.5 |
D-2010 | .00219 | -37.2 |
D-2013 | .00524 | -31.5 |
D-2014 | .00148 | -37.6 |
D-2017 | .00333 | -37.6 |
D-2018 | .00315 | -21.3 |
D-2020 | > 0.5 | 6 |
D-2021 | > 0.5 | -1.6 |
D-2022 | .00272 | -30.9 |
D-2023 | > 0.5 | 24 |
D-2025 | .0101 | -30.3 |
D-2026 | .00551 | -23 |
D-2413 | -45.832 | |
D-2419 | > 0.5 | -4.545 |
D-2426 | > 0.1 | -15.207 |
D-2454 | 0.00187 | -51.735 |
D-2473 | > 0.1 | -21.333 |
D-2522 | 0.00504 | -43.036 |
D-2523 | 0.0122 | -52.084 |
D-2564 | 0.01 | -63.199 |
D-2565 | 0.00938 | -58.392 |
D-2566 | 0.00343 | -61.484 |
D-2567 | 0.0175 | -53.489 |
D-2568 | > 0.1 | -18.367 |
D-2569 | 0.0195 | -62.568 |
D-2570 | 0.0127 | -77.141 |
D-2571 | > 0.1 | -15.922 |
D-2572 | > 0.1 | -12.434 |
D-2573 | > 0.1 | -14.649 |
D-2574 | 0.0215 | -52.515 |
D-2575 | 0.0203 | -53.175 |
D-2576 | 0.018 | -48.137 |
D-2577 | > 0.1 | -16.105 |
D-2578 | > 0.1 | -21.309 |
D-2579 | > 0.1 | -17.510 |
D-2580 | > 0.1 | -24.616 |
D-2581 | > 0.1 | -13.987 |
D-2582 | 0.0574 | -30.543 |
D-2583 | > 0.1 | -23.990 |
D-2584 | > 0.1 | -6.715 |
D-2585 | > 0.1 | -17.518 |
D-2586 | > 0.1 | -24.518 |
D-2587 | 0.0391 | -58.478 |
D-2588 | 0.0218 | -56.609 |
D-2589 | > 0.1 | -17.418 |
D-2590 | > 0.1 | -21.161 |
D-2591 | 0.0167 | -59.366 |
D-2592 | 0.0104 | -61.548 |
D-2593 | 未定義 | -61.879 |
D-2594 | 0.0211 | -43.856 |
D-2595 | 0.0272 | -63.020 |
D-2596 | > 0.1 | -10.278 |
D-2597 | 0.0546 | -31.743 |
D-2598 | 未定義 | -47.517 |
D-2599 | 0.00489 | -70.825 |
D-2600 | > 0.1 | -8.522 |
D-2601 | 0.0364 | -31.836 |
D-2602 | 0.00577 | -65.062 |
D-2603 | 0.01 | -58.287 |
D-2604 | 0.00353 | -40.649 |
D-2605 | 0.0113 | -50.691 |
D-2606 | > 0.1 | -5.097 |
D-2607 | 0.0261 | -49.898 |
D-2608 | > 0.1 | -23.747 |
D-2609 | > 0.1 | -23.804 |
D-2620 | > 0.5 | -21.969 |
D-2637 | >0.1 | -12.63 |
D-2638 | >0.1 | -16.25 |
D-2639 | >0.1 | -22.905 |
D-2640 | >0.1 | -14.572 |
D-2666 | > 0.5 | -14.898 |
D-2667 | > 0.5 | -14.537 |
D-2668 | > 0.5 | -18.779 |
D-2669 | > 0.5 | -8.624 |
D-2670 | > 0.5 | -16.641 |
D-2671 | > 0.5 | -8.665 |
D-2672 | > 0.5 | 4.92 |
D-2673 | > 0.5 | -8.024 |
D-2675 | > 0.5 | -17.999 |
D-2676 | > 0.5 | -22.989 |
D-2677 | > 0.5 | -11.245 |
D-2678 | > 0.5 | -10.209 |
D-2679 | -46.578 | |
D-2680 | > 0.5 | -12.289 |
D-2681 | > 0.5 | -9.564 |
D-2682 | 0.184 | -57.848 |
D-2683 | > 0.5 | -19.024 |
D-2684 | > 0.5 | -5.026 |
D-2685 | 0.0395 | -34.673 |
D-2686 | > 0.5 | -12.298 |
D-2687 | > 0.5 | -7.153 |
D-2688 | > 0.5 | -12.895 |
D-2689 | > 0.5 | -20.429 |
D-2690 | > 0.5 | -8.608 |
D-2691 | > 0.5 | -7.608 |
D-2692 | > 0.5 | -7.763 |
D-2693 | > 0.5 | -12.294 |
D-2694 | > 0.5 | -10.718 |
D-2695 | -25.618 | |
D-2696 | > 0.5 | -7.666 |
D-2697 | > 0.5 | -11.529 |
D-2698 | > 0.5 | -11.875 |
D-2699 | > 0.5 | -4.132 |
D-2700 | -34.399 | |
D-2701 | > 0.5 | -4.434 |
D-2702 | > 0.5 | -9.919 |
D-2703 | > 0.5 | -9.667 |
D-2704 | > 0.5 | -1.989 |
D-2705 | > 0.5 | -8.5 |
D-2706 | >0.167 | |
D-2707 | >0.167 | |
D-2708 | 0.103 | -32.624 |
D-2709 | > 0.5 | -2.042 |
D-2710 | > 0.5 | -5.079 |
D-2712 | > 0.5 | -1.399 |
D-2717 | > 0.5 | -1.399 |
D-2718 | 0.00521 | -38.552 |
D-2719 | 0.013 | |
D-2720 | > 0.5 | -2.52 |
D-2721 | > 0.5 | -3.882 |
D-2722 | > 0.5 | -6.527 |
D-2723 | > 0.5 | -16.917 |
D-2724 | 0.0227 | -46.386 |
D-2725 | 0.0187 | |
D-2726 | 0.0105 | |
D-2727 | 0.0124 | -38.592 |
D-2728 | 0.00741 | -30.796 |
D-2729 | 0.0152 | -36.3 |
D-2730 | > 0.5 | -17.996 |
D-2731 | 0.00433 | -45.007 |
D-2732 | > 0.5 | -4.662 |
D-2733 | > 0.5 | -8.838 |
D-2734 | > 0.5 | -11.614 |
D-2735 | > 0.5 | -15.778 |
D-2736 | > 0.5 | -17.337 |
D-2737 | > 0.5 | -11.139 |
D-2717 | > 0.5 | -6.604 |
D-2738 | 0.00878 | -30.636 |
D-2739 | > 0.5 | -12.614 |
D-2740 | 0.013 | -57.307 |
D-2741 | 0.00938 | -48.272 |
D-2742 | 0.0271 | |
D-2743 | 0.00623 | -41.449 |
D-2744 | 0.015 | -37.951 |
D-2745 | 0.00611 | -53.267 |
D-2746 | 0.0108 | -63.434 |
D-2747 | > 0.5 | -13.578 |
D-2748 | 0.0382 | -38.04 |
D-2749 | > 0.5 | -8.412 |
D-2750 | > 0.5 | -2.25 |
D-2751 | > 0.5 | -12.014 |
D-2752 | > 0.5 | 10.68 |
D-2753 | > 0.5 | -3.443 |
D-2754 | 0.0704 | -40.897 |
D-2755 | > 0.5 | -9.405 |
D-2756 | > 0.5 | -18.327 |
D-2757 | > 0.5 | -10.531 |
D-2758 | > 0.5 | -13.929 |
D-2759 | 0.0174 | -39.948 |
D-2760 | 0.048 | -66.415 |
D-2761 | > 0.5 | -14.128 |
D-2762 | 0.0132 | -61.074 |
D-2763 | 0.0333 | -67.071 |
D-2764 | > 0.5 | -10.771 |
D-2413 | -45.832 | |
D-2419 | > 0.5 | -4.545 |
按照製造商方案,在OptiThaw培養基(Xenotech公司目錄號K8000)中解凍人原代肝細胞(Xenotech公司 / 積水化學工業株式會社(Sekisui)供體批號#HC3-38),將細胞離心且在培養基抽吸後,重懸浮於OptiPlate肝細胞培養基(Xenotech公司目錄號K8200)中,且塗到96孔膠原蛋白塗佈之平板中(葛萊娜公司(Greiner)目錄號655950)。在溫育2-4小時後,除去培養基且用OptiCulture肝細胞培養基(Xenotech公司目錄號K8300)替換。加入OptiCulture培養基後2-4小時,藉由自由攝取(無轉染試劑)將經GalNAc綴合之siRNA遞送至細胞。將細胞在37°C及5% CO2下溫育24-72小時。然後用Qiagen RLT緩衝液 (79216)+ 1% 2-巰基乙醇(西格瑪公司(Sigma),M-3148)裂解細胞,且將裂解物儲存於 -20°C。根據製造商說明書,使用Qiagen QIACube HT儀器(9001793)及Qiagen RNeasy 96 QIACube HT套組(74171)純化RNA。使用QIAxpert系統(9002340)分析樣本。使用Applied Biosystems High Capacity cDNA Reverse Transcription套組(4368813)由RNA樣本合成cDNA,根據製造商說明書組裝反應,輸入隨樣本而變化的RNA濃度。在以下條件下在BioRad四分體熱循環儀(型號#PTC-0240G)上進行逆轉錄:25°C 10分鐘、37°C 120分鐘、85°C 5分鐘、然後(視情況)4°C無限保持。
根據製造商說明書,使用BioRad之QX200 AutoDG微滴式數位PCR系統進行微滴式數位PCR(ddPCR)。使用BioRad ddPCR Supermix for Probes(1863010)將反應裝配到Eppendorf透明96孔PCR板(951020303)中,且使用螢光標記PNPA3(IDT Hs.PT.58.21464637,引子與探針比率3.6 : 1及TBP(IDT) Hs.PT.53a.20105486,探針比率為3.6 : 1)及無RNA酶的水(Ambion公司,AM9937)之qPCR測定。最終引子/探針濃度分別為900 nM/250 nM,輸入在孔中不同之cDNA濃度。使用BioRad Auto DG微滴發生器(1864101)形成微滴,該發生器設置有製造商推薦之消耗品(BioRad DG32濾筒1864108、BioRad尖端1864121、Eppendorf blue 96孔PCR板951020362、BioRad用於探針之微滴生成油1864110、及BioRad微滴板組裝)。使用以下條件在BioRad C1000觸摸熱循環儀(1851197)上擴增微滴:在95°C下酶活化10分鐘、在94°C下變性30秒、然後在60°C下退火/延伸1分鐘、使用2°C/秒升溫速率進行40個循環,在98°C下酶失活10分鐘、然後(視情況)在4°C下無限保持。然後在BioRad QX200微滴讀出器上讀取樣本,該讀出器量測與PNPLA3或TBP濃度相關之FAM/HEX信號。使用BioRad之QuantaSoft軟體包分析數據。藉由通道(螢光標記)對樣本進行閘控以判定各樣本之濃度。然後將各樣本表示為目標基因(PNPLA3)濃度/持家基因(TBP)濃度之比率以便控制樣本加載差異。然後將數據導入Genedata Screener,其中將各測試siRNA標準化至中性對照孔(僅緩衝液)之中值。IC50值在表7中報道。
[表7]. 對原代肝細胞之ddPCR測定
實例 4 :在人源化小鼠模型中所選擇 PNPLA3 siRNA 分子之功效篩選
雙鏈體編號 | IC50 (μM) | % PNPLA3敲低 |
D-2068 | .0339 | -49.628 |
D-2069 | .00408 | -52.997 |
D-2070 | .00433 | -42.193 |
D-2072 | .00884 | -53.16 |
D-2073 | > 2.0 | -7.435 |
D-2078 | .0044 | -43.123 |
D-2084 | 0.00499 | -38.791 |
D-2085 | 0.00539 | -64.312 |
D-2086 | > 2.0 | -14.938 |
D-2087 | > 2.0 | -25.465 |
D-2088 | 0.207 | -34.944 |
D-2089 | 0.0107 | -38.791 |
D-2090 | 0.0218 | -38.977 |
D-2091 | 0.0508 | -41.209 |
D-2092 | 0.00192 | -44 |
D-2093 | 0.00634 | -30.233 |
D-2094 | > 2.0 | 4.93 |
D-2095 | 0.00181 | -59.814 |
D-2096 | 0.0181 | -52.807 |
D-2099 | 0.00549 | -39.296 |
D-2100 | 0.0142 | -55.281 |
D-2158 | 0.0681 | -48.649 |
D-2159 | 0.0325 | -36.036 |
D-2160 | > 0.667 | -13.514 |
D-2161 | > 2.0 | -24.229 |
D-2162 | 0.0726 | -28.634 |
D-2163 | > 0.667 | -16.3 |
D-2164 | > 2.0 | -15.418 |
D-2165 | 0.00644 | -26.872 |
D-2166 | 0.00192 | -30.045 |
D-2167 | > 0.667 | -6.726 |
D-2168 | > 2.0 | -15.418 |
D-2169 | > 2.0 | -13.004 |
D-2170 | > 2.0 | -9.417 |
D-2171 | 0.00505 | -44.395 |
D-2172 | 0.003 | -55.336 |
D-2173 | 0.00598 | -46.188 |
D-2174 | > 2.0 | -9.009 |
D-2175 | 0.017 | -27.928 |
D-2176 | 0.00452 | -35.426 |
D-2177 | > 2.0 | 4.5 |
D-2178 | > 2.0 | 1.8 |
D-2179 | > 2.0 | -6.306 |
D-2180 | 0.00546 | -40.969 |
D-2181 | 0.00152 | -43.119 |
D-2182 | 0.00317 | -54.128 |
D-2183 | 0.00232 | -53.211 |
D-2184 | 0.0109 | -50.459 |
