CN106086022B - 纤维症预防或者治疗剂 - Google Patents
纤维症预防或者治疗剂 Download PDFInfo
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- CN106086022B CN106086022B CN201610411134.1A CN201610411134A CN106086022B CN 106086022 B CN106086022 B CN 106086022B CN 201610411134 A CN201610411134 A CN 201610411134A CN 106086022 B CN106086022 B CN 106086022B
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Abstract
本发明提供对治疗纤维症有效的siRNA和使用了它的医药品。一种siRNA,其以选自序列号1中的碱基序号为1285~1318号的碱基、1398~1418号的碱基、1434~1463号的碱基、1548~1579号的碱基、1608~1628号的碱基、1700~1726号的碱基、1778~1798号的碱基以及1887~1907号的碱基中的连续的17~23个碱基为靶序列,全长为30个核苷酸以下。
Description
本发明专利申请是针对申请日为2011年10月14日、申请号为201180044862.9、发明名称为“纤维症预防或者治疗剂”的申请提出的分案申请。
技术领域
本发明涉及纤维症治疗剂。具体而言,涉及以编码转化生长因子(TGF)-β1的基因为靶的小干扰RNA(siRNA)和使用了它的医药品。
背景技术
肺纤维症是指由于过量的胶原和其它细胞外基质的蓄积而导致肺组织纤维化的症状。在肺纤维症中,特发性肺纤维症是中位生存期平均为3年、5年生存率为20~40%的预后不良的慢性难治性疾病。作为肺纤维症的治疗,可使用类固醇药物、免疫抑制剂,但目前没有可改善预后这样的有效的治疗法,因此需要开发新的治疗药。
近年来,逐渐清楚许多疾病是由于基因而发病的,关于肺纤维症,也报告了许多基因的参与(专利文献1~4、非专利文献1~6)。作为参与肺纤维症的主要因子,报告有TGF-β1(专利文献1~2、非专利文献2~6)、Smad3(非专利文献1)、MCP-1(专利文献3)等。
另一方面,核酸特别是siRNA引起与细胞中存在的特定序列相同或者几乎相同的基因的mRNA的降解,阻碍靶基因的表达(RNA干扰)。因此,由RNA干扰所产生的阻碍靶基因表达的功能在减少或治疗由特定的基因或基因组的异常表达所引起的疾病症状方面是有用的。关于肺纤维症相关基因,也进行了使用siRNA来尝试抑制该基因的表达的报告(专利文献1~4、非专利文献1~6)。
然而,至今为止报告的技术仅显示了siRNA序列对实验动物(小鼠、大鼠)的疾病相关基因的抑制效果,而对人类基因没有充分地显示出特异的效果。另外,关于siRNA的抑制效果,也仅显示出在200nM(非专利文献1)、20~500nM(非专利文献5)的效果,并没有示出在低浓度下可效率良好抑制肺纤维症相关基因的表达的核酸分子。
关于以TGF-β1基因为靶的siRNA,至今为止报告有数十种siRNA(专利文献1、5~7),但TGF-β1的基因的全长为2346个碱基(GenBank Accession No.NM_000660.3),从该基因的全长选出约20mer左右的序列的组合存在无数个,因此并不容易从中设计出可效率良好抑制该基因的表达的dsRNA、siRNA分子。
专利文献
专利文献1:国际公开第2007/109097号小册子
专利文献2:国际公开第2003/035083号小册子
专利文献3:日本特开2007-119396号公报
专利文献4:日本特表2009-516517号公报
专利文献5:国际公开第2009/061417号小册子
专利文献6:国际公开第2008/109548号小册子
专利文献7:国际公开第2007/79224号小册子
非专利文献
非专利文献1:Wang Z,Gao Z,Shi Y,Sun Y,Lin Z,Jiang H,Hou T,Wang Q,YuanX,Zhu X,Wu H,Jin Y,J Plast Reconstr Aesthet Surg,1193-1199,60,2007
非专利文献2:Hwang M,Kim HJ,Noh HJ,Chang YC,Chae YM,Kim KH,Jeon JP,LeeTS,Oh HK,Lee YS,Park KK,Exp Mol Pathol,48-54,81,2006
非专利文献3:Takabatake Y,Isaka Y,Mizui M,Kawachi H,Shimizu F,Ito T,Hori M,Imai E,Gene Ther,965-973,12,2005
非专利文献4:Xu W,Wang LW,Shi JZ,Gong ZJ,Hepatobiliary Pancreat DisInt,300-308,8,2009
非专利文献5:Liu XJ,Ruan CM,Gong XF,Li XZ,Wang HL,Wang MW,Yin JQ,Biotechnol Lett,1609-1615,27,2005
非专利文献6:福元重太郎,末次彩子,原田知佳,河口知允,滨田直树,前山隆茂,桑野和善,中西洋一,日本呼吸器学会杂志,185,46,2008
发明内容
本发明涉及提供对治疗纤维症有效的siRNA和使用它的医药品的技术。
本发明人等为了解决上述课题而进行了深入研究,结果发现以人类TGF-β1基因为靶的特定的siRNA在人体细胞中能够有效地抑制TGF-β1的表达,进而在肺纤维症模型小鼠的肺中,能够在不诱导干扰素反应的情况下,改善肺纤维症的症状。
即,本发明涉及以下1)~19)的内容。
1)一种siRNA,以选自序列号1中的碱基序号为1285~1318号的碱基、1398~1418号的碱基、1434~1463号的碱基、1548~1579号的碱基、1608~1628号的碱基、1700~1726号的碱基、1778~1798号的碱基、1806~1826号的碱基以及1887~1907号的碱基中的连续的17~23个碱基为靶序列,全长为30个核苷酸以下。
2)根据上述1)的siRNA,其中,siRNA选自以下(a)~(s)。
(a)由序列号2所示的正义序列和序列号3所示的反义序列形成的siRNA
(b)由序列号4所示的正义序列和序列号5所示的反义序列形成的siRNA
(c)由序列号6所示的正义序列和序列号7所示的反义序列形成的siRNA
(d)由序列号8所示的正义序列和序列号9所示的反义序列形成的siRNA
(e)由序列号10所示的正义序列和序列号11所示的反义序列形成的siRNA
(f)由序列号12所示的正义序列和序列号13所示的反义序列形成的siRNA
(g)由序列号14所示的正义序列和序列号15所示的反义序列形成的siRNA
(h)由序列号16所示的正义序列和序列号17所示的反义序列形成的siRNA
(i)由序列号18所示的正义序列和序列号19所示的反义序列形成的siRNA
(j)由序列号20所示的正义序列和序列号21所示的反义序列形成的siRNA