D-2185 | > 2.0 | -7.339 |
D-2186 | 0.0021 | -48.624 |
D-2187 | > 2.0 | -11.009 |
D-2188 | > 2.0 | 1.32 |
D-2191 | 0.0984 | -66.923 |
D-2192 | 0.124 | -64.231 |
D-2193 | 0.138 | -60.606 |
D-2194 | 0.0478 | -54.182 |
D-2195 | 0.0801 | -47.515 |
D-2199 | 0.0517 | -62.973 |
D-2201 | 0.0165 | -72.404 |
D-2202 | 0.00946 | -49.459 |
D-2203 | 0.0241 | -58.545 |
D-2204 | 0.0382 | -45.576 |
D-2205 | 0.0222 | -50.946 |
D-2206 | 0.0459 | -46.081 |
D-2209 | 0.0867 | -46.622 |
D-2212 | 0.358 | -60 |
D-2216 | 0.0826 | -58.942 |
D-2218 | 0.0242 | -63.462 |
D-2220 | 0.0113 | -69.333 |
D-2221 | 0.138 | -55.541 |
D-2224 | 0.0198 | -66.486 |
D-2225 | 0.245 | -68.077 |
D-2228 | 0.155 | -44.606 |
D-2229 | 0.0651 | -38.909 |
D-2231 | 0.0512 | -56.892 |
D-2232 | 0.0678 | -67.981 |
D-2233 | 0.00802 | -57.182 |
D-2234 | 0.00473 | -55.947 |
D-2235 | 0.00816 | -62.115 |
D-2236 | 0.00245 | -51.542 |
D-2237 | 0.00495 | -60 |
D-2238 | 0.00561 | -63.017 |
D-2239 | 0.00453 | -55.537 |
D-2240 | 0.00584 | -56.116 |
D-2241 | 0.00755 | -54.76 |
D-2242 | 0.0137 | -56.332 |
D-2243 | 0.00329 | -57.118 |
D-2244 | 0.0127 | -56.909 |
D-2245 | 0.00697 | -58.364 |
D-2246 | 0.00713 | -56.828 |
D-2247 | 0.00875 | -57.797 |
D-2248 | 0.0098 | -58.59 |
D-2249 | 0.00603 | -57.759 |
D-2250 | 0.0105 | -62.155 |
D-2251 | 0.00521 | -59.914 |
D-2252 | 0.00988 | -58.678 |
D-2253 | 0.00481 | -57.118 |
D-2254 | 0.00721 | -56.332 |
D-2255 | 0.00788 | -52.838 |
D-2256 | 0.00831 | -55.455 |
D-2257 | 0.00503 | -54.545 |
D-2258 | 0.00626 | -54.545 |
D-2259 | 0.00401 | -55.947 |
D-2260 | 0.00379 | -52.423 |
D-2261 | 0.00151 | -54.31 |
D-2262 | 0.00292 | -53.448 |
D-2263 | 0.00607 | -59.483 |
D-2264 | 0.00703 | -59.504 |
D-2265 | > 4.0 | -20.524 |
D-2266 | 0.0129 | -32.727 |
D-2267 | 0.107 | -27.273 |
D-2275 | 0.00359 | -45.701 |
D-2276 | 0.00416 | -43.891 |
D-2277 | 0.00218 | -49.14 |
D-2278 | 0.00743 | -42.986 |
D-2279 | 0.0116 | -53.846 |
D-2280 | 0.00347 | -36.652 |
D-2281 | 0.0134 | -37.557 |
D-2282 | 0.00864 | -34.842 |
D-2283 | 0.00738 | -49.558 |
D-2284 | 0.0202 | -38.053 |
D-2285 | 0.00543 | -45.487 |
D-2286 | 0.00934 | -47.611 |
D-2287 | 0.00652 | -55.575 |
D-2288 | 0.0259 | -61.593 |
D-2289 | 0.00549 | -53.805 |
D-2290 | 0.00476 | -51.062 |
D-2291 | 0.0105 | -42.584 |
D-2292 | 0.0059 | -45.455 |
D-2293 | 0.0117 | -45.646 |
D-2294 | 0.0109 | -52.823 |
D-2295 | 0.01246 +/- 0.015 | -59.847 +/- 15.2 |
D-2296 | 0.0279 | -41.346 |
D-2297 | 0.00529 | -55.926 |
D-2298 | 0.00838 | -35.577 |
D-2299 | 0.00832 | -40.865 |
D-2300 | 0.00371 | -40.096 |
D-2301 | 0.00563 | -38.365 |
D-2302 | 0.00639 | -40.385 |
D-2303 | 0.00669 | -41.25 |
D-2304 | 0.00212 | -38.462 |
D-2305 | 0.00573 | -37.736 |
D-2306 | 0.0645 | -33.962 |
D-2307 | 0.00232 | -33.019 |
D-2308 | 0.00181 | -31.132 |
D-2309 | 0.0447 | -45.283 |
D-2310 | 0.00655 | -42.453 |
D-2311 | 0.00613 | -41.509 |
D-2312 | 0.00941 | -50 |
D-2313 | 0.0218 | -41.784 |
D-2314 | 0.0142 | -42.723 |
D-2315 | 0.0182 | -31.455 |
D-2316 | > 4.0 | -23.005 |
D-2317 | 0.0228 | -37.089 |
D-2318 | 0.00809 | -45.352 |
D-2319 | 0.0165 | -44.601 |
D-2320 | 0.0184 | -38.373 |
D-2321 | 0.00766 | -41.627 |
D-2322 | 0.00815 | -46.507 |
D-2323 | 0.0168 | -48.325 |
D-2324 | 0.00663 | -62.254 |
D-2325 | 0.00716 | -39.367 |
D-2326 | 0.044 | -52.036 |
D-2327 | 0.00282 | -65.701 |
D-2328 | 0.00411 | -53.991 |
D-2329 | 0.012 | -48.357 |
D-2330 | 0.019 | -42.593 |
D-2331 | 0.00448 | -47.418 |
D-2332 | 0.00944 | -37.327 |
D-2333 | 0.00514 | -37.327 |
D-2334 | 0.154 | -38.249 |
D-2335 | 0.0089 | -40.092 |
D-2336 | 0.0169 | -48.148 |
D-2337 | 0.00274 | -46.296 |
D-2338 | 0.0225 | -42.723 |
D-2339 | 0.00222 +/- 0.00136 | -43.218 +/- 4.81 |
D-2340 | 0.0136 | -56.561 |
D-2341 | 0.0222 | -50.226 |
D-2342 | 0.0273 | -55.385 |
D-2343 | 0.0164 | -41.784 |
D-2344 | 0.0314 | -60.282 |
D-2345 | 0.0103 | -65.1 |
D-2346 | 0.0427 | -63.9 |
D-2347 | 0.00446 | -49.4 |
D-2348 | 0.113 | -53.4 |
D-2349 | 0.0176 | -56.3 |
D-2350 | 0.00467 | -49.4 |
將在磷酸鹽緩衝鹽水(賽默飛世爾科技公司(Thermo Fisher Scientific),14190-136)中稀釋至每隻動物4e11至1e12病毒顆粒的相關腺病毒(AAV;血清型AAV8或AAV7; 無內毒素,由美商安進公司(Amgen)內部製備)靜脈內注入C57BL/6NCrl雄性小鼠(查理斯河實驗室有限公司(Charles River Laboratories Inc.))之尾靜脈,以驅動人PNPLA3WT
(PNPLA3-WT)
、PNPLA3rs738409
(PNPLA3-I148M
)、或PNPLA3rs738409-rs738408
(PNPLA3-I148M DM
) 在肝臟中之表現。小鼠通常為10-12週齡,且每組包括n = 4-6隻動物。每輪篩選包括至少兩個經媒介物處理之對照組:用媒介物處理之AAV-空載體及AAV-PNPLA3WT
或PNPLA3rs738409
及PNPLA3rs738409-rs738408
。針對AAV-PNPLA3WT
、PNPLA3rs738409
及/或PNPLA3rs738409-rs738408
測試所有siRNA。在AAV注射後兩週,將小鼠藉由皮下注射,以用磷酸鹽緩衝鹽水(賽默飛世爾科技公司(Thermo Fisher Scientific),14190-136)稀釋之0.5、1.0、3.0或5.0毫克/千克動物之單劑量的siRNA D-2324(0.5mM)處理。在siRNA注射後8、15、22、28或42天,自動物收集肝臟,在液氮中快速冷凍,根據製造商說明書,使用QIACube HT儀器(凱傑公司(Qiagen),9001793)及RNeasy 96 QIACube HT套組(凱傑公司(Qiagen),74171)處理經純化之RNA。使用QIAxpert系統(凱傑公司(Qiagen),9002340)分析樣本。用RQ1無RNA酶 DNA酶(普洛麥格公司(Promega),M6101)處理RNA,且使用TaqManTM
RNA-to-CT TM
1-Step套組(應用生物系統公司(Applied Biosystems),4392653)製備即時qPCR。即時qPCR在QuantStudio Real-Time PCR機器上運行。結果基於人PNPLA3之基因表現,其標準化至小鼠Gapdh(來自英傑公司(Invitrogen)之TaqManTM
測定,分別為hs00228747_m1及4352932E),且表示為人PNPLA3 mRNA表現與經媒介物處理之對照動物相比之相對敲低。判定內源性小鼠Pnpla3表現以用於比較(英傑公司(Invitrogen),Mm00504420_m1)。
對於肝臟甘油三酯含量分析,將來自動物之約0.05-0.1毫克冷凍肝臟在一毫升異丙醇中勻漿化。在冰上溫育一小時後,將樣本在微量離心機中以10,000 rpm旋轉,且將上清液轉移至清潔的深孔96孔板中。根據製造商說明書,使用比色Infinity甘油三酯試劑(賽默飛世爾科技公司(Thermo Fisher Scientific),TR22421)及甘油三酯標準品(普安特科技公司(Pointe Scientific),T7531-STD)判定甘油三酯含量。結果表示為每毫克組織的甘油三酯毫克數。
圖 1A-D
. 在活體內針對劑量依賴性mRNA敲低及功能耐久性來篩選的5種siRNA分子之實例。在靜脈內AAV注射後兩週,用siRNA處理表現人PNPLA3rs738409-rs738408
之小鼠。每組N = 6隻小鼠;數據表示為平均值及平均值之標準誤差。(A) 將siRNA以0.5、1.0、3.0或5.0毫克/千克體重皮下注射到小鼠腹部。在siRNA處理四週後,處死小鼠,且收集肝臟並處理以用於基因表現分析。數據表示在相對於經媒介物處理對照組設定之各組中之人PNPLA3rs738409-rs738408
之平均相對敲低。(B) 來自相同四週處理組之肝臟亦針對甘油三酯含量進行處理以獲得功能效力。數據表示每克經處理組織的甘油三酯之平均毫克數。(C) 將siRNA以1.0及3.0毫克/千克體重皮下注射到並行動物群之腹部。在siRNA處理後六週收穫小鼠以比較活體內siRNA分子之耐久性。收集肝臟且進行處理以進行基因表現分析。數據表示在相對於經媒介物處理對照組設定之各組中之人PNPLA3rs738409-rs738408
之平均相對敲低。(D) 來自相同六週處理組之肝臟亦針對甘油三酯含量進行處理以隨時間獲得功能效力。數據表示每克經處理組織的甘油三酯之平均毫克數。
相對敲低之數據顯示在表8-12中,期分別顯示第8、15、22、28、及42天之相對敲低及各種劑量。PNPLA3敲低為百分比,其中負值表明PNPLA3水平降低。
[表8].第8天PNPLA3敲低測定
[表9]. 第15天PNPLA3敲低測定
[表10]. 第22天PNPLA3敲低測定
[表11]. 第28天PNPLA3敲低測定
[表12]. 第42天PNPLA3敲低測定
實例 5 :在人源化 PNPLA3rs738409-rs738408 小鼠模型中藉由 siRNA 分子預防及拯救 NAFLD
雙鏈體編號 | AAV載體 | AAV顆粒/動物 | 經投與之劑量 (mg/kg) | PNPLA3敲低 (%) |
D-2092 | PNPLA3-I148M | 1.00E+12 | 3 | -91.51 |
D-2092 | PNPLA3-I148M | 1.00E+12 | 5 | -92.64 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 3 | -79.21 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 5 | -86.38 |
D-2068 | PNPLA3-I148M | 4.00E+11 | 5 | -34.15 |
D-2069 | PNPLA3-I148M | 4.00E+11 | 5 | -82.74 |
D-2070 | PNPLA3-I148M | 4.00E+11 | 5 | -79.64 |
D-2071 | PNPLA3-I148M | 4.00E+11 | 5 | -48.80 |
D-2071 | PNPLA3-WT | 4.00E+11 | 5 | -59.43 |
D-2075 | PNPLA3-I148M | 4.00E+11 | 5 | -79.10 |
D-2075 | PNPLA3-WT | 4.00E+11 | 5 | -60.60 |
D-2079 | PNPLA3-I148M | 4.00E+11 | 5 | -50.08 |
D-2072 | PNPLA3-I148M | 4.00E+11 | 5 | -44.40 |
D-2072 | PNPLA3-WT | 4.00E+11 | 5 | -43.75 |
D-2077 | PNPLA3-I148M | 4.00E+11 | 5 | -28.46 |
D-2076 | PNPLA3-I148M | 4.00E+11 | 5 | -83.25 |
D-2078 | PNPLA3-I148M | 4.00E+11 | 5 | -49.10 |
D-2078 | PNPLA3-WT | 4.00E+11 | 5 | -3.45 |
D-2073 | PNPLA3-I148M | 4.00E+11 | 5 | -39.42 |
D-2074 | PNPLA3-I148M | 4.00E+11 | 5 | -46.68 |
D-2074 | PNPLA3-WT | 4.00E+11 | 5 | -5.11 |
D-2084 | PNPLA3-I148M | 4.00E+11 | 5 | -87.49 |
D-2084 | PNPLA3-I148M | 8.00E+11 | 5 | -88.75 |