(k)由序列号22所示的正义序列和序列号23所示的反义序列形成的siRNA
(l)由序列号24所示的正义序列和序列号25所示的反义序列形成的siRNA
(m)由序列号26所示的正义序列和序列号27所示的反义序列形成的siRNA
(n)由序列号28所示的正义序列和序列号29所示的反义序列形成的siRNA
(o)由序列号30所示的正义序列和序列号31所示的反义序列形成的siRNA
(p)由序列号32所示的正义序列和序列号33所示的反义序列形成的siRNA
(q)由序列号34所示的正义序列和序列号35所示的反义序列形成的siRNA
(r)由序列号36所示的正义序列和序列号37所示的反义序列形成的siRNA
(s)由序列号54所示的正义序列和序列号55所示的反义序列形成的siRNA
3)根据上述1)或2)的siRNA,其中,从上述siRNA的正义链的3’末端的末端侧起除去悬突核苷酸的连续的1~10个核苷酸被转换成DNA。
4)根据上述1)~3)的siRNA,其中,从上述siRNA的反义链的5’末端的末端侧起连续的1~10个核苷酸被转换成DNA。
5)根据上述1)~4)的siRNA,其中,从上述siRNA的正义链的3’末端的末端侧起除去悬突核苷酸的连续的1~10个核苷酸被转换成DNA,且从反义链的5’末端的末端侧起连续的1~10个核苷酸被转换成DNA。
6)根据上述1)~5)的siRNA,反义链的5’末端被单磷酸化或者单硫代磷酸化。
7)一种医药组合物,含有上述1)~6)中任一项的siRNA。
8)一种TGF-β1基因表达抑制剂,含有上述1)~6)中任一项的siRNA作为有效成分。
9)一种纤维症预防或者治疗剂,含有上述1)~6)中任一项的siRNA作为有效成分。
10)一种肺纤维症或肺癌的预防或者治疗剂,含有上述1)~6)中任一项的siRNA作为有效成分。
11)上述1)~6)的siRNA在制造TGF-β1基因表达抑制剂中的应用。
12)上述1)~6)的siRNA在制造纤维症预防或者治疗剂中的应用。
13)上述1)~6)的siRNA在制造肺纤维症或肺癌的预防或者治疗剂中的应用。
14)用于抑制TGF-β1基因表达的上述1)~6)的siRNA。
15)用于预防或者治疗纤维症的上述1)~6)的siRNA。
16)用于预防或者治疗肺纤维症或肺癌的上述1)~6)的siRNA。
17)一种TGF-β1基因表达抑制方法,其特征在于,将上述1)~6)的siRNA给予人或动物。
18)一种预防或者治疗纤维症的方法,其特征在于,将上述1)~6)的siRNA给予人或动物。
19)一种预防或者治疗肺纤维症或肺癌的方法,其特征在于,将上述1)~6)的siRNA给予人或动物。
本发明的siRNA能够以低浓度效率良好地抑制或者阻碍TGF-β1的表达,因此作为用于预防或者治疗纤维症的医药品是有用的。
附图说明
图1是表示siRNA引起的TGF-β1mRNA的表达抑制率的图。
图2是表示嵌合体型siRNA引起的TGF-β1mRNA的表达抑制率的图。
图3是表示键合有磷酸或者硫代磷酸的嵌合体型siRNA引起的TGF-β1mRNA的表达抑制率的图。
图4是表示肺纤维症模型的TGF-β1的表达抑制率的图。
图5是肺组织切片(H.E.染色和马松三色染色)的光学显微镜照片(倍率:5倍)。
具体实施方式
本发明的作为siRNA的靶的序列为选自序列号1中的碱基序号为1285~1318号的碱基、1398~1418号的碱基、1434~1463号的碱基、1548~1579号的碱基、1608~1628号的碱基、1700~1726号的碱基、1778~1798号的碱基、1806~1826号的碱基以及1887~1907号的碱基中的连续的17~23个碱基,但是,其中,作为选自各组的连续的17~23个碱基,优选为19~23个碱基,更优选为21个碱基。
序列号1所示的碱基序列为TGF-β1的mRNA的碱基序列,该序列信息在GenBank中,被注册为GenBank Accession No.NM_000660.3。
本发明的siRNA是作为与TGF-β1的mRNA的上述靶序列互补的序列的反义链与作为与该反义链互补的序列的正义链杂交而成的,具有切断该TGF-β1的mRNA的活性(RNA干扰作用),具有阻止该mRNA的翻译的能力,即阻碍该TGF-β1基因的表达的能力。
就本发明的siRNA的核苷酸长度而言,正义链、反义链可以为相同的核苷酸长度,也可以为不同的核苷酸长度,其全长为30个核苷酸以下,优选为25个核苷酸以下,更优选为23个核苷酸以下、或者21个核苷酸。
另外,正义链和反义链的两端可以为平滑末端,各自链的3’侧也可以为悬突(突出末端)。在此,“平滑末端”是指在双链RNA的末端部分,正义链的末端区域和与其配对的反义链的末端区域在不形成单链部分的情况下配对的结构。另外,“悬突”也被称为悬垂端,是指在双链RNA的末端部分的正义链的末端区域或者与其配对的反义链的末端区域,不存在配对的碱基,因此无法形成双链而存在单链部分(突出末端)的结构。
突出末端部分的碱基数为1~10个核苷酸,优选为1~4个核苷酸,进一步优选为1~2个核苷酸。应予说明,突出末端的长度在两条链之间是没有关系的,两条链可以为相互不同的长度。突出末端部分的核苷酸可以为RNA,也可以为DNA,优选与作为靶的TGF-β1的mRNA互补的碱基,但只要保持上述RNA干扰能力,也可以为非互补的碱基。
本发明的siRNA可以为由2条分别的链构成的1个双链RNA,除此之外,也可以为1条链通过采用茎-环结构而形成的双链RNA。即,在本发明的siRNA中,也包括在正义链的5’末端和反义链的3’末端形成了由2~4个核苷酸构成的环的RNA、在正义链的3’末端和反义链的5’末端形成了由2~4个核苷酸构成的环的RNA。进而,还包括在正义链的5’末端和反义链的3’末端以及正义链的3’末端和反义链的5’末端这两端形成了由2~4个核苷酸构成的环的RNA。
本发明的siRNA与靶序列优选相同,但在可诱导上述RNA干扰的范围内,可以实质上相同、即同源的序列。具体而言,只要本发明的siRNA的反义链序列与靶序列杂交,就可以有1个或数个(例如,2、3、4个)的错配。即,在本发明的siRNA中,包括相对于靶序列而言取代、添加或者缺失了1个或数个碱基且可诱导RNA干扰的siRNA,或者与靶序列具有85%以上、优选为90%以上、更优选为95%以上,进一步优选为98%以上的序列同源性且可诱导RNA干扰的siRNA。
应予说明,就此时的杂交条件而言,将本发明的siRNA给予体内作为医药品使用时,为体内的条件,将本发明的siRNA在体外作为试剂使用时,为中度严谨的条件或者高度严谨的条件,作为这样的条件,例如,可举出400mM的NaCl、40mM的PIPES pH值6.4、1mM的EDTA、在50℃~70℃、12~160小时下的杂交条件。关于这些条件,为本领域技术人员所公知,在Sambrook et al.(Molecular Cloning:A Laboratory Manual second edition,Cold Spring Harbor Laboratory Press,New York,USA,1989)中记载。
另外,序列同源性通过Lipman-Pearson法(Science,227,1435,(1985))等计算即可,例如,使用遗传信息处理软件Genetyx-Win(Ver.5.1.1;软件开发)的同源性分析(Search homology)程序,使Unit size to compare(ktup)为2,进行查询,从而算出。