D-2084 | PNPLA3-I148M | 8.00E+11 | 1 | -16.49 |
D-2084 | PNPLA3-WT | 8.00E+11 | 1 | -15.42 |
D-2085 | PNPLA3-I148M | 4.00E+11 | 5 | -77.14 |
D-2085 | PNPLA3-I148M | 8.00E+11 | 5 | -84.42 |
D-2085 | PNPLA3-I148M | 8.00E+11 | 1 | -25.19 |
D-2085 | PNPLA3-WT | 8.00E+11 | 1 | -21.14 |
D-2086 | PNPLA3-I148M | 4.00E+11 | 5 | -64.54 |
D-2086 | PNPLA3-I148M | 4.00E+11 | 5 | -52.95 |
D-2087 | PNPLA3-I148M | 4.00E+11 | 5 | -20.15 |
D-2088 | PNPLA3-I148M | 4.00E+11 | 5 | -47.18 |
D-2088 | PNPLA3-I148M | 8.00E+11 | 5 | -66.96 |
D-2089 | PNPLA3-I148M | 8.00E+11 | 5 | -85.47 |
D-2089 | PNPLA3-WT | 8.00E+11 | 1 | -21.01 |
D-2089 | PNPLA3-I148M | 8.00E+11 | 1 | -34.21 |
D-2089 | PNPLA3-I148M | 1.00E+12 | 5 | -90.55 |
D-2090 | PNPLA3-I148M | 8.00E+11 | 5 | -89.58 |
D-2090 | PNPLA3-I148M | 8.00E+11 | 1 | -50.13 |
D-2090 | PNPLA3-WT | 8.00E+11 | 1 | 8.76 |
D-2091 | PNPLA3-WT | 8.00E+11 | 5 | -35.70 |
D-2092 | PNPLA3-I148M | 8.00E+11 | 5 | -92.34 |
D-2092 | PNPLA3-I148M | 8.00E+11 | 1 | -51.35 |
D-2092 | PNPLA3-WT | 8.00E+11 | 1 | -42.88 |
D-2093 | PNPLA3-I148M | 8.00E+11 | 5 | -83.87 |
D-2094 | PNPLA3-I148M | 8.00E+11 | 5 | -70.12 |
D-2095 | PNPLA3-I148M | 8.00E+11 | 1 | -29.95 |
D-2095 | PNPLA3-WT | 8.00E+11 | 1 | 67.40 |
D-2095 | PNPLA3-I148M | 8.00E+11 | 5 | -85.42 |
D-2096 | PNPLA3-WT | 8.00E+11 | 5 | -90.44 |
D-2081 | PNPLA3-I148M | 8.00E+11 | 5 | 6.25 |
D-2081 | PNPLA3-I148M | 8.00E+11 | 5 | -11.81 |
D-2097 | PNPLA3-I148M | 8.00E+11 | 5 | -87.61 |
D-2098 | PNPLA3-I148M | 8.00E+11 | 5 | -79.84 |
D-2099 | PNPLA3-I148M | 8.00E+11 | 5 | -84.36 |
D-2100 | PNPLA3-I148M | 8.00E+11 | 5 | -79.67 |
D-2101 | PNPLA3-I148M | 8.00E+11 | 5 | -89.64 |
D-2102 | PNPLA3-I148M | 8.00E+11 | 5 | -61.49 |
D-2103 | PNPLA3-I148M | 8.00E+11 | 5 | -19.65 |
D-2104 | PNPLA3-I148M | 8.00E+11 | 5 | -79.70 |
D-2104 | PNPLA3-I148M | 1.00E+12 | 5 | -82.87 |
D-2105 | PNPLA3-I148M | 8.00E+11 | 5 | -84.49 |
D-2105 | PNPLA3-I148M | 1.00E+12 | 5 | -87.71 |
D-2152 | PNPLA3-I148M | 1.00E+12 | 5 | -39.35 |
D-2153 | PNPLA3-I148M | 1.00E+12 | 5 | -79.04 |
D-2154 | PNPLA3-I148M | 1.00E+12 | 5 | -66.72 |
D-2155 | PNPLA3-I148M | 1.00E+12 | 5 | -44.68 |
D-2156 | PNPLA3-I148M | 1.00E+12 | 5 | -84.72 |
D-2157 | PNPLA3-I148M | 1.00E+12 | 5 | -17.25 |
D-2280 | PNPLA3-I148M DM | 1.00E+12 | 5 | -99.70 |
D-2280 | PNPLA3 WT | 1.00E+12 | 5 | -51.13 |
D-2295 | PNPLA3-WT | 1.00E+12 | 5 | -35.90 |
D-2295 | PNPLA3-I148M DM | 1.00E+12 | 5 | -94.68 |
D-2296 | PNPLA3-WT | 1.00E+12 | 5 | -23.24 |
D-2296 | PNPLA3-I148M DM | 1.00E+12 | 5 | -92.78 |
D-2297 | PNPLA3-WT | 1.00E+12 | 5 | 43.71 |
D-2297 | PNPLA3-I148M DM | 1.00E+12 | 5 | -94.59 |
D-2324 | PNPLA3-WT | 1.00E+12 | 5 | 6.53 |
D-2324 | PNPLA3-I148M DM | 1.00E+12 | 5 | -97.39 |
D-2326 | PNPLA3-WT | 1.00E+12 | 5 | -8.25 |
D-2326 | PNPLA3-I148M DM | 1.00E+12 | 5 | -77.82 |
D-2328 | PNPLA3-WT | 1.00E+12 | 5 | -2.12 |
D-2328 | PNPLA3-I148M DM | 1.00E+12 | 5 | -92.49 |
雙鏈體編號 | AAV載體 | AAV顆粒/動物 | 經投與之劑量 (mg/kg) | PNPLA3敲低 (%) |
D-2092 | PNPLA3-I148M | 1.00E+12 | 3 | -87.58 |
D-2092 | PNPLA3-I148M | 1.00E+12 | 5 | -93.96 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 3 | -81.73 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 5 | -72.99 |
D-2131 | PNPLA3-I148M | 1.00E+12 | 5 | -66.02 |
D-2186 | PNPLA3-I148M | 1.00E+12 | 5 | -87.67 |
D-2089 | PNPLA3-I148M | 1.00E+12 | 5 | -95.01 |
D-2104 | PNPLA3-I148M | 1.00E+12 | 5 | -76.01 |
D-2105 | PNPLA3-I148M | 1.00E+12 | 5 | -72.67 |
D-2123 | PNPLA3-I148M | 1.00E+12 | 5 | -56.93 |
D-2128 | PNPLA3-I148M | 1.00E+12 | 5 | -37.26 |
D-2138 | PNPLA3-I148M | 1.00E+12 | 5 | -62.16 |
D-2149 | PNPLA3-I148M | 1.00E+12 | 5 | -72.34 |
D-2156 | PNPLA3-I148M | 1.00E+12 | 5 | -75.81 |
D-2259 | PNPLA3-I148M | 1.00E+12 | 5 | -79.15 |
D-2260 | PNPLA3-I148M | 1.00E+12 | 5 | -69.97 |
D-2261 | PNPLA3-I148M | 1.00E+12 | 5 | -50.40 |
D-2262 | PNPLA3-I148M | 1.00E+12 | 5 | -84.36 |
D-2263 | PNPLA3-I148M | 1.00E+12 | 5 | -77.08 |
D-2264 | PNPLA3-I148M | 1.00E+12 | 5 | -40.86 |
D-2269 | PNPLA3-I148M | 1.00E+12 | 5 | -37.55 |
D-2270 | PNPLA3-I148M | 1.00E+12 | 5 | -77.42 |
D-2271 | PNPLA3-I148M | 1.00E+12 | 5 | -26.87 |
D-2272 | PNPLA3-I148M | 1.00E+12 | 5 | -56.05 |
D-2280 | PNPLA3-I148M DM | 1.00E+12 | 3 | -86.30 |
D-2287 | PNPLA3-I148M DM | 1.00E+12 | 3 | -94.73 |
D-2289 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.48 |
D-2292 | PNPLA3-I148M DM | 1.00E+12 | 3 | -82.48 |
D-2297 | PNPLA3-I148M DM | 1.00E+12 | 3 | -72.61 |
D-2322 | PNPLA3-I148M DM | 1.00E+12 | 3 | -32.92 |
D-2324 | PNPLA3-I148M DM | 1.00E+12 | 3 | -87.56 |
D-2327 | PNPLA3-I148M DM | 1.00E+12 | 3 | -86.70 |
D-2345 | PNPLA3-WT | 1.00E+12 | 5 | -83.48 |
D-2346 | PNPLA3-WT | 1.00E+12 | 5 | -75.93 |
D-2347 | PNPLA3-WT | 1.00E+12 | 5 | -22.83 |
D-2348 | PNPLA3-WT | 1.00E+12 | 5 | -20.09 |
D-2349 | PNPLA3-WT | 1.00E+12 | 5 | -67.70 |
D-2350 | PNPLA3-WT | 1.00E+12 | 5 | -57.51 |
D-2351 | PNPLA3-WT | 1.00E+12 | 5 | -56.11 |
D-2352 | PNPLA3-I148M DM | 1.00E+12 | 3 | -92.21 |
D-2353 | PNPLA3-I148M DM | 1.00E+12 | 3 | -89.55 |
D-2354 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.21 |
D-2358 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.10 |
D-2359 | PNPLA3-I148M DM | 1.00E+12 | 3 | -91.17 |
D-2360 | PNPLA3-I148M DM | 1.00E+12 | 3 | -63.98 |
D-2361 | PNPLA3-I148M DM | 1.00E+12 | 3 | -92.47 |
D-2362 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.10 |
D-2364 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.31 |
D-2370 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.99 |
D-2370 | PNPLA3 WT | 1.00E+12 | 3 | 11.88 |
D-2371 | PNPLA3-I148M DM | 1.00E+12 | 3 | -91.14 |
D-2372 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.71 |
D-2373 | PNPLA3-I148M DM | 1.00E+12 | 3 | -73.80 |
D-2374 | PNPLA3-I148M DM | 1.00E+12 | 3 | -75.98 |
D-2375 | PNPLA3-I148M DM | 1.00E+12 | 3 | -96.68 |
D-2376 | PNPLA3-I148M DM | 1.00E+12 | 3 | -96.78 |
D-2377 | PNPLA3-I148M DM | 1.00E+12 | 3 | -96.88 |
D-2378 | PNPLA3-I148M DM | 1.00E+12 | 3 | -97.60 |
D-2379 | PNPLA3-I148M DM | 1.00E+12 | 3 | -94.73 |
D-2380 | PNPLA3-I148M DM | 1.00E+12 | 3 | -94.73 |
D-2381 | PNPLA3-I148M DM | 1.00E+12 | 3 | -76.24 |
D-2382 | PNPLA3-I148M DM | 1.00E+12 | 3 | -80.33 |
D-2383 | PNPLA3-I148M DM | 1.00E+12 | 3 | -71.98 |
D-2384 | PNPLA3-I148M DM | 1.00E+12 | 3 | -87.24 |
D-2385 | PNPLA3-I148M DM | 1.00E+12 | 3 | -78.77 |
D-2386 | PNPLA3-I148M DM | 1.00E+12 | 3 | -70.58 |
D-2387 | PNPLA3-I148M DM | 1.00E+12 | 3 | -67.09 |
D-2390 | PNPLA3-I148M DM | 1.00E+12 | 3 | -92.97 |
D-2391 | PNPLA3-I148M DM | 1.00E+12 | 3 | -94.10 |
D-2392 | PNPLA3-I148M DM | 1.00E+12 | 3 | -92.14 |
D-2395 | PNPLA3-I148M DM | 1.00E+12 | 3 | -85.63 |
D-2396 | PNPLA3-I148M DM | 1.00E+12 | 3 | -94.63 |
D-2397 | PNPLA3-I148M DM | 1.00E+12 | 3 | -92.90 |
D-2398 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.48 |
D-2399 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.40 |
D-2400 | PNPLA3-I148M DM | 1.00E+12 | 3 | -97.55 |
D-2401 | PNPLA3-I148M DM | 1.00E+12 | 3 | -96.98 |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 3 | -97.25 |
D-2403 | PNPLA3 WT | 1.00E+12 | 3 | 36.90 |
D-2404 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.08 |
D-2405 | PNPLA3-I148M DM | 1.00E+12 | 3 | -96.93 |
D-2406 | PNPLA3 WT | 1.00E+12 | 3 | 28.80 |
D-2395 | PNPLA3-I148M DM | 1.00E+12 | 3 | -17.25 |
D-2396 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.70 |
D-2413 | PNPLA3-I148M DM | 1.00E+12 | 3 | -94.80 |
D-2415 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.95 |
D-2418 | PNPLA3-I148M DM | 1.00E+12 | 3 | -88.45 |
D-2419 | PNPLA3 WT | 1.00E+12 | 3 | 43.72 |
D-2453 | PNPLA3 WT | 1.00E+12 | 3 | -81.33 |
D-2454 | PNPLA3 WT | 1.00E+12 | 3 | -83.38 |
D-2455 | PNPLA3 WT | 1.00E+12 | 3 | -68.85 |
D-2456 | PNPLA3 WT | 1.00E+12 | 3 | -89.03 |
D-2460 | PNPLA3 WT | 1.00E+12 | 3 | -68.65 |
D-2461 | PNPLA3 WT | 1.00E+12 | 3 | -47.85 |
雙鏈體編號 | AAV載體 | AAV顆粒/動物 | 經投與之劑量 (mg/kg) | PNPLA3敲低 (%) |
D-2092 | PNPLA3-I148M | 1.00E+12 | 3 | -74.61 |
D-2092 | PNPLA3-I148M | 1.00E+12 | 5 | -85.78 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 3 | -27.78 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 5 | -33.60 |
D-2324 | PNPLA3-I148M DM | 1.00E+12 | 3 | -56.68 |
D-2324 | PNPLA3-I148M DM | 1.00E+12 | 5 | -88.02 |
D-2089 | PNPLA3-I148M | 1.00E+12 | 5 | -80.86 |
D-2104 | PNPLA3-I148M | 1.00E+12 | 5 | -65.61 |
D-2105 | PNPLA3-I148M | 1.00E+12 | 5 | -39.67 |
D-2280 | PNPLA3-I148M DM | 1.00E+12 | 5 | -84.12 |
D-2297 | PNPLA3-I148M DM | 1.00E+12 | 3 | -58.01 |
D-2297 | PNPLA3-I148M DM | 1.00E+12 | 5 | -76.43 |
D-2352 | PNPLA3-I148M DM | 1.00E+12 | 5 | -92.74 |
D-2353 | PNPLA3-I148M DM | 1.00E+12 | 5 | -84.54 |
D-2354 | PNPLA3-I148M DM | 1.00E+12 | 5 | -89.06 |
D-2355 | PNPLA3-I148M DM | 1.00E+12 | 5 | -95.53 |
D-2356 | PNPLA3-I148M DM | 1.00E+12 | 5 | -94.99 |
D-2357 | PNPLA3-I148M DM | 1.00E+12 | 5 | -97.37 |
雙鏈體編號 | AAV載體 | AAV顆粒/動物 | 經投與之劑量 (mg/kg) | PNPLA3敲低 (%) |
D-2092 | PNPLA3-I148M | 1.00E+12 | 3 | -72.57 |
D-2092 | PNPLA3-I148M | 1.00E+12 | 5 | -82.74 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 3 | -32.93 |
D-2095 | PNPLA3-I148M | 1.00E+12 | 5 | -55.31 |
D-2089 | PNPLA3-I148M | 1.00E+12 | 5 | -63.49 |
D-2104 | PNPLA3-I148M | 1.00E+12 | 5 | -44.39 |
D-2105 | PNPLA3-I148M | 1.00E+12 | 5 | -39.80 |
D-2370 | PNPLA3-I148M DM | 1.00E+12 | 3 | -89.28 |
D-2400 | PNPLA3-I148M DM | 1.00E+12 | 3 | -88.23 |
D-2401 | PNPLA3-I148M DM | 1.00E+12 | 3 | -91.95 |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 0.5 | -50.65 |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 1 | -69.37 |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 3 | -96.43 |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 3 | -85.44 |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.52 |
D-2404 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.67 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 0.5 | -68.83 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 1 | -76.88 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 3 | -97.53 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.95 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 3 | -89.66 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.25 |
D-2420 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.93 |
D-2421 | PNPLA3-I148M DM | 1.00E+12 | 0.5 | -50.01 |
D-2421 | PNPLA3-I148M DM | 1.00E+12 | 1 | -66.30 |
D-2421 | PNPLA3-I148M DM | 1.00E+12 | 3 | -94.99 |
D-2421 | PNPLA3-I148M DM | 1.00E+12 | 3 | -80.05 |
D-2421 | PNPLA3 WT | 1.00E+12 | 3 | -36.65 |
D-2421 | PNPLA3-I148M DM | 1.00E+12 | 3 | -91.08 |
D-2425 | PNPLA3-I148M DM | 1.00E+12 | 3 | -16.07 |
D-2426 | PNPLA3-I148M DM | 1.00E+12 | 3 | -37.54 |
D-2427 | PNPLA3-I148M DM | 1.00E+12 | 3 | -25.19 |
D-2428 | PNPLA3-I148M DM | 1.00E+12 | 3 | -16.71 |
D-2437 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.78 |
D-2438 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.63 |
D-2439 | PNPLA3-I148M DM | 1.00E+12 | 3 | -88.10 |
D-2440 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.25 |
D-2441 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.13 |
D-2442 | PNPLA3-I148M DM | 1.00E+12 | 3 | -57.24 |
D-2443 | PNPLA3-I148M DM | 1.00E+12 | 3 | -95.43 |
D-2444 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.58 |
D-2445 | PNPLA3-I148M DM | 1.00E+12 | 3 | -84.55 |
D-2446 | PNPLA3-I148M DM | 1.00E+12 | 3 | -81.50 |
D-2427 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.09 |
D-2462 | PNPLA3-I148M DM | 1.00E+12 | 3 | -89.20 |
D-2463 | PNPLA3-I148M DM | 1.00E+12 | 3 | -41.25 |
D-2464 | PNPLA3-I148M DM | 1.00E+12 | 3 | -60.53 |
D-2465 | PNPLA3-I148M DM | 1.00E+12 | 3 | -91.35 |
D-2466 | PNPLA3-I148M DM | 1.00E+12 | 3 | -93.68 |
D-2467 | PNPLA3-I148M DM | 1.00E+12 | 3 | -85.15 |
D-2472 | PNPLA3-I148M DM | 1.00E+12 | 0.5 | -46.58 |
D-2472 | PNPLA3-I148M DM | 1.00E+12 | 1 | -74.47 |
D-2472 | PNPLA3-I148M DM | 1.00E+12 | 3 | -88.79 |
D-2472 | PNPLA3 WT | 1.00E+12 | 3 | -23.16 |
D-2472 | PNPLA3-I148M DM | 1.00E+12 | 3 | -90.54 |
D-2473 | PNPLA3-I148M DM | 1.00E+12 | 0.5 | -57.95 |
D-2473 | PNPLA3-I148M DM | 1.00E+12 | 1 | -71.96 |
D-2473 | PNPLA3-I148M DM | 1.00E+12 | 3 | -91.70 |
D-2473 | PNPLA3-I148M DM | 1.00E+12 | 3 | -85.94 |
D-2473 | PNPLA3 WT | 1.00E+12 | 3 | -18.70 |
D-2675 | PNPLA3-I148M DM | 1.00E+12 | 3 | -25.9 |
D-2677 | PNPLA3-I148M DM | 1.00E+12 | 3 | -3.807 |
D-2678 | PNPLA3-I148M DM | 1.00E+12 | 3 | -31.76 |
D-2679 | PNPLA3-I148M DM | 1.00E+12 | 3 | -88.48 |
D-2680 | PNPLA3-I148M DM | 1.00E+12 | 3 | -56.44 |
D-2681 | PNPLA3-I148M DM | 1.00E+12 | 3 | -35.71 |
D-2682 | PNPLA3-I148M DM | 1.00E+12 | 3 | -71.43 |
D-2683 | PNPLA3-I148M DM | 1.00E+12 | 3 | -68.62 |
D-2685 | PNPLA3-I148M DM | 1.00E+12 | 3 | -29.93 |
D-2687 | PNPLA3-I148M DM | 1.00E+12 | 3 | -75.05 |
D-2689 | PNPLA3-I148M DM | 1.00E+12 | 3 | -81.12 |
D-2690 | PNPLA3-I148M DM | 1.00E+12 | 3 | -79.08 |
D-2694 | PNPLA3-I148M DM | 1.00E+12 | 3 | -4.531 |
D-2695 | PNPLA3-I148M DM | 1.00E+12 | 3 | 29.23 |
D-2668 | PNPLA3-I148M DM | 1.00E+12 | 3 | -65.16 |
D-2669 | PNPLA3-I148M DM | 1.00E+12 | 3 | -78.36 |
D-2670 | PNPLA3-I148M DM | 1.00E+12 | 3 | -56.05 |
D-2671 | PNPLA3-I148M DM | 1.00E+12 | 3 | -56.87 |
雙鏈體編號 | AAV載體 | AAV顆粒/動物 | 經投與之劑量 (mg/kg) | PNPLA3敲低 (%) |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 1 | -57.74 |
D-2402 | PNPLA3-I148M DM | 1.00E+12 | 3 | -83.75 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 1 | -71.07 |
D-2419 | PNPLA3-I148M DM | 1.00E+12 | 3 | -70.8 |
D-2421 | PNPLA3-I148M DM | 1.00E+12 | 1 | -62.21 |
D-2421 | PNPLA3-I148M DM | 1.00E+12 | 3 | -80.12 |
D-2472 | PNPLA3-I148M DM | 1.00E+12 | 1 | -60.54 |
D-2472 | PNPLA3-I148M DM | 1.00E+12 | 3 | -74.77 |
D-2473 | PNPLA3-I148M DM | 1.00E+12 | 1 | -54.55 |
D-2473 | PNPLA3-I148M DM | 1.00E+12 | 3 | -81.13 |
藉由用反式脂肪(佔脂肪總量之45%)及糖較高之飲食(Tetri 2008)餵養小鼠來開發用於NAFLD/NASH之「經美國生活方式誘導之肥胖症候群」或ALIOS小鼠模型。對於該等研究,如前所述,向八至十週齡之C57BL / 6NCrl雄性小鼠(查理斯河實驗室有限公司(Charles River Laboratories Inc.))注射AAV空載體或AAV8-PNPLA3rs738409-rs738408
。在AAV注射時,小鼠保持正常食物或接受ALIOS飲食(Envigo公司,TD.06303),配有由55%果糖及45%葡萄糖組成(分別為西格瑪公司(Sigma),F0127及G7021)之飲用水直到收穫。在先前實驗中,確定PNPLA3rs738409-rs738408
之過表現在此情況下加速及惡化NAFLD表型(數據未顯示)。
在AAV注射及飲食開始後兩週,經由皮下注射,以用磷酸鹽緩衝鹽水(賽默飛世爾科技公司(Thermo Fisher Scientific),14190-136)稀釋之5.0毫克/千克動物之單劑量的siRNA D-2324(0.5mM)或媒介物對照處理小鼠。每兩週重複給藥直至收穫。在收穫時,收集體重,然後在異氟烷麻醉下藉由心臟穿刺來收集血清,然後獲得肝重。經由10%中性緩衝福爾馬林固定中葉,然後進行石蠟處理及包埋。如前所述,將肝臟其餘部分快速冷凍以用於含量及基因表現分析。
如前所述,處理快速冷凍肝組織以用於RNA及基因表現分析。結果顯示為標準化至小鼠Gapdh
之指定基因之原始Ct值及相對mRNA表現。(來自英傑公司(Invitrogen)之TaqManTM
:人PNPLA3
, hs00228747_m1;小鼠Pnpla3
, Mm00504420_m1;小鼠Gapdh
, 4352932E)。
根據製造商說明書處理經福爾馬林固定之組織以便進行蘇木精及伊紅染色(分別為達科公司(Dako),CS70030-2,CS70130-2)。藉由經委員會認證之病理學家對脂肪變性及炎症進行評分。
血清分析包括TIMP1,其為一種與NASH及NASH相關性纖維化相關之生物標誌物(Youssani 2011)。根據製造商說明書進行TIMP1 ELISA(研究開發系統公司(R&D Systems),MTM100)。
圖 2A-G.