另外,在本发明的siRNA中,只要可诱导上述RNA干扰,就包括将正义链或者反义链中的任一方的核苷酸全部转换成DNA的siRNA(杂合体型)、将正义链和/或反义链的一部分核苷酸转换成DNA的siRNA(嵌合体型)。
在此,从RNA核苷酸向DNA的转换是指从AMP向dAMP、从GMP向dGMP、从CMP向dCMP、从UMP向dTMP转换。
作为杂合体型,优选将正义链的核苷酸转换成DNA的siRNA。作为嵌合体型,可举出将下游侧(正义链的3’末端侧,反义链的5’末端侧)的一部分核苷酸转换成DNA的siRNA。具体而言,可举出将正义链的3’末端侧和反义链的5’末端侧的核苷酸一并转换成DNA的siRNA、将正义链的3’末端侧或者反义链的5’末端侧中任一方的核苷酸转换成DNA的siRNA。另外,转换的核苷酸长度优选为直到与RNA分子的1/2长度相当的核苷酸为止的任意长度,例如可举出从末端起,1~13个核苷酸、优选1~10个核苷酸。从RNA干扰效果、RNA分子的稳定性、安全性等角度出发,作为优选的嵌合体型siRNA,例如可举出核苷酸长度分别为19~23个核苷酸且将下述长度的核苷酸以任意数目连续地转换成DNA的siRNA,即,从正义链的3’末端侧起除去了悬突核苷酸的1~10个核苷酸、优选1~8个核苷酸,更优选1~6个核苷酸以及从反义链的5’末端侧起1~10个核苷酸、优选1~8个核苷酸,更优选1~6个核苷酸(参照后述〔表2〕)。另外,此时,更优选正义链(排除悬突核苷酸)与反义链的DNA转换数目相同。
另外,就本发明的siRNA而言,只要可诱导上述RNA干扰,其核苷酸(核糖核苷酸、脱氧核糖核苷酸)就可以为糖、碱基和/或磷酸盐被化学修饰过的核苷酸类似物。作为碱基被修饰过的核苷酸类似物,例如,可举出5位修饰尿嘧啶或者胞嘧啶(例如,5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、5-甲基胞嘧啶、5-甲基尿嘧啶、5-(2-氨基)丙基尿嘧啶、5-卤代胞嘧啶、5-卤代尿嘧啶、5-甲氧基尿嘧啶等);8位修饰腺嘌呤或者鸟嘌呤(例如,8-溴鸟嘌呤等);脱氮核苷酸(例如,7-脱氮腺嘌呤等);O-和N-烷基化核苷酸(例如,N6-甲基腺嘌呤等)等。
另外,作为糖被修饰过的核苷酸类似物,例如,可举出核糖核苷酸的2’-OH被H、OR、R、卤原子、SH、SR、NH2、NHR、NR2、或CN(在此,R表示碳原子数为1~6的烷基、烯基或者炔基)等取代的2’位糖取代体、5’末端的-OH发生单磷酸化或者单硫代磷酸化的5’末端磷酸化体、5’末端单硫代磷酸化体。
作为磷酸盐被修饰过的核苷酸类似物,可举出用硫代磷酸酯基取代硫酸酯基而成的核苷酸类似物,所述硫酸酯基将邻接的核糖核苷酸进行键合。
另外,本发明的siRNA与上述核苷酸类似物不同,特定的取代基或功能性分子可以与从正义链的5’末端侧或者3’末端侧起第1~6号核苷酸(5’末端、3’末端、或者末端以外的内部的碱基、糖)中的至少一个直接或者介由接头键合,优选该取代基或功能性分子与从正义链的5’末端侧起第1~6号,优选第1~4号核苷酸中的至少一个键合。
在此,作为取代基而言,作为一个例子,可以举出氨基;巯基;硝基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的烷基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的氨基烷基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的硫代烷基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的烷氧基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的氨基烷氧基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的硫代烷氧基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的单或二烷基氨基;碳原子数为1~40(优选为2~20,进一步优选为4~12)的烷基巯基;碳原子数为2~40(优选为2~20,进一步优选为4~12)的聚环氧乙烷基;碳原子数为3~39(优选为3~21,进一步优选为3~12)的聚环氧丙烷基等。通过键合这些取代基,从而能够显著增强RNA干扰效果。
作为功能性分子,可例示糖、蛋白质、肽、氨基酸、DNA、RNA(包括tRNA)、核酸适体、修饰核苷酸、低分子有机·无机材料、胆固醇、树枝状大分子、脂质、高分子材料等。通过附加功能性分子,从而能够兼备优异的RNA干扰效果和基于该功能性分子的有用效果。
作为上述糖,例如,可举出葡萄糖、半乳糖、氨基葡萄糖、氨基半乳糖等单糖,任意组合这些单糖而成的寡糖或者多糖等。
作为上述蛋白质,可以使用存在于体内的蛋白质、具有药理作用的蛋白质、具有分子识别作用的蛋白质等,作为该蛋白质的一个例子,可以举出输入蛋白b蛋白质、抗生物素蛋白、抗体等。
作为上述DNA,具体而言,可例示碱基长度为5~50的DNA,优选碱基长度为5~25的DNA。
作为上述肽,例如,可举出八聚精氨酸肽R8、核定位信号肽序列(HIV-1Tat、SV40T抗原等)、核输出信号肽(HIV-1Rev、MAPKK等)、细胞膜融合肽等。作为上述修饰核苷酸,例如,可举出修饰硫代磷酸酯型、硼烷磷酸酯型DNA/RNA等的磷酸骨架而成的修饰核苷酸;2’-OMe修饰RNA、2’-F修饰RNA等2’修饰核苷酸;交联LNA(Locked Nucleic Acid)、ENA(2’-O,4’-C-Ethylene-bridged nucleic acids)等核苷酸的糖分子而成的修饰核苷酸;PNA(肽核酸)、吗啉代核苷酸等基本骨架不同的修饰核苷酸等(参照国际公开第2008/140126号小册子、国际公开第2009/123185号小册子)。
作为上述低分子有机·无机材料,例如,可举出Cy3、Cy5等荧光物质;生物素;量子点;金微粒等。作为上述树枝状大分子,例如,可举出聚酰胺-胺型树枝状大分子等。作为上述脂质,例如,可举出亚油酸、DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)等,除此之外,也可举出在国际公开第2009/123185号小册子中记载的具有2个疏水基团的双链脂质。作为上述高分子材料,例如,可举出聚乙二醇、聚乙烯亚胺等。
在此,作为具有功能性分子的基团,可以为功能性分子的残基本身,另外,也可以为双官能接头中的一个官能团与功能性分子的残基键合而成的基团。换言之,在前者的情况下,功能性分子直接键合在上述正义链RNA的规定部位,在后者的情况下,功能性分子介由双官能接头而键合在上述正义链RNA的规定部位。在此,作为双官能接头,只要是含有2个官能团的接头,就没有特别限制,例如,可使用3-(2-吡啶二硫)丙酸N-羟基琥珀酰亚胺酯、N-4-马来酰亚胺丁酸、S-(2-吡啶二硫)半胱胺、碘代乙酸琥珀酰亚胺酯、N-(4-马来酰亚胺丁酰氧基)琥珀酰亚胺、N-[5-(3’-马来酰亚胺丙酰胺基)-1-羧基戊基]亚氨基二乙酸、N-(5-氨基戊基)-亚氨基二乙酸等。