為評估PNPLA3rs738409-rs738408
特異性siRNA分子D-2324防止與NAFLD相關之表型發展及PNPLA3rs738409-rs738408
過表現的能力,小鼠接受AAV8-空載體(EV)或AAV8-PNPLA3rs738409-rs738408
或媒介物,且保持正常飲食或過渡到ALIOS飲食。AAV注射後兩週,每隔一週用siRNA或媒介物處理小鼠,持續六週;共有三輪注射。在八週時間點收穫小鼠。結果表示為組平均值及標準誤差,每組N = 8。星號代表AAV8-PNPLA3rs738409-rs738408
群組之統計學顯著性,其藉由使用Dunnett氏多重比較檢驗之單向ANOVA產生。(A) 收穫時肝重(克)與體重(克)之比率。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,** = 0.0018,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(B) 藉由qPCR確認肝臟中之人PNPLA3 mRNA表現及緘默。標準化至小鼠Gapdh
之(左)原始Ct值及(右)相對倍數mRNA表現;將經ALIOS餵養之PNPLA3rs738409-rs738408
+ 媒介物及PNPLA3rs738409-rs738408
+ siRNA組進行比較。(C) 藉由qPCR分析肝臟中之小鼠Pnpla3
mRNA表現,表明內源性Pnpla3未被經AAV介導之過表現或siRNA緘默顯著改變。標準化至小鼠Gapdh
之(左)原始Ct值及(右)相對倍數mRNA表現;將經食物餵養之無AAV組與經ALIOS餵養之PNPLA3rs738409-rs738408
+媒介物及PNPLA3rs738409-rs738408
+ siRNA組進行比較。(D) 肝臟甘油三酯含量表示為每克肝組織之甘油三酯毫克數。經調整之P值:無AAV+媒介物組,**** < 0.0001,AAV-EV + 媒介物,* = 0.0393,AAV-PNPLA3rs738409-rs738408
+ siRNA,** = 0.0063。(E) 血清TIMP1表示為每毫升血清之皮克數。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,**** < 0.0001,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(F) 基於H&E染色之脂肪變性的組織學指征,評分為:在正常限度內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,不顯著性,AAV-PNPLA3rs738409-rs738408
+ siRNA,** = 0.0012。(G) 基於H&E染色之炎症的組織學指征,評分為:在正常限度內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,**** < 0.0001,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。
圖 3A-G.
為評估PNPLA3rs738409-rs738408
特異性siRNA分子在疾病發作後阻止經PNPLA3rs738409-rs738408
介導之疾病進一步發展的能力,小鼠接受AAV8-空載體(EV)或AAV8-PNPLA3rs738409-rs738408
或媒介物,且保持常規的飲食或過渡到ALIOS飲食。在AAV注射及飲食改變八週後,每隔一週用siRNA或媒介物處理小鼠,再持續八週;共有四輪注射。在十六週時間點收穫小鼠。儘管在脂肪變性方面沒有觀察到變化,但若在疾病誘導後開始siRNA治療,則一些其他疾病相關終點顯著減少。結果表示為平均值及標準誤差,每組N = 8。星號代表AAV8-PNPLA3rs738409-rs738408
群組之統計學顯著性,其藉由使用Dunnett氏多重比較檢驗之單向ANOVA產生。(A) 在收穫時肝重(克)與體重(克)之比率。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,不顯著,AAV-PNPLA3rs738409-rs738408
+ siRNA,*** = 0.0006。(B) 藉由qPCR確認肝臟中之人PNPLA3 mRNA表現及緘默。標準化至小鼠Gapdh
之(左)原始Ct值及(右)相對倍數mRNA表現;將經ALIOS餵養之PNPLA3rs738409-rs738408
+ 媒介物及PNPLA3rs738409-rs738408
+ siRNA組進行比較。(C) 藉由qPCR分析肝臟中之小鼠Pnpla3
mRNA表現,表明內源性Pnpla3未被經AAV介導之過表現或siRNA緘默顯著改變。標準化至小鼠Gapdh
之(左)原始Ct值及(右)相對倍數mRNA表現;將經食物餵養之無AAV組與經ALIOS餵養之PNPLA3rs738409-rs738408
+ 媒介物及PNPLA3rs738409-rs738408
+ siRNA組進行比較。(D) 肝臟甘油三酯含量表示為每克肝組織之甘油三酯毫克數。經調整之P值:AAV-EV + 媒介物,不顯著,AAV-PNPLA3rs738409-rs738408
+ siRNA,* = 0.0403。(E) 血清TIMP1表示為每毫升血清之皮克數。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,** = 0.0027,AAV-PNPLA3rs738409-rs738408
+ siRNA,** = 0.002。(F) 基於H&E染色之脂肪變性的組織學指征,評分為:在正常限度內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,不顯著,AAV-PNPLA3rs738409-rs738408
+ siRNA,不顯著。(G) 基於H&E染色之炎症的組織學指征,評分為:在正常限度內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,不顯著,AAV-PNPLA3rs738409-rs738408
+ siRNA,** = 0.0068。
圖 4A-D.
為評估PNPLA3rs738409-rs738408
特異性siRNA分子拯救由於過表現PNPLA3rs738409-rs738408
導致之疾病相關表型的能力,將來自經ALIOS餵養之經八週AAV8-PNPLA3rs738409-rs738408
-媒介物處理之小鼠的肝臟及血清與來自經ALIOS餵養之經十六週媒介物或siRNA處理之AAV8-PNPLA3rs738409-rs738408
小鼠的血清及肝臟進行比較。雖然在此時間點使用siRNA處理未觀察到脂肪變性之變化,但是與八週時之媒介物對照相比,在十六週時肝臟甘油三酯、血清TIMP1及炎症在統計學上均較低。結果表示為平均值及標準誤差,每組N = 8。星號代表經八週AAV8-PNPLA3rs738409-rs738408
媒介物處理之群組的統計學顯著性,其藉由使用Dunnett氏多重比較檢驗之單向ANOVA產生。(A) 肝臟甘油三酯含量表示為每克肝臟組織之甘油三酯毫克數。經調整之P值:16WK AAV-PNPLA3rs738409-rs738408
+ 媒介物,不顯著;16WK AAV-PNPLA3rs738409-rs738408
+ siRNA,** = 0.0011。(B) 血清Timp1表示為每毫升血清之皮克數。經調整之P值:16WK AAV-PNPLA3rs738409-rs738408
+ 媒介物,不顯著;16WK AAV-PNPLA3rs738409-rs738408
+ siRNA,* = 0.0134。(C) 基於H&E染色之脂肪變性的組織學指征,評分為:在正常範圍內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:16WK AAV-PNPLA3rs738409-rs738408
+ 媒介物,不顯著;16WK AAV-PNPLA3rs738409-rs738408
+ siRNA,不顯著。(D) 基於H&E染色之炎症的組織學指征,評分為:在正常限度內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:16WK AAV-PNPLA3rs738409-rs738408
+ 媒介物,不顯著;16WK AAV-PNPLA3rs738409-rs738408
+ siRNA,* = 0.0112。實例 6 :在人源化 PNPLA3rs738409-rs738408 小鼠模型中藉由 siRNA 分子預防肝纖維化
由阿米林製藥公司(Amylin Pharmaceuticals)開發的「AMLN」飲食(Clapper 2013)為ALIOS飲食之改良版。飼料包括膽固醇(2%)及額外蔗糖之十倍增加。接受「AMLN」飲食之小鼠在20-30週後發展為輕度至中度纖維化(Clapper,Mells及Kristiansen論文)。對於該研究,如上所述,向八至十週齡之C57BL / 6NCrl雄性小鼠(查理斯河實驗室有限公司(Charles River Laboratories Inc.))注射AAV-空載體或AAV-PNPLA3rs738409-rs738408
,以加速疾病發作。在注射AAV時,小鼠繼續正常飲食或接受Envigo飲食TD.170748,配有由55%果糖及45%葡萄糖(分別為西格瑪公司(Sigma),F0127及G7021)組成之飲用水,直至收穫。
AAV注射及飲食開始後兩週,藉由皮下注射以用磷酸鹽緩衝鹽水(賽默飛世爾科技公司(Thermo Fisher Scientific),14190-136)稀釋之5.0毫克/千克動物之單劑量的siRNA D-2324(0.5mM)或媒介物對照處理小鼠。每兩週重複給藥直至收穫。在收穫時,收集體重,然後在異氟烷麻醉下藉由心臟穿刺收集血清,然後獲得肝重。經由10%中性緩衝福爾馬林固定中葉,然後進行石蠟處理及包埋。快速冷凍肝臟其餘部分以用於基因表現分析。
如前所述,處理快速冷凍肝組織以用於RNA及基因表現分析。結果顯示為標準化至小鼠Gapdh
之指定基因的原始Ct值及相對mRNA表現。(來自英傑公司(Invitrogen)之TaqManTM
測定:人PNPLA3
, hs00228747_m1;小鼠Pnpla3
, Mm00504420_m1;小鼠Col1a1
, Mm00801666_g1;小鼠Col3a1
, Mm01254471_g1;Col4a1
, Mm01210125_m1;小鼠Gapdh
, 4352932E)。Col1a1、Col3a1及Col4a1為與肝星狀細胞活化及肝纖維化相關之細胞外基質標記物(Baiocchini 2016)。
根據製造商說明書處理經福爾馬林固定之組織以用於蘇木精、伊紅及Masson氏三色染色(分別為達科公司(Dako),CS70030-2、CS70130-2、AR17311-2)。在無抗原修復之情況下且使用達科公司(DAKO)自動染色器進行抗平滑肌肌動蛋白染色。使用Peroxidazed 1及Sniper(分別為Biocare,PX968及BS966)處理載玻片,且用單株抗肌動蛋白、α-平滑肌抗體(西格瑪公司(Sigma),F3777)染色,然後用兔抗-FITC(英傑公司(Invitrogen),711900)、Envision-兔HRP聚合物(達科公司(Dako),K4003)、DAB +(達科公司(Dako),K3468)、及蘇木精染色。藉由經委員會認證之病理學家對脂肪變性、炎症、卵圓細胞/膽管增生及aSMA陽性細胞之量進行評分。
根據製造商說明書分析血清之小鼠TIMP1(研究開發系統公司(R&D Systems),MTM100)及小鼠細胞角蛋白18-M30(華美生物(Cusabio),CSB-E14265m)。除TIMP1外,細胞介素18-M30已被確定為NAFLD / NASH之潛在生物標誌物,包括早期纖維化(Neuman 2014及Yang 2015)。圖 5A-L.
為評估PNPLA3rs738409-rs738408
特異性siRNA分子預防早期纖維化發展的能力,小鼠接受AAV8-空載體(EV)或AAV8-PNPLA3rs738409-rs738408
或媒介物,且保持常規食物或過渡到AMLN飲食。在AAV注射後兩週,每隔一週用siRNA、D-2324或媒介物處理小鼠,再持續十週;共有六輪注射。在十週時間點收穫小鼠。結果表示為平均值及標準誤差,經食物餵養之無AAV + 媒介物及經AMLN餵養之AAV8-PNPLA3rs738409-rs738408
+ 媒介物,每組N = 8; 經AMLN餵養之AAV8-PNPLA3rs738409-rs738408
+ 媒介物及AAV8-PNPLA3rs738409-rs738408
+ siRNA,每組N = 12。星號代表經AAV8-PNPLA3rs738409-rs738408
-媒介物處理之群組的統計學顯著性,其藉由使用Dunnett氏多重比較檢驗之單向ANOVA產生。(A) 在收穫時肝重(克)與體重(克)之比率。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,不顯著,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(B) 藉由qPCR確認肝臟中之人PNPLA3
mRNA表現及緘默。標準化至小鼠Gapdh
之(左)原始Ct值及(右)相對倍數mRNA表現;將PNPLA3rs738409-rs738408
+ 媒介物及PNPLA3rs738409-rs738408
+ siRNA組進行比較。(C) 藉由qPCR分析肝臟中之小鼠Pnpla3
mRNA表現,表明內源性Pnpla3未被經AAV介導之過表現或siRNA緘默顯著改變。標準化至小鼠Gapdh
之(左)原始Ct值及(右)相對倍數mRNA表現;將經食物餵養之無-AAV組與經AMLN餵養之PNPLA3rs738409-rs738408
+ 媒介物及PNPLA3rs738409-rs738408
+ siRNA組進行比較。(D) 血清Timp1表示為每毫升血清之皮克數。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,**** < 0.0001,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(E) 血清CK18m30表示為每毫升血清之皮克數。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,**** < 0.0001,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(F) 基於H&E染色之炎症的組織學指征,評分為:在正常限度內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,* = 0.0108,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(G) 基於H&E染色之卵圓細胞/膽管增生之組織學指征,評分為:在正常範圍內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,不顯著,AAV-PNPLA3rs738409-rs738408
+ siRNA,** = 0.0081。(H) 抗平滑肌肌動蛋白之免疫組織化學染色,評分為:在正常限度內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,* = 0.0101,AAV-PNPLA3rs738409-rs738408
+ siRNA,*** = 0.0002。(I) 纖維化之Masson氏三色染色,評分為:在正常範圍內(0)、最小(1)、輕度(2)、中度(3)、及嚴重(4)。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,不顯著,AAV-PNPLA3rs738409-rs738408
+ siRNA,不顯著。(J) 藉由qPCR獲得肝臟中之小鼠Col1a1
mRNA表現。標準化至小鼠Gapdh
之相對倍數mRNA表現。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,**** < 0.0001,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(K) 藉由qPCR獲得肝臟中之小鼠Col3a1
mRNA表現。標準化至小鼠Gapdh
之相對倍數mRNA表現。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,**** < 0.0001,AAV-PNPLA3rs738409-rs738408
+ siRNA,**** < 0.0001。(L) 藉由qPCR獲得肝臟中之小鼠Col4a1 mRNA表現。標準化至小鼠Gapdh
之相對倍數mRNA表現。經調整之P值:無AAV + 媒介物組,**** < 0.0001,AAV-EV + 媒介物,*** < 0.0005,AAV-PNPLA3rs738409-rs738408
+ siRNA,** < 0.0041。實例 7 :使用生物發光成像小鼠模型篩選 PNPLA3 siRNA 分子
向通常在10-12週大的BALB/c雄性小鼠(查理斯河實驗室有限公司(Charles River Laboratories Inc.))注射用磷酸鹽緩衝鹽水(賽默飛世爾科技公司(Thermo Fisher Scientific),14190-136)稀釋之相關腺病毒(AAV;血清型AAVDJ8;無內毒素,由美商安進公司(Amgen)內部製備)。以從5e11至7.5e11個病毒顆粒向每隻動物投與AAV,並且靜脈內注入尾靜脈中。使用鼠巨細胞病毒(CMV)啟動子和螢火蟲螢光素酶報導基因設計AAV構建體,其中在3'端一串核苷酸含有人PNPLA3WT
(參考等位基因)或人PNPLA3rs738409-rs738408
(次要等位基因)siRNA靶序列的區段,以及其他非SNP跨越人PNPLA3目標靶序列,如圖6和7所示。
在AAV注射後兩週,根據製造商的說明書給小鼠注射RediJect D-Luciferin(珀金埃爾默公司(PerkinElmer),770504)。使用IVIS Spectrum In Vivo Imaging System(珀金埃爾默公司(PerkinElmer))捕獲小鼠體內的生物發光信號,並使用Living Image軟體(珀金埃爾默公司(PerkinElmer))進行分析。然後根據肝臟區域的總通量[光子/秒]信號將小鼠隨機分為n = 5組。隨機分到處理組後,向小鼠給藥用磷酸鹽緩衝鹽水(賽默飛世爾科技公司(Thermo Fisher Scientific),14190-136)稀釋之單劑量的siRNA(0.5 mM)。經由皮下注射以指定的毫克/千克動物劑量投與siRNA。針對每輪篩選和每種AAV類型,包括一個經媒介物處理的對照組。
siRNA注射後第1、2、3和4週,對小鼠進行重新成像,並使用為基線讀數建立的相同定義的目的地區域收集總通量[p/s]測量結果。對於每隻動物,藉由計算第1、2、3或4週的總通量相對於動物基線總通量的百分比變化來判定相對敲低百分比,將其標準化至相同時間點的媒介物對照組基線總通量的平均變化。例如,用siRNA處理的動物的相對敲低計算如下:(第2週時的動物總通量)/(基線時的動物總通量),標準化為平均值(第2週時媒介物動物的總通量/基線時媒介物動物的總通量)。圖8描述了在siRNA處理之前和之後,注射有表現人PNPLA3次要等位基因靶序列或參考等位基因靶序列的AAV的動物以及總通量隨時間的變化的代表性圖像。
[表13]. PNPLA3敲低測定
實例 8 :嵌合人源化肝小鼠模型中 PNPLA3rs738409-rs738408 選擇性 siRNA 分子的功效確認
雙鏈體編號 | AAV 載體 | AAV 顆粒/動物 | 經投與之劑量 (mg/kg) | 第8天的PNPLA3敲低 (%) | 第15天的PNPLA3敲低 (%) | 第22天的PNPLA3敲低 (%) | 第29天的PNPLA3敲低 (%) | 第43天的PNPLA3敲低 (%) |
D-2324 | PNPLA3-I148M DM | 5E+11 | 5 | -98.03 | -94.77 | -94.15 | -65.40 | |
D-2370 | PNPLA3-I148M DM | 5E+11 | 5 | -96.48 | -96.52 | -95.40 | -69.31 | |
D-2419 | PNPLA3-I148M DM | 5E+11 | 5 | -97.66 | -97.09 | -96.84 | -90.52 | |
D-2324 | PNPLA3-I148M DM | 5E+11 | 3 | -96.39 | -91.43 | -87.71 | -61.75 | |
D-2370 | PNPLA3-I148M DM | 5E+11 | 3 | -97.42 | -95.98 | -95.51 | -85.66 | |
D-2419 | PNPLA3-I148M DM | 5E+11 | 3 | -97.37 | -95.32 | -95.52 | -81.81 | |
D-2421 | PNPLA3-I148M DM | 5E+11 | 3 | -96.13 | -93.99 | -95.26 | -85.49 | |
D-2404 | PNPLA3-I148M DM | 5E+11 | 3 | -97.77 | -96.82 | -96.83 | ||
D-2402 | PNPLA3-I148M DM | 5E+11 | 3 | -97.69 | -96.69 | -96.81 | -55.46 | |
D-2472 | PNPLA3-I148M DM | 5E+11 | 3 | -95.89 | -93.61 | -92.05 | -68.74 | |
D-2443 | PNPLA3-I148M DM | 5E+11 | 3 | -97.33 | -96.51 | -95.82 | -84.56 | |
D-2466 | PNPLA3-I148M DM | 5E+11 | 3 | -97.96 | -97.13 | -96.75 | -79.62 | |
D-2473 | PNPLA3-I148M DM | 5E+11 | 3 | -97.18 | -95.95 | -95.79 | -68.96 | |
D-2324 | PNPLA3-I148M DM | 5E+11 | 1 | -90.86 | -82.53 | -80.56 | -74.32 | |
D-2370 | PNPLA3-I148M DM | 5E+11 | 1 | -93.49 | -90.34 | -89.17 | -56.45 | |
D-2419 | PNPLA3-I148M DM | 5E+11 | 1 | -89.12 | -84.16 | -87.52 | -47.52 | |
D-2421 | PNPLA3-I148M DM | 5E+11 | 1 | -83.03 | -70.41 | -74.45 | -26.47 | |
D-2404 | PNPLA3-I148M DM | 5E+11 | 1 | -94.34 | -91.18 | -90.01 | -59.48 | |
D-2402 | PNPLA3-I148M DM | 5E+11 | 1 | -87.41 | -79.59 | -84.95 | -32.94 | |
D-2472 | PNPLA3-I148M DM | 5E+11 | 1 | -87.28 | -79.86 | -76.77 | -33.42 | |
D-2443 | PNPLA3-I148M DM | 5E+11 | 1 | -48.96 | -46.63 | -69.39 | -34.70 | |
D-2466 | PNPLA3-I148M DM | 5E+11 | 1 | -90.36 | -83.03 | -85.21 | -44.20 | |
D-2473 | PNPLA3-I148M DM | 5E+11 | 1 | -80.89 | -76.26 | -84.45 | -56.63 | |
D-2454 | PNPLA3 WT | 5E+11 | 3 | -97.89 | -85.94 | -92.41 | -44.16 | |
D-2453 | PNPLA3 WT | 5E+11 | 3 | -98.67 | -90.86 | -94.56 | -72.71 | |
D-2456 | PNPLA3 WT | 5E+11 | 3 | -97.75 | -84.03 | -87.90 | -40.07 | |
D-2455 | PNPLA3 WT | 5E+11 | 3 | -89.75 | -61.34 | -80.50 | -38.33 | |
D-2454 | PNPLA3 WT | 5E+11 | 1 | -94.43 | -73.24 | -89.57 | -60.76 | |
D-2453 | PNPLA3 WT | 5E+11 | 1 | -94.15 | -72.63 | -80.99 | 5.10 | |
D-2456 | PNPLA3 WT | 5E+11 | 1 | -79.90 | -50.52 | -69.95 | -20.01 | |
D-2455 | PNPLA3 WT | 5E+11 | 1 | -71.82 | -1.32 | -55.65 | 17.39 | |
D-2419 | PNPLA3-I148M DM | 7.5E+11 | 3 | -98.00 | -97.61 | -96.67 | -91.38 | |
D-2666 | PNPLA3-I148M DM | 7.5E+11 | 3 | -94.32 | -94.07 | -90.69 | -80.56 | |
D-2667 | PNPLA3-I148M DM | 7.5E+11 | 3 | -95.49 | -94.77 | -93.58 | -85.70 | |
D-2676 | PNPLA3-I148M DM | 7.5E+11 | 3 | -94.63 | -91.64 | -89.02 | -72.10 | |
D-2699 | PNPLA3-I148M DM | 7.5E+11 | 3 | -90.85 | -85.48 | -57.10 | ||
D-2700 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.65 | -96.86 | -93.76 | -87.13 | |
D-2701 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.37 | -96.59 | -95.00 | -88.29 | |
D-2705 | PNPLA3-I148M DM | 7.5E+11 | 3 | -95.74 | -91.67 | -86.98 | -75.38 | |
D-2706 | PNPLA3-I148M DM | 7.5E+11 | 3 | -95.51 | -91.01 | -85.19 | -68.36 | |
D-2707 | PNPLA3-I148M DM | 7.5E+11 | 3 | -94.33 | -89.29 | -85.91 | -66.08 | |
D-2708 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.72 | -96.34 | -95.70 | -89.66 | |
D-2684 | PNPLA3-I148M DM | 7.5E+11 | 3 | -94.66 | -89.59 | -85.28 | -55.31 | |
D-2686 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.29 | -94.61 | -89.34 | -51.49 | |