关于上述正义链RNA中的取代基或者功能性分子或者连接它们的接头的键合部位,没有特别限定,但优选它们取代构成正义链RNA的规定的核苷酸的磷酸部分的羟基的氢原子而进行键合。
本发明的siRNA,具体而言,例如可例示以下(a)~(s)所示的由正义序列和反义序列形成的双链RNA分子。
表1
另外,作为嵌合体型,可优选举出将从正义链的3’末端侧起1~8个核苷酸以及从反义链的5’末端侧起1~6个核苷酸以任意数目连续地转换成DNA的siRNA,例如若使用上述(l)的情况例示,则如下所示。
表2
表中,大写字母表示RNA,小写字母表示DNA,下划线表示悬突的位置。
本发明的siRNA的制造方法没有特别限定,可以利用公知的制造方法来合成,例如,可以在体外进行化学合成、或者利用使用了启动子和RNA聚合酶的转录体系来合成。
化学合成可以以含有作为siRNA构成要素的核酸分子的Amidite树脂为原料,利用核酸合成装置来合成。
利用转录体系的合成可以通过将发夹型RNA进行修整的试管内转录法来合成双链RNA。
如后述实施例所示,这样得到的本发明的siRNA在来自人肺泡上皮细胞的细胞中能够以mRNA水平有效地抑制TGF-β1的表达,进而在肺纤维症模型小鼠肺中能够在不诱导干扰素反应的情况下,发挥改善肺纤维症的症状的效果。
因此,本发明的siRNA和可将该siRNA在给予对象内表达的表达载体作为用于向人或动物给予的医药品(医药组合物)是有用的。具体而言,作为用于抑制TGF-β1基因表达的医药品、用于预防·治疗由TGF-β1的过度表达所引起的疾病,例如纤维症的医药品,即纤维症的预防或者治疗剂是有用的。
在此,作为引起肺纤维化的疾病,种类广泛,有间质性肺炎、囊肿性纤维化症、慢性阻塞性肺病(COPD:Chronic obstructive pulmonary disease)、急性呼吸窘迫综合征(ARDS:Acute respiratory distress syndrome)、炎症性肺部疾病、肺部感染症、放射性肺炎、试剂性间质性肺炎、伴有胶原病的间质性肺炎等,但是特别优选属于原因无法确定的间质性肺炎的特发性间质性肺炎(IIPs)中的特发性肺纤维症(IPF)。特发性间质性肺炎(IIPs)中,作为临床病理学的疾病,包括特发性肺纤维症(IPF)、非特异性间质性肺炎(NSIP)、特发性机化性肺炎(COP/BOOP)、急性间质性肺炎(AIP)、脱屑性间质性肺炎(DIP)、伴有呼吸性细支气管炎的间质性肺病(RB-ILD)、淋巴细胞间质性肺炎(LIP)等。
另外,就TGF-β1而言,报告有促进癌、特别是肺腺癌的浸润、转移(Mol CellBiochem(2011)355:309.314,Cancer Genomics Proteomics2010,7,217);TGF-β1在癌治疗后的正常组织的损伤部位过度表达,通过以TGF-β1路径为靶而能够抑制该正常组织的损伤(The Oncologist2010;15:350.359)等,因此本发明的siRNA和可将该siRNA在给予对象内表达的表达载体作为癌、特别是肺癌的预防或者治疗剂也是有用的。
将本发明的siRNA用作医药品时,可以直接使用,但也可以与高度分支环状糊精或者环糊精形成复合体。在此,高度分支环状糊精是指使分支酶与支链淀粉作用而生产的具有内分支环状结构部分和外分支结构部分的聚合度为50~5000的葡聚糖。在此,内分支环状结构部分是指由α-1,4-糖苷键和α-1,6-糖苷键形成的环状结构部分,而且外分支结构部分是与该内分支环状结构部分键合了的非环状结构部分。作为高度分支环状糊精的优选形态,可例示上述葡聚糖的内分支环状结构部分的聚合度为10~100的形态、上述葡聚糖的外分支结构部分的聚合度为40以上的形态、上述外分支结构部分的各单元链的聚合度平均从10到20的形态。另外,高度分支环状糊精已市售,在本发明中也可使用市售品。
环糊精是葡萄糖利用α-1,4键进行键合的环状α-1,4-葡聚糖,在螺旋结构的内侧具有立体且有深度的空洞部分。作为在本发明中使用的环糊精中的葡萄糖的聚合度,没有特别限制,例如例示10~500,优选10~100,进一步优选22~50。环糊精可以利用麦芽糖转葡糖基酶等酶而由葡萄糖制备。另外,环糊精已市售,在本发明中也可以使用市售品(国参照际公开第2009/61003号小册子)。
本发明的siRNA可以使用1个以上的药学上允许的载体或者赋形剂,利用常法进行制剂化而制成医药组合物。该医药组合物可以是经肺给予、经鼻给予、经口给予、直肠给予、注射等任意给予形态的组合物,给予方式可以是全身性或者局部给予中的任一种。其剂型根据其给予路径,可以是液体制剂、悬浮剂、乳剂、片剂、丸剂、颗粒、胶囊剂、散剂、缓释制剂、栓剂、气雾剂、喷雾剂等适合使用的任何剂型。
例如,经鼻给予时,可以将有效成分溶解于适当的溶剂中(生理盐水、醇等),通过将该溶液注入到鼻内或者点鼻来送达。或者,经肺给予或者经鼻给予时,将有效成分以下述方式方便地送达,即,使用适当的喷雾剂,例如二氯二氟甲烷、三氯氟甲烷、二氯四氟乙烷、二氧化碳、或者其它适当的气体,从加压包装或者喷雾器中喷出气雾剂。在加压气雾剂的情况下,投药单位量可以通过以送达计量份的方式设置阀来决定。进而,也可制成粉末吸入剂给予。
在注射的情况下,有效成分可以配方成例如通过团注或者连续注入的非经口给予(即静脉内或者肌肉内给予)用溶剂,优选配方成汉克氏液或林格氏液、生理盐水等具有生理学上的相容性的缓冲液作为处方。该溶剂可以含有悬浮剂、稳定剂和/或分散剂等可配方的试剂。或者,为了在使用前与例如灭菌的不含发热性物质的水等适当的赋形剂一起再次构成,可以将有效成分制成粉末形态。注射用制剂可以添加保存剂,例如制成安瓿或者多次给予容器中的单位给予剂型而提供。
经口给予时,本发明的治疗剂可例如为片剂、颗粒、散剂、乳剂、胶囊、糖浆、水性或油性悬浮液、或酏剂的形态。在片剂或者丸药形态的情况下,为了延迟在胃肠道内的分散和吸收,由此带来长时间持续的作用,可以将组合物进行包衣。
作为在药学上允许的载体或者赋形剂,没有限定,可举出液体(例如水、油、生理盐水、葡萄糖水溶液、乙醇等)、固体(例如***树胶、明胶、淀粉、葡萄糖、乳糖、蔗糖、滑石、硬脂酸钠、单硬脂酸甘油酯、角蛋白、胶体状二氧化硅、干燥脱脂乳、甘油等)。另外,本发明的治疗剂可以含有通常的医药组合物中所配合的助剂、防腐剂、稳定剂、增稠剂、润滑剂、着色剂、湿润剂、乳化剂以及pH缓冲剂等中的适当的添加剂。
在本发明的医药组合物中,可以含有0.001~50质量%,优选为0.01~10质量%,进一步优选为0.1~1质量%的本发明的siRNA。
就本发明的医药组合物的给予量而言,只要应用有效量,就没有特别限制,例如,每1kg体重,优选为0.0001~100mg,进一步优选为0.002~1mg。
在本发明的医药组合物中,也可以使用可在给予对象内表达该siRNA的表达载体来代替本发明的siRNA。
此时的表达载体,例如,可以通过将可编码本发明的siRNA的DNA***到适当的基因治疗用载体中来构建,所述基因治疗用载体例如可举出腺病毒载体、腺相关病毒(AAV)载体或者慢病毒载体等。
实施例
以下,使用实施例来进一步详细说明本发明,但本发明的技术范围不限于这些实施例。