D-2688 | PNPLA3-I148M DM | 7.5E+11 | 3 | -95.97 | -94.43 | -93.01 | -82.63 | |
D-2691 | PNPLA3-I148M DM | 7.5E+11 | 3 | -94.47 | -86.71 | -81.93 | -64.98 | |
D-2692 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.52 | -94.01 | -89.97 | -80.83 | |
D-2696 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.94 | -94.79 | -92.89 | -83.76 | |
D-2697 | PNPLA3-I148M DM | 7.5E+11 | 3 | -91.39 | -83.82 | -75.89 | -51.50 | |
D-2419 | PNPLA3-I148M DM | 7.5E+11 | 3 | -95.52 | -93.54 | -91.46 | -74.42 | |
D-2523 | PNPLA3-I148M DM | 7.5E+11 | 3 | -94.27 | -91.79 | -66.89 | -50.87 | |
D-2413 | PNPLA3-I148M DM | 7.5E+11 | 3 | -94.04 | -95.17 | -76.04 | -53.22 | |
D-2523 | PNPLA3-I148M DM | 7.5E+11 | 1 | -65.61 | -86.75 | 78.32 | 163.55 | |
D-2413 | PNPLA3-I148M DM | 7.5E+11 | 1 | -83.37 | -85.51 | -26.85 | -9.13 | |
D-2667 | PNPLA3-I148M DM | 7.5E+11 | 1 | -58.87 | -59.41 | -4.15 | 35.34 | |
D-2419 | PNPLA3-I148M DM | 7.5E+11 | 1 | -91.58 | -90.56 | -73.21 | -54.90 | |
D-2688 | PNPLA3-I148M DM | 7.5E+11 | 1 | -73.57 | -84.01 | -37.46 | -1.49 | |
D-2708 | PNPLA3-I148M DM | 7.5E+11 | 1 | -84.06 | -86.91 | -65.11 | -62.60 | |
D-2669 | PNPLA3-I148M DM | 7.5E+11 | 1 | -0.36 | 15.49 | 220.82 | 268.45 | |
D-2711 | PNPLA3-I148M DM | 7.5E+11 | 3 | -0.01 | 49.59 | 238.44 | 212.77 | |
D-2713 | PNPLA3-I148M DM | 7.5E+11 | 3 | -91.72 | -90.51 | -63.93 | -30.60 | |
D-2716 | PNPLA3-I148M DM | 7.5E+11 | 3 | -86.43 | -87.62 | -53.78 | -44.36 | |
D-2674 | PNPLA3-I148M DM | 7.5E+11 | 3 | -80.59 | -81.95 | -36.01 | 0.59 | |
D-2698 | PNPLA3-I148M DM | 7.5E+11 | 3 | -85.22 | -71.73 | 483.18 | 807.64 | |
D-2693 | PNPLA3-I148M DM | 7.5E+11 | 3 | -75.98 | -59.22 | 39.49 | 110.57 | |
D-2456 | PNPLA3 WT | 7.5E+11 | 3 | -85.45 | -78.57 | -73.40 | -58.06 | |
D-2523 | PNPLA3 WT | 7.5E+11 | 3 | -27.40 | -30.01 | -40.22 | -49.59 | |
D-2413 | PNPLA3 WT | 7.5E+11 | 3 | -36.68 | -27.93 | -10.77 | 12.54 | |
D-2419 | PNPLA3 WT | 7.5E+11 | 3 | 115.08 | 100.96 | 101.39 | 64.16 | |
D-2679 | PNPLA3 WT | 7.5E+11 | 3 | -34.16 | -6.82 | 17.45 | 17.46 | |
D-2688 | PNPLA3 WT | 7.5E+11 | 3 | 135.68 | 67.66 | 73.67 | 48.09 | |
D-2708 | PNPLA3 WT | 7.5E+11 | 3 | 69.43 | 40.96 | 50.89 | 52.15 | |
D-2669 | PNPLA3 WT | 7.5E+11 | 3 | 189.68 | 50.72 | 148.32 | 147.67 | |
D-2711 | PNPLA3 WT | 7.5E+11 | 3 | 109.67 | 114.26 | 75.39 | 93.47 | |
D-2713 | PNPLA3 WT | 7.5E+11 | 3 | 115.32 | 48.86 | 18.67 | 1.36 | |
D-2716 | PNPLA3 WT | 7.5E+11 | 3 | 205.93 | 114.38 | 114.09 | 83.88 | |
D-2674 | PNPLA3 WT | 7.5E+11 | 3 | 81.00 | 67.18 | 109.44 | 65.24 | |
D-2698 | PNPLA3 WT | 7.5E+11 | 3 | 105.88 | 76.39 | 62.37 | 37.65 | |
D-2693 | PNPLA3 WT | 7.5E+11 | 3 | 148.52 | 125.48 | 116.64 | 61.43 | |
D-2690 | PNPLA3 WT | 7.5E+11 | 3 | 260.84 | 226.65 | 132.56 | 143.47 | |
D-2419 | PNPLA3-I148M DM | 7.5E+11 | 1 | -93.56 | -89.52 | -84.43 | -63.34 | |
D-2717 | PNPLA3-I148M DM | 7.5E+11 | 1 | -80.51 | -67.95 | -58.97 | -22.43 | |
D-2718 | PNPLA3-I148M DM | 7.5E+11 | 1 | -71.69 | -55.51 | -36.08 | 38.24 | |
D-2721 | PNPLA3-I148M DM | 7.5E+11 | 1 | -63.71 | -62.50 | -48.52 | -12.97 | |
D-2719 | PNPLA3-I148M DM | 7.5E+11 | 1 | -78.95 | -70.97 | -65.38 | -13.45 | |
D-2713 | PNPLA3-I148M DM | 7.5E+11 | 1 | -70.43 | -64.51 | -43.23 | -0.48 | |
D-2712 | PNPLA3-I148M DM | 7.5E+11 | 1 | -79.63 | -68.64 | -51.82 | -8.94 | |
D-2720 | PNPLA3-I148M DM | 7.5E+11 | 1 | -65.95 | -86.70 | -40.92 | 2.24 | |
D-2722 | PNPLA3-I148M DM | 7.5E+11 | 1 | -80.32 | -75.04 | -63.28 | -34.56 | |
D-2723 | PNPLA3-I148M DM | 7.5E+11 | 1 | -72.69 | -55.59 | -39.98 | 35.01 | |
D-2724 | PNPLA3-I148M DM | 7.5E+11 | 1 | -87.97 | -78.29 | -73.04 | -42.89 | |
D-2725 | PNPLA3-I148M DM | 7.5E+11 | 1 | -79.65 | -70.69 | -32.50 | -35.65 | |
D-2726 | PNPLA3-I148M DM | 7.5E+11 | 1 | -40.03 | -38.86 | -14.74 | 55.71 | |
D-2716 | PNPLA3-I148M DM | 7.5E+11 | 1 | 34.75 | 92.81 | 181.00 | 440.62 | |
D-2454 | PNPLA3 WT | 7.5E+11 | 3 | -98.16 | -95.32 | -90.10 | -88.50 | |
D-2523 | PNPLA3 WT | 7.5E+11 | 3 | -44.09 | -18.41 | 133.97 | 45.50 | |
D-2413 | PNPLA3 WT | 7.5E+11 | 3 | -64.12 | -40.89 | 38.07 | -3.05 | |
D-2717 | PNPLA3 WT | 7.5E+11 | 3 | -47.67 | -19.71 | 109.49 | 42.75 | |
D-2718 | PNPLA3 WT | 7.5E+11 | 3 | -77.04 | -60.05 | -3.84 | -40.18 | |
D-2721 | PNPLA3 WT | 7.5E+11 | 3 | -47.41 | -49.33 | 10.37 | -41.12 | |
D-2719 | PNPLA3 WT | 7.5E+11 | 3 | -29.15 | -27.68 | 72.52 | -20.99 | |
D-2720 | PNPLA3 WT | 7.5E+11 | 3 | -67.46 | -46.37 | 66.09 | -20.66 | |
D-2726 | PNPLA3 WT | 7.5E+11 | 3 | -83.36 | -81.38 | -37.21 | -61.84 | |
D-2723 | PNPLA3 WT | 7.5E+11 | 3 | -76.09 | -57.32 | -7.17 | -45.85 | |
D-2419 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.66 | -97.62 | -93.93 | -88.77 | |
D-2765 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.64 | -97.81 | -95.29 | -87.53 | |
D-2766 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.30 | -98.15 | -95.74 | -90.07 | |
D-2724 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.41 | -97.40 | -94.77 | -74.92 | |
D-2768 | PNPLA3-I148M DM | 7.5E+11 | 3 | -93.09 | -95.62 | -90.36 | -74.62 | |
D-2769 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.94 | -97.93 | -95.17 | -89.60 | |
D-2770 | PNPLA3-I148M DM | 7.5E+11 | 3 | -89.23 | -94.14 | -90.41 | -79.60 | |
D-2771 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.22 | -97.80 | -95.95 | -90.02 | |
D-2772 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.47 | -97.84 | -95.09 | -92.03 | |
D-2773 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.51 | -97.95 | -96.18 | -93.12 | |
D-2774 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.78 | -97.86 | -95.36 | -91.98 | |
D-2413 | PNPLA3-I148M DM | 7.5E+11 | 3 | -97.94 | -98.01 | -96.24 | -91.62 | |
D-2419 | PNPLA3-I148M DM | 7.5E+11 | 3 | -95.82 | -97.64 | -93.55 | -89.59 | |
D-2717 | PNPLA3-I148M DM | 7.5E+11 | 3 | -96.64 | -97.39 | -95.19 | -90.49 | |
D-2453 | PNPLA3 WT | 7.5E+11 | 1.5 | -94.74 | -92.67 | -88.92 | -82.19 | |
D-2724 | PNPLA3 WT | 7.5E+11 | 3 | 20.64 | 74.00 | 49.42 | 77.34 | |
D-2522 | PNPLA3 WT | 7.5E+11 | 3 | -27.08 | 53.78 | 58.28 | 17.26 | |
D-2765 | PNPLA3 WT | 7.5E+11 | 3 | -65.40 | -34.32 | -35.49 | -46.32 | |
D-2766 | PNPLA3 WT | 7.5E+11 | 3 | -11.64 | 30.74 | 34.37 | 27.78 | |
D-2767 | PNPLA3 WT | 7.5E+11 | 3 | 14.66 | 60.91 | 75.79 | 39.35 | |
D-2768 | PNPLA3 WT | 7.5E+11 | 3 | 50.61 | 196.44 | 345.66 | 149.29 | |
D-2769 | PNPLA3 WT | 7.5E+11 | 3 | 30.51 | 68.93 | 90.25 | 35.98 | |
D-2770 | PNPLA3 WT | 7.5E+11 | 3 | 25.01 | 69.81 | 95.00 | 99.26 | |
D-2771 | PNPLA3 WT | 7.5E+11 | 3 | 60.93 | 158.57 | 281.70 | 147.49 | |
D-2772 | PNPLA3 WT | 7.5E+11 | 3 | -3.32 | 58.08 | 89.41 | 85.72 | |
D-2773 | PNPLA3 WT | 7.5E+11 | 3 | -36.28 | -20.77 | 21.91 | -22.36 | |
D-2774 | PNPLA3 WT | 7.5E+11 | 3 | 2.83 | 51.07 | 71.49 | 65.75 | |
D-2413 | PNPLA3 WT | 7.5E+11 | 3 | -5.20 | 44.59 | 50.74 | 28.08 | |
D-2419 | PNPLA3 WT | 7.5E+11 | 3 | 101.03 | 115.15 | 32.98 | -16.47 | |
D-2717 | PNPLA3 WT | 7.5E+11 | 1.5 | 21.80 | 107.39 | 173.59 | 87.94 |