实施例1siRNA的制作
设计下述表3所示的以TGF-β1基因为靶的siRNA分子,通过化学合成法来合成各siRNA寡核苷酸,用HPLC精制法进行精制来使用。
表3
实施例2TGF-β1的表达抑制效果的评价(体外)
(1)细胞
使用来自人肺泡上皮细胞的A549细胞(DS Pharma Biomedical株式会社)。
(2)培养条件
将1×105个细胞播种于含有10%胎牛血清的D-MEM培养基(Dulbecco’s modifiedEagle’s Medium,含有100unit/mL青霉素、100μg/mL链霉素,12孔板)。在37℃、5%CO2的条件下培养一晚后,将成为40%铺满的A549细胞的培养基更换成无血清培养基。
(3)前处理·siRNA添加量
使用上述表3所示的寡核苷酸作为siRNA,在细胞成为40%铺满的时刻,使用Lipofectamine 2000(Invitrogen公司)进行向上述细胞的导入。
具体而言,以每孔2.0μL将Lipofectamine2000添加到98μL OPTI-MEM(Invitrogen)中,在室温下培养5分钟(A溶液)。
将0.625μL的0.2μM siRNA溶液添加到99.375μL的OPTI-MEM中(B溶液)。混合A和B溶液,在室温下培养20分钟。培养后,在12-孔板的各孔中添加AB混合液。以siRNA的最终浓度成为0.1nM的方式进行添加。
(4)后处理
(i)细胞因子处理
添加siRNA和Lipofectamine的混合液6小时后,将培养基换成含有0.1%BSA(牛血清白蛋白)和细胞因子(1ng/mL的IL-1β和1ng/mL的TNF-α)的D-MEM培养基,培养12小时。对培养后上清进行取样。
(ii)细胞中总RNA提取
细胞中总RNA提取中,使用自动核酸提取装置QuickGene-810(FUJI FILM株式会社)和作为QuickGene-810的专用试剂盒的QuickGene RNA cultured cell kitS(FUJIFILM株式会社)。用1.0mL PBS清洗细胞,添加0.5mL细胞溶解液,进行细胞中含有的总RNA的提取。用跷跷板型振荡机,将添加有0.5mL溶解液(LRC,添加完巯基乙醇)的12孔板搅拌5分钟。利用移液管,使溶解液往复5-6次而充分混合,将液体移到Eppendorf管中。添加420μL乙醇,用旋涡混合器搅拌15秒钟后,用QuickGene-810进行处理。在用QuickGene-810的处理中,添加DNase(RQ1RNase-free DNase,Promega)。提取的总RNA样品保存在-80℃冰箱,直至进行下一处理。
(iii)总RNA的cDNA化
由在260nm处的吸光度的测定值来计算从培养细胞提取的总RNA样品中的RNA浓度(μg/mL)(对照:TE缓冲液)。基于该值,对于各样品,将与RNA量为0.1μg相当的量的溶液加入96孔板中。向其中加入蒸馏水,使得总量为12μL,再加入2μL的QuantiTect ReverseTranscription Kit(QIAGEN)中所含的gDNA Wipeout Buffer,进行旋涡混合后,在42℃下培养2分钟,其后冷却至4℃。向其中加入QuantiTect Reverse Transcription Kit(QIAGEN)中所含的Quantiscript Reverse Transcriptase 1μL、Quantiscript RT Buffer4μL以及RT Primer Mix 1μL,进行混合,在42℃下培养15分钟。接着为了使QuantiscriptReverse Transcriptase失活,在95℃下加热3分钟后,冷却至4℃。
用TE缓冲液将该制备液(cDNA制备原液)稀释5倍,制成以靶基因(TGF-β1)为靶的PCR用cDNA溶液。
另外,用TE缓冲液将cDNA制备原液稀释50倍,选定GAPDH作为内标基因,制成PCR用cDNA溶液。应予说明,用TE缓冲液将对照样品(siRNA非给予)的cDNA制备原液稀释1、10、100、以及1000倍,制成以TGF-β1为靶的PCR标准曲线用样品。同样,用TE缓冲液将对照样品cDNA制备原液稀释10、100、1000以及10000倍,制成以GAPDH为靶的PCR标准曲线用样品。
(5)TGF-β1表达量的测定方法
将2.5μL的来自TGF-β1的cDNA产物作为模板,使用12.5μL的QuantiFast SYBRGreen PCR Master Mix(QIAGEN)和来自人类TGF-β1基因或者来自人类的GAPDH基因的2.5μL QuantiTect Primer Assay(QIAGEN),制备用灭菌蒸馏水使得最终容量为25μL的PCR反应溶液。将制成的溶液用Applied Biosystems 7500(Life Technologies Japan)在95℃下加热5分钟后,重复40次1)95℃,10秒、2)60℃,35秒的循环,然后从95℃缓慢冷却至60℃,进行热解离测定。基于来自PCR扩增过程的Ct(Threshold Cycle)值,修正基于GAPDH基因的Ct值的各靶基因的扩增比例,评价靶基因的mRNA抑制效果。将结果示于图1和表4。
表4
可知siRNA号为d、p、r、f、c、g、q、j、n、i、h、e、a、k、l、o的siRNA即便在0.1nM的浓度下也具有40%以上的TGF-β1的表达抑制效率。特别是,siRNA号为l和o的siRNA即便在0.1nM也显示80%以上的抑制效率。进而,该序列即便在0.01nM下也显示出显著的抑制效果。
实施例3嵌合体型siRNA的制作
基于siRNA号(l),设计下述表5所示的以TGF-β1基因为靶的嵌合体型siRNA分子,通过化学合成法合成各嵌合体型siRNA寡核苷酸,用HPLC精制法进行精制来使用。
表5
表中,大写字母表示RNA,小写字母表示DNA,下划线表示悬突的位置。
实施例4TGF-β1的表达抑制效果的评价
使用表5所示的寡核苷酸(浓度:10nM或0.1nM),用与实施例2同样的方法评价TGF-β1的表达抑制效果。将结果示于图2和表6。
表6
实施例5键合有磷酸或者硫代磷酸的嵌合体型siRNA的合成
基于siRNA号(l),设计下述表7所示的以TGF-β1基因为靶的键合有磷酸或者硫代磷酸的嵌合体型siRNA分子,通过化学合成法合成各嵌合体型siRNA寡核苷酸,用HPLC精制法进行精制来使用。
表7
表中,大写字母表示RNA,小写字母表示DNA,下划线表示悬突的位置。
另外,P-表示5'末端磷酸化,PS-表示5'末端硫代磷酸化
实施例6TGF-β1的表达抑制效果的评价
使用表7所示的寡核苷酸,用与实施例2同样的方法评价TGF-β1的表达抑制效果。将结果示于图3和表8。
表8
实施例7在悬突部分具有DNA的siRNA的合成
基于siRNA号(l),设计下述表9所示的以TGF-β1基因为靶的在悬突部分具有DNA的siRNA分子,通过化学合成法合成siRNA寡核苷酸,用HPLC精制法进行精制来使用。
表9
表中,大写字母表示RNA,小写字母表示DNA,下划线表示悬突的位置。
实施例8TGF-β1的表达抑制效果的评价
使用表9所示的寡核苷酸,用与实施例2同样的方法评价TGF-β1的表达抑制效果。将结果示于表10。
表10
实施例9肺纤维症模型中的效力的评价(体外试验)
基于作为小鼠与人的共有序列的siRNA号(q)(序列号34和35),设计嵌合体型siRNA(q-C8:正义链(5’→3’):CCAAGGGCUACCAtgccaact(序列号52)、反义链(5’→3’):ttggcaUGGUAGCCCUUGGGC(序列号53),与实施例3同样地合成嵌合体型siRNA寡核苷酸,用HPLC精制法进行精制。将其作为被检物质使用,用博来霉素肺纤维症模型小鼠进行效力评价。
实验对象:小鼠(C57BL/6NCrSlc(SLC)、雌性、13周龄),将戊巴比妥(大日本住友制药株式会社制)给予到小鼠腹腔内后,在麻醉下的小鼠背部皮下埋入ALZETTM渗透泵(model2001,DURECT Corporation)而制成。应予说明,在ALZETTM渗透泵中预先注入200μL约10mg/mL的博来霉素生理盐水溶液,博来霉素使用日本化药株式会社的制品。
将被检物质在ALZETTM渗透泵埋入后第3、7、14天,用蒸馏水(大塚制药株式会社)溶解,使用MicroSprayerTM(model IA-1C,PENN CENTURY,Inc.)以每次100μg/body进行气管内给予。就给予容量而言,博来霉素给予开始后第3、7天以75μL/body进行给予,第14天以50μL/body进行给予。
应予说明,作为比较对照,设定下述两组,即在ALZETTM渗透泵中仅注入200μL生理盐水,给予蒸馏水作为被检物质的组;以及在ALZETTM渗透泵中注入200μL的约10mg/mL的博来霉素生理盐水溶液,给予蒸馏水作为被检物质的组。
在ALZETTM渗透泵埋入后第21天,向小鼠腹腔内给予戊巴比妥,剥离麻醉下的小鼠颈部皮肤和肌肉,露出气管。从颈静脉放血致死后,使用留置针,分3次向气管内注入2mL生理盐水(大塚制药株式会社),回收约2mL BALF(bronchoalveolar lavage fluid)。接着,开胸,在左心耳切入,用约1mL生理盐水由右心室进行灌流,摘除肺。为了进行组织学评价,将摘除的肺的左叶浸渍在10%的中性缓冲***固定液(和光纯药工业株式会社)中。
将采集的BALF进行离心分离(2000rpm,4℃,10分钟),用ELISA法测定上清的TGF-β1蛋白量。将结果示于图4。图中,SAL表示生理盐水(saline),DW表示蒸馏水(distilledwater),BLM表示博来霉素(bleomycin)。
将用10%中性缓冲***固定液固定的肺组织进行石蜡包埋,制成组织切片后,进行H.E.(Hematoxilin-Eosin)染色和马松三色染色。将组织图示于图5。
由图4可知,被检物质给予组(BLM/TGF-β1)中,BALF中的TGF-β1量显著下降。该效果在比较对照的蒸馏水给予组(BLM/DW)中未观察到。另外,由图5的组织图可知,被检物质给予组中,炎症和纤维化的程度降低。
Claims (7)
1.siRNA在制造肺纤维症或肺癌的预防或者治疗剂中的应用,其中,所述siRNA选自以下(a)~(n):
(a)由序列号2所示的正义序列和序列号3所示的反义序列形成的siRNA
(e)由序列号10所示的正义序列和序列号11所示的反义序列形成的siRNA
(h)由序列号16所示的正义序列和序列号17所示的反义序列形成的siRNA
(i)由序列号18所示的正义序列和序列号19所示的反义序列形成的siRNA
(j)由序列号20所示的正义序列和序列号21所示的反义序列形成的siRNA
(k)由序列号22所示的正义序列和序列号23所示的反义序列形成的siRNA
(n)由序列号28所示的正义序列和序列号29所示的反义序列形成的siRNA。
2.根据权利要求1所述的应用,其中,所述siRNA是从正义链的3’末端的末端侧起除去悬突核苷酸的连续的1~10个核苷酸被转换成DNA的siRNA。
3.根据权利要求1所述的应用,其中,所述siRNA是从反义链的5’末端的末端侧起连续的1~10个核苷酸被转换成DNA的siRNA。
4.根据权利要求1所述的应用,其中,所述siRNA是从正义链的3’末端的末端侧起除去悬突核苷酸的连续的1~10个核苷酸被转换成DNA,且从反义链的5’末端的末端侧起连续的1~10个核苷酸被转换成DNA的siRNA。
5.根据权利要求1所述的应用,其中,所述siRNA是反义链的5’末端被单磷酸化或者单硫代磷酸化的siRNA。
6.根据权利要求1所述的应用,其中,所述siRNA是在0.1nM的浓度下具有40%以上的TGF-β1表达抑制率的siRNA。
7.siRNA在制造肺纤维症或肺癌的预防或者治疗剂中的应用,其中,所述siRNA选自以下(a)~(n),以下(a)~(n)的siRNA的核苷酸是糖、碱基和/或磷酸盐被化学修饰过的核苷酸类似物,
(a)由序列号2所示的正义序列和序列号3所示的反义序列形成的siRNA
(e)由序列号10所示的正义序列和序列号11所示的反义序列形成的siRNA
(h)由序列号16所示的正义序列和序列号17所示的反义序列形成的siRNA
(i)由序列号18所示的正义序列和序列号19所示的反义序列形成的siRNA
(j)由序列号20所示的正义序列和序列号21所示的反义序列形成的siRNA
(k)由序列号22所示的正义序列和序列号23所示的反义序列形成的siRNA
(n)由序列号28所示的正义序列和序列号29所示的反义序列形成的siRNA。
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WO2015093495A1 (ja) * | 2013-12-16 | 2015-06-25 | 株式会社ボナック | TGF-β1遺伝子発現制御のための一本鎖核酸分子 |
JP6726105B2 (ja) | 2014-12-15 | 2020-07-22 | 株式会社ボナック | TGF−β1発現抑制のための一本鎖核酸分子 |
WO2017043490A1 (ja) * | 2015-09-07 | 2017-03-16 | 協和発酵バイオ株式会社 | 自然免疫誘導効果が増強した二重鎖リボ核酸 |
RU2714257C2 (ru) * | 2015-10-30 | 2020-02-13 | Бонак Корпорейшн | КОМПОЗИЦИЯ, СОДЕРЖАЩАЯ В СТАБИЛЬНОМ СОСТОЯНИИ ОДНОЦЕПОЧЕЧНУЮ МОЛЕКУЛУ НУКЛЕИНОВОЙ КИСЛОТЫ, КОТОРАЯ ПОДАВЛЯЕТ ЭКСПРЕССИЮ ГЕНА TGF-β1 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007109097A2 (en) * | 2006-03-16 | 2007-09-27 | Alnylam Pharmaceuticals, Inc. | RNAi MODULATION OF TGF-BETA AND THERAPEUTIC USES THEREOF |
WO2009061417A1 (en) * | 2007-11-06 | 2009-05-14 | Sirnaomics, Inc. | Multi-targeted rnai therapeutics for scarless wound healing of skin |