為了評估PNPLA3次要等位基因選擇性siRNA在活體內人肝細胞中的功效,使用了來自費尼克斯生物公司(PhoenixBio Co., Ltd)(日本)的嵌合人源化肝PXB小鼠(Miyamoto等人, (2017) Xenobiotica 47(12):1052-1063;Tateno等人 (2015) PLoS One 10(11):e0142145. doi:10.1371/journal.pone.0142145)。研究開始時,雄性小鼠大約四個月大;移植後至少三個月。藉由PhoenixBio,判定小鼠的人肝細胞置換指數為90%-95%,並且根據先前的基因分型,小鼠對PNPLA3rs738409
(批號BD195)是雜合的。到達後,按照費尼克斯生物公司(PhoenixBio)的建議,將小鼠置於ProLab RMH 3000食物上。經過一星期的馴化週期後,將飲食改為高脂肪果糖含量的NASH誘導飲食(研究飲食公司(Research Diets),D19021301)。在NASH飲食一週後,根據體重測量結果將小鼠隨機分組。經由皮下注射,以用磷酸鹽緩衝鹽水(賽默飛世爾科技公司(Thermo Fisher Scientific),14190-136)稀釋之3.0或10.0毫克/千克動物之單劑量的siRNA(0.5mM)或僅接受媒介物來處理小鼠。在siRNA注射後兩週或四週,自動物收集肝臟,在液氮中快速冷凍,根據製造商說明書,使用QIAcube Automated DNA/RNA Isolation Purification System(凱傑公司(Qiagen)及RNeasy Mini QIAcube套組(凱傑公司(Qiagen),74116)處理經純化之RNA。使用NanoDrop™ 8000分光光度計(賽默科技公司(Thermo Scientific),ND-8000-GL)分析樣本。用RQ1無RNA酶 DNA酶(普洛麥格公司(Promega),M6101)處理RNA,並根據製造商的說明進行數位微滴式PCR(ddPCR)的製備。AccuScript高保真第1鏈cDNA合成套組(賽默飛世爾公司(Thermo Fisher),200820)用於逆轉錄反應,並使用ddPCR Supermix for Probes(伯樂公司(BioRad),1863010)組裝PCR反應。使用AutoDG液滴微滴式數位PCR系統(伯樂公司(BioRad),QX200)進行ddPCR。以下TaqMan™測定購自英傑公司(Invitrogen):人PNPLA3(Hs00228747_m1),人ASGR1(Hs1005019_m1),小鼠Asgr1(Mm01245581_m1),和人PNPLA3 rs738409次要/參考等位基因辨別測定(C______7241_10)。以下測定購自集成DNA技術公司(Integrated DNA Technologies Inc.):人TBP(Hs. PT 53a.20105486;引物與探針的比率為3.6 : 1),人HPRT1(Hs.PT.39a.22214821;引物與探針的比率為3.6 : 1),以及小鼠Hprt(Mm.PT.39a.22214828;引物與探針的比率為3.6 : 1)。人PNPLA3 、 HPRT
和ASGR1
以及小鼠Hprt
和Asgr1
的結果表示為拷貝/20微升反應,其標準化至人TBP。人PNPLA3
、人HPRT
和小鼠Hprt
的數據也表示為與經媒介物處理的對照動物相比的mRNA表現的相對敲低百分比。
對於肝臟甘油三酯含量分析,將來自小鼠之約0.05-0.1毫克冷凍肝臟在一毫升異丙醇中勻漿化。在冰上溫育一小時後,將樣本在微量離心機中以10,000 rpm旋轉,且將上清液轉移至清潔的深孔96孔板中。根據製造商說明書,使用比色Infinity甘油三酯試劑(賽默飛世爾科技公司(Thermo Fisher Scientific),TR22421)及甘油三酯標準品(普安特科技公司(Pointe Scientific),T7531-STD)以及帶有SoftMax Pro6軟體的SpectraMax Plus酶標儀(分子裝置公司(Molecular Devices))判定甘油三酯含量。結果表示為每克肝組織的甘油三酯毫克數。
圖9顯示了siRNA分子D-2419的實例,展示了活體內劑量依賴性和等位基因選擇性mRNA的敲低及功能效力。在NASH飲食一週後,用siRNA或媒介物處理對人PNPLA3rs738409-rs738408
雜合的小鼠。(A) 將siRNA分子D-2419以3.0或10.0毫克/千克體重皮下注射到小鼠腹部。在siRNA處理兩週和四週後,處死小鼠,且收集肝臟並處理以用於分析。使用ddPCR和等位基因特異性TaqMan®兩染料試劑來辨別PNPLA3的次要等位基因與參考等位基因,數據表明PNPLA3rs738409-rs738408
的劑量依賴性和等位基因選擇性敲低,而PNPLA3WT
沒有可量的變化。每組N = 5隻小鼠;數據表示為平均值及平均值之標準誤差。雙向ANOVA,** 0.001,*** < 0.001,**** < 0.0001,NS = 不顯著。(B) 數據表示在相對於經媒介物處理對照組設定之人PNPLA3rs738409-rs738408
等位基因相比PNPLA3WT
的平均相對mRNA敲低百分比及平均值之標準誤差。相對於兩週媒介物對照平均值的值均標準化至人TBP
。(C) 來自兩週處理組之肝臟針對甘油三酯含量進行處理以評估功能效力。數據表示每克肝臟的甘油三酯之毫克數。每組N = 5隻小鼠;數據表示為平均值及平均值之標準誤差。單向ANOVA,** 0.01,NS = 不顯著。(D) 為了控制有效的GalNAc介導的siRNA遞送,以10毫克/千克遞送D-2787(一種分別對人和小鼠HPRT和Hprt具有交叉反應性的siRNA),並且在兩週後收穫肝臟。數據表示在經D-2787處理的小鼠(N = 4)相比經媒介物處理的小鼠(N = 5)中HPRT
mRNA和Hprt
mRNA的拷貝。數據表示為平均值及平均值之標準誤差。單向ANOVA,* 0.01。(E) 數據表示在相對於經媒介物處理對照組設定之人HPRT
和小鼠Hprt
mRNA的平均相對mRNA敲低百分比及平均值之標準誤差;其全部標準化至人TBP。(F) 為了確認GalNAc受體在PXB小鼠(PXB mice)®
肝細胞上的表現,在不存在和存在D-2419的情況下、在siRNA注射後兩週和四週時,評估小鼠Asgr1
mRNA和人ASGR1
mRNA水平。每組N = 5隻小鼠;數據表示為平均值及平均值之標準誤差。
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[圖1A-D]展示針對活體內劑量依賴性mRNA敲低及功能耐久性之五種siRNA分子的篩選。
[圖2A-G]展示PNPLA3 siRNA分子在小鼠活體內對於肝重量、人PNPLA3表現之確認、肝甘油三酯含量、血清TIMP1水平及脂肪變性或炎症之組織學指征的作用。
[圖3A-G]展示PNPLA3 siRNA分子在活體內對於肝重量、人PNPLA3表現之確認、肝甘油三酯含量、血清TIMP1水平及脂肪變性或炎症之組織學指征的作用。
[圖4A-D]展示PNPLA3rs738409-rs738408
特異性siRNA分子挽救由於PNPLA3rs738409-rs738408
過表現而導致的疾病相關表型、肝臟甘油三酯含量、血清TIMP1水平及脂肪變性或炎症之組織學指征的能力。
[圖5A-L]展示PNPLA3rs738409-rs738408
特異性siRNA分子預防早期纖維化發展的能力。
[圖6a和6b]展示含有PNPLA3次要等位基因靶序列的AAV質體的完整序列。在含有鼠CMV啟動子、螢火蟲螢光素酶報導基因和靶序列的部分標有底線。
[圖7a和7b]展示含有PNPLA3參照等位基因靶序列的AAV質體的完整序列。在含有鼠CMV啟動子、螢火蟲螢光素酶報導基因和靶序列的部分標有底線。
[圖8]展示注射有表現人PNPLA3次要等位基因靶序列的AAV的小鼠(上部行)與注射有表現人PNPLA3參考等位基因靶序列的AAV的小鼠(下部行)相比的示例圖像。在獲取基線圖像(第一列)後,向兩隻小鼠注射相同的siRNA分子(D-2878,3 mpk)。第2列至第5列分別是在第1、2、3和4週捕獲的圖像。已使用Living Image®軟體將圖像轉換為灰度圖。較亮區是低總通量[p/s]的區域,而較暗區是高總通量[p/s]的區域。在插圖中(第2行和第4行)顯示了在每週一次時間點處,經siRNA處理的小鼠的相對百分比敲低,其標準化至經媒介物處理的小鼠。
[圖9]顯示了siRNA分子D-2419的實例,展示了活體內劑量依賴性和等位基因選擇性mRNA的敲低及功能效力。(A) 將siRNA分子D-2419以3.0或10.0毫克/千克體重皮下注射到小鼠腹部。(B) 數據表示在相對於經媒介物處理對照組設定之人PNPLA3rs738409-rs738408
等位基因相比PNPLA3WT
的平均相對mRNA敲低百分比及平均值之標準誤差。(C) 來自兩週處理組之肝臟針對甘油三酯含量進行處理以評估功能效力。(D) 為了控制有效的GalNAc介導的siRNA遞送,以10毫克/千克遞送D-2787(一種分別對人和小鼠HPRT和Hprt具有交叉反應性的siRNA),並且在兩週後收穫肝臟。數據表示在經D-2787處理的小鼠(N = 4)相比經媒介物處理的小鼠(N = 5)中HPRT
mRNA和Hprt
mRNA的拷貝。(E) 數據表示在相對於經媒介物處理對照組設定之人HPRT
和小鼠Hprt
mRNA的平均相對mRNA敲低百分比及平均值之標準誤差;其全部標準化至人TBP。(F) 為了確認GalNAc受體在PXB小鼠(PXB mice)®
肝細胞上的表現,在不存在和存在D-2419的情況下評估小鼠Asgr1
mRNA和人ASGR1
mRNA水平。
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Claims (43)
- 一種RNAi構建體,其包含有義鏈及反義鏈,其中該反義鏈包含具有至少15個連續核苷酸之區域,該等核苷酸與表1或2中列出之反義序列相差不超過3個核苷酸,且其中該RNAi構建體抑制含有Patatin樣磷脂酶結構域3(PNPLA3)之表現。
- 如請求項1之RNAi構建體,其中該反義鏈包含與PNPLA3 mRNA序列互補之區域。
- 如上述請求項中任一項之RNAi構建體,其中該有義鏈包含具有至少15個連續核苷酸之區域,該等核苷酸與表1或2中列出之反義序列相差不超過3個核苷酸。
- 如上述請求項中任一項之RNAi構建體,其中該構建體優先抑制PNPLA3-rs738409次要等位基因。
- 如請求項4之RNAi構建體,其中與主要等位基因相比,該構建體對PNPLA3-rs738409次要等位基因之抑制大至少10%。
- 如上述請求項中任一項之RNAi構建體,其中該構建體優先抑制PNPLA3-rs738408次要等位基因。
- 如請求項6之RNAi構建體,其中與主要等位基因相比,該構建體對PNPLA3-rs738409-rs738408雙重次要等位基因之抑制大至少10%。
- 如上述請求項中任一項之RNAi構建體,其中該有義鏈包含與該反義鏈之序列充分互補之序列,以形成長度為約15至約30個鹼基對之雙鏈體區。
- 如請求項8之RNAi構建體,其中該雙鏈體區之長度為約17至約24個鹼基對。
- 如請求項8之RNAi構建體,其中該雙鏈體區之長度為約19至約21個鹼基對。
- 如請求項10之RNAi構建體,其中該雙鏈體區之長度為19個鹼基對。
- 如請求項10之RNAi構建體,其中該雙鏈體區之長度為20個鹼基對。
- 如請求項10之RNAi構建體,其中該雙鏈體區之長度為21個鹼基對。
- 如請求項8至13中任一項之RNAi構建體,其中該有義鏈及該反義鏈之長度各為約15至約30個核苷酸。
- 如請求項14之RNAi構建體,其中該有義鏈及該反義鏈之長度各為約19至約27個核苷酸。
- 如請求項14之RNAi構建體,其中該有義鏈及該反義鏈之長度各為約21至約25個核苷酸。
- 如請求項14之RNAi構建體,其中該有義鏈及該反義鏈之長度各為約21至約23個核苷酸。
- 如請求項1至17中任一項之RNAi構建體,其中該RNAi構建體包含至少一個鈍端。
- 如請求項1至17中任一項之RNAi構建體,其中該RNAi構建體包含至少一個具有1至4個未配對核苷酸之核苷酸突出端。
- 如請求項19之RNAi構建體,其中該核苷酸突出端具有2個未配對核苷酸。
- 如請求項19或20之RNAi構建體,其中該RNAi構建體在該有義鏈之3'端、該反義鏈之3'端或該有義鏈及該反義鏈兩者之3'端處包含核苷酸突出端。
- 如請求項19至21中任一項之RNAi構建體,其中該核苷酸突出端包含5'-UU-3'二核苷酸或5'-dTdT-3'二核苷酸。
- 如請求項1至22中任一項之RNAi構建體,其中該RNAi構建體包含至少一個經修飾之核苷酸。
- 如請求項23之RNAi構建體,其中該經修飾之核苷酸為經2'-修飾之核苷酸。
- 如請求項23之RNAi構建體,其中該經修飾之核苷酸為經2'-氟修飾之核苷酸、經2'-O-甲基修飾之核苷酸、經2'-O-甲氧基乙基修飾之核苷酸、經2'-O-烯丙基修飾之核苷酸、雙環核酸(BNA)、二醇核酸、反向鹼基、或其組合。
- 如請求項25之RNAi構建體,其中該經修飾之核苷酸為經2'-O-甲基修飾之核苷酸、經2'-O-甲氧基乙基修飾之核苷酸、經2'-氟修飾之核苷酸、或其組合。
- 如請求項23之RNAi構建體,其中該有義鏈及該反義鏈中之所有該等核苷酸均為經修飾之核苷酸。
- 如請求項27之RNAi構建體,其中該等經修飾之核苷酸為經2'-O-甲基修飾之核苷酸、經2'-氟修飾之核苷酸、或其組合。
- 如請求項1至28中任一項之RNAi構建體,其中該RNAi構建體包含至少一個硫代磷酸酯核苷酸間鍵。
- 如請求項29之RNAi構建體,其中該RNAi構建體在該反義鏈之3'端處包含兩個連續硫代磷酸酯核苷酸間鍵。
- 如請求項29之RNAi構建體,其中該RNAi構建體在該反義鏈之3'端及5'端處包含兩個連續硫代磷酸酯核苷酸間鍵,且在該有義鏈之5'端處包含兩個連續硫代磷酸酯核苷酸間鍵。
- 如請求項1至31中任一項之RNAi構建體,其中該反義鏈包含選自表1或表2中列出之該等反義序列的序列。
- 如請求項32之RNAi構建體,其中該有義鏈包含選自表1或表2中列出之該等有義序列的序列。
- 如請求項1至33中任一項之RNAi構建體,其中該RNAi構建體為表1至2中之任一者中列出的雙鏈體化合物中之任一者。
- 如請求項1至34中任一項之RNAi構建體,其中與已經與對照RNAi構建體一起溫育之肝細胞中之PNPLA3表現水平相比,在與該RNAi構建體一起溫育之後,該RNAi構建體降低肝細胞中之PNPLA3表現水平。
- 如請求項29之RNAi構建體,其中該等肝細胞為Hep3B或HepG2細胞。
- 如請求項1至36中任一項之RNAi構建體,其中該RNAi構建體在活體外在Hep3B細胞中以5 nM抑制至少10% PNPLA3表現。
- 如請求項1至36中任一項之RNAi構建體,其中該RNAi構建體在活體外在HepG2細胞中以5 nM抑制至少10% PNPLA3表現。
- 如請求項1至36中任一項之RNAi構建體,其中該RNAi構建體以小於約1 nM之IC50抑制Hep3B細胞中之PNPLA3表現。
- 如請求項1至36中任一項之RNAi構建體,其中該RNAi構建體以小於約1 nM之IC50抑制HepG2細胞中之PNPLA3表現。
- 一種藥物組成物,其包含如請求項1至40中任一項之RNAi構建體及藥學上可接受之載體、賦形劑或稀釋劑。
- 一種用於降低有需要的患者中之PNPLA3表現的方法,其包括向該患者投與如請求項1至40中任一項之RNAi構建體。
- 如請求項42之方法,其中與未接受該RNAi構建體之患者中之PNPLA3表現水平相比,在投與該RNAi構建體後,在該患者中肝細胞中之PNPLA3表現水平降低。
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TW202317762A (zh) * | 2021-06-02 | 2023-05-01 | 美商艾拉倫製藥股份有限公司 | 含有類PATATIN磷脂酶結構域3(PNPLA3)的iRNA組成物及其使用方法 |
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