Family Cites Families (41)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19956568A1 (de) | 1999-01-30 | 2000-08-17 | Roland Kreutzer | Verfahren und Medikament zur Hemmung der Expression eines vorgegebenen Gens |
US7829693B2 (en) | 1999-11-24 | 2010-11-09 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
DE10100586C1 (de) | 2001-01-09 | 2002-04-11 | Ribopharma Ag | Verfahren zur Hemmung der Expression eines Ziegens |
WO2003035869A1 (de) | 2001-10-26 | 2003-05-01 | Ribopharma Ag | Verwendung einer doppelsträngigen ribonukleinsäure zur gezielten hemmung der expression eines vorgegebenen zielgens |
US7423142B2 (en) | 2001-01-09 | 2008-09-09 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US8546143B2 (en) | 2001-01-09 | 2013-10-01 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
US7767802B2 (en) | 2001-01-09 | 2010-08-03 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of anti-apoptotic genes |
DE10163098B4 (de) | 2001-10-12 | 2005-06-02 | Alnylam Europe Ag | Verfahren zur Hemmung der Replikation von Viren |
US7745418B2 (en) | 2001-10-12 | 2010-06-29 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting viral replication |
CN1604783A (zh) | 2001-10-26 | 2005-04-06 | 里伯药品公司 | 通过rna干扰治疗纤维化疾病的药物 |
US20050119202A1 (en) | 2001-10-26 | 2005-06-02 | Roland Kreutzer | Medicament to treat a fibrotic disease |
US20040121348A1 (en) | 2001-10-26 | 2004-06-24 | Ribopharma Ag | Compositions and methods for treating pancreatic cancer |
DE10230997A1 (de) | 2001-10-26 | 2003-07-17 | Ribopharma Ag | Medikament zur Erhöhung der Wirksamkeit eines Rezeptor-vermittelt Apoptose in Tumorzellen auslösenden Arzneimittels |
ATE536408T1 (de) * | 2003-04-02 | 2011-12-15 | Dharmacon Inc | Modifizierte polynukleotide zur verwendung bei rna-interferenz |
US7668257B2 (en) * | 2003-10-01 | 2010-02-23 | Samsung Electronics Co., Ltd. | Transmissions with reduced code rate in 8VSB digital television |
KR101147147B1 (ko) | 2004-04-01 | 2012-05-25 | 머크 샤프 앤드 돔 코포레이션 | Rna 간섭의 오프 타겟 효과 감소를 위한 변형된폴리뉴클레오타이드 |
JP2007119396A (ja) | 2005-10-28 | 2007-05-17 | Hosokawa Funtai Gijutsu Kenkyusho:Kk | 核酸化合物封入ナノ粒子を含む経肺投与用医薬製剤 |
US20070269815A1 (en) | 2005-11-21 | 2007-11-22 | Rivory Laurent P | Multitargeting interfering RNAs having two active strands and methods for their design and use |
CA2674210A1 (en) | 2005-12-30 | 2007-07-12 | Intradigm Corporation | Sirna compositions promoting scar-free wound healing of skin and methods for wound treatment |
JP2010519905A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Akt遺伝子の発現を抑制するための核酸化合物およびその使用 |
US20080299659A1 (en) | 2007-03-02 | 2008-12-04 | Nastech Pharmaceutical Company Inc. | Nucleic acid compounds for inhibiting apob gene expression and uses thereof |
JP2010519913A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Wnt遺伝子の発現を抑制するための核酸化合物およびその使用 |
US20080286866A1 (en) | 2007-03-02 | 2008-11-20 | Nastech Pharmaceutical Company Inc. | Nucleic acid compounds for inhibiting vegf gene expression and uses thereof |
WO2008109548A2 (en) | 2007-03-02 | 2008-09-12 | Mdrna, Inc. | Nucleic acid compounds for inhibiting tgfb gene expression and uses thereof |
US20080287383A1 (en) | 2007-03-02 | 2008-11-20 | Nastech Pharmaceutical Company Inc. | Nucleic acid compounds for inhibiting erbb gene expression and uses thereof |
EP2121925A2 (en) | 2007-03-02 | 2009-11-25 | MDRNA, Inc. | Nucleic acid compounds for inhibiting ras gene expression and uses thereof |
JP2010519907A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Vegfファミリー遺伝子の発現を抑制するための核酸化合物およびその使用 |
WO2008109449A1 (en) | 2007-03-02 | 2008-09-12 | Mdrna Inc. | Nucleic acid compounds for inhibiting bcl2 gene expression and uses thereof |
WO2008109381A2 (en) | 2007-03-02 | 2008-09-12 | Mdrna, Inc. | Nucleic acid compounds for inhibiting hif1a gene expression and uses thereof |
JP2010519911A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Myc遺伝子の発現を抑制するための核酸化合物およびその使用 |
EP2121923A1 (en) | 2007-03-02 | 2009-11-25 | MDRNA, Inc. | Nucleic acid compounds for inhibiting erbb family gene expression and uses thereof |
US20100105134A1 (en) | 2007-03-02 | 2010-04-29 | Mdrna, Inc. | Nucleic acid compounds for inhibiting gene expression and uses thereof |
DK2494993T3 (en) * | 2007-05-04 | 2018-11-12 | Marina Biotech Inc | Amino acid lipids and uses thereof |
JP5296328B2 (ja) | 2007-05-09 | 2013-09-25 | 独立行政法人理化学研究所 | 1本鎖環状rnaおよびその製造方法 |
UA97559C2 (uk) | 2007-11-08 | 2012-02-27 | Оцука Фармасьютікал Ко., Лтд. | Комплекс нуклеїнової кислоти і композиція для доставки нуклеїнової кислоти |
WO2009082817A1 (en) * | 2007-12-27 | 2009-07-09 | Protiva Biotherapeutics, Inc. | Silencing of polo-like kinase expression using interfering rna |
US8188060B2 (en) * | 2008-02-11 | 2012-05-29 | Dharmacon, Inc. | Duplex oligonucleotides with enhanced functionality in gene regulation |
BRPI0910446A8 (pt) | 2008-03-31 | 2016-07-26 | Nat Inst Of Advanced Ind Scien | rna modificado por lipídeo de filamento duplo tendo alto efeito de interferência de rna |
CN110042099A (zh) | 2010-03-24 | 2019-07-23 | 菲奥医药公司 | 皮肤与纤维化症候中的rna干扰 |
WO2011119871A1 (en) * | 2010-03-24 | 2011-09-29 | Rxi Phrmaceuticals Corporation | Rna interference in ocular indications |
AR083445A1 (es) | 2010-10-14 | 2013-02-27 | Univ Mie | siARN CONTRA LA FIBROSIS |
-
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007109097A2 (en) * | 2006-03-16 | 2007-09-27 | Alnylam Pharmaceuticals, Inc. | RNAi MODULATION OF TGF-BETA AND THERAPEUTIC USES THEREOF |
WO2009061417A1 (en) * | 2007-11-06 | 2009-05-14 | Sirnaomics, Inc. | Multi-targeted rnai therapeutics for scarless wound healing of skin |
Non-Patent Citations (2)
Title |
---|
Functional dissection of sirna sequence by systematic DNA substitution:modified siRNA with a DNA seed arm is a powerful tool for mammalian gene siliencing with significantly reduced off-target effect;UI-TEI K et al.;《Nucleic Acids Res.》;20081231;第36卷(第7期);2136-2151 * |
Silencing of transforming growth factor-β1 in situ by rna interference for breast cancer:implication for proliferation and migration in vitro and metastasis in vivo;MOORE LD et al.;《Clin.Cancer Res.》;20081231;第14卷(第15期);4961-4970 * |
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