CN115916978A - 寡核苷酸 - Google Patents
寡核苷酸 Download PDFInfo
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- CN115916978A CN115916978A CN202180049291.1A CN202180049291A CN115916978A CN 115916978 A CN115916978 A CN 115916978A CN 202180049291 A CN202180049291 A CN 202180049291A CN 115916978 A CN115916978 A CN 115916978A
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Abstract
本发明涉及维持Toll样受体7(TLR7)应答和/或增强Toll样受体8(TLR8)感测的寡核苷酸。
Description
相关申请的交叉引用
本申请要求2020年5月19日提交的澳大利亚临时专利申请号2020901606的优先权,其内容以引用的方式整体并入本文。
技术领域
本发明涉及维持Toll样受体7(TLR7)应答和/或增强Toll样受体8(TLR8)感测的寡核苷酸。
背景技术
随着8种基于寡核苷酸的治疗药物在美国和欧盟获得批准(Yin和Rogge,2019;AlShaer等人,2020),以及当前III期研究中更多药物的商业化的前景(Coutinho等人,2019),信使RNA(mRNA)的治疗靶向将在疾病管理中发挥重要作用。虽然已经开发了不同的策略来影响mRNA翻译,例如招募RNAse-H1(用反义寡核苷酸[ASO]如inotersen或volanesorsen)或Ago2(用小干扰RNA[siRNA]如patisiran、inclisiran或givosiran)以主动降解靶mRNA,或促进剪接调节(用ASO如依特立生(eteplirsen)和诺西那生钠(nusinersen)),值得注意的是,迄今为止批准的所有治疗性寡核苷酸均依赖于广泛的化学修饰。这种修饰对于防止核酸酶降解至关重要,也可能影响与靶mRNA的结合亲和力。如硫代磷酸酯(PS)主链修饰所示,这些修饰可用于稳定磷酸二酯(PO)核苷酸间连接,或通过糖修饰(例如,用2’-O-甲基[2’OMe]、2’-甲氧基乙基[2’MOE]、2’-氟[2’F]或锁核酸[LNA])稳定碱基(Yin和Rogge,2019)。
在哺乳动物中,外源性核酸的识别是对病原体的免疫应答的关键组分,并且通过多种先天免疫传感器实现,其中包括Toll样受体(TLR),如TLR7、TLR8和TLR9、维甲酸诱导基因-I(RIG-I)样受体、NOD样受体和环-GMP-AMP合成酶(cGAS)途径。因此,并不令人惊讶是发现选择性寡核苷酸治疗药物通过直接接触此类传感器激发强效免疫应答(Hornung等人,2005;Kleinman等人,2008;Krieg等人,1995;Pichlmair等人,2006),指导行业在临床前和临床开发期间仔细考虑和监测此类免疫应答(Frazier等人,2015)。然而,先天免疫传感器对自身和非自身核酸的区分可通过在病原体中很少遇到的核酸修饰的存在来调节,如在人核糖体RNA中丰度是细菌RNA的25倍的2’-O甲基化(2’OMe)核苷所示的(Kariko等人,2005)。TLR7和TLR8选择性检测RNA分子和碱基类似物(如咪唑喹啉和核苷类似物),并被2’OMe碱基抑制,促进自身和非自身RNA之间的分子区分(Kariko等人,2005)。因此,在治疗性寡核苷酸中掺入选择性碱基修饰(包括2’OMe)是帮助减轻TLR7和TLR8的异常免疫应答的有用策略(Kariko等人,2005;Hamm等人,2010),并广泛应用于治疗性siRNA(Coutinho等人,2019)。
然而,这种方法也可能导致非预期的免疫抑制作用,如在含有特异性2’OMe基序的寡核苷酸序列拮抗TLR7和TLR8的情况下观察到的(Sarvestani等人,2015)。类似地,据报道,PS修饰的DNA寡核苷酸以序列依赖性方式拮抗TLR9(Gursel等人,2003)、TLR7(Beignon等人,2005)、AIM2(Kaminkski等人,2013)和cGAS(Steinhagen等人,2018)的感测(Bayik等人,2016)。关键地,考虑到目前批准或正在研究的大多数治疗性寡核苷酸联合PS和碱基修饰,目前尚不确定此类组合是否影响免疫抑制的频率。
因此,需要对Toll样受体7(TLR7)和/或TLR8应答具有有限免疫抑制作用的寡核苷酸。
发明内容
在设计和测试寡核苷酸时,发明人观察到有助于维持Toll样受体7(TLR7)反应的结构特征。
因此,在一个方面,本发明提供了一种寡核苷酸,其包含在寡核苷酸的5’端和/或3’端的7个碱基内的三个连续嘧啶碱基。
在另一方面,本发明涉及一种寡核苷酸,所述寡核苷酸包含位于或靠近寡核苷酸5’端的两个连续胞嘧啶碱基。适当地,两个连续胞嘧啶碱基中的一个或两个被修饰和/或具有修饰的主链。
在一个实施方案中,上述方面的寡核苷酸包含:
a)包含修饰的和/或具有修饰的主链的碱基的5’区域,
b)包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,和
c)包含修饰的和/或具有修饰的主链的碱基的3’区域。
在相关方面,本发明提供了一种寡核苷酸,所述寡核苷酸包含5’区域、3’区域和中间区域,所述中间区域包含核糖核酸、脱氧核糖核酸或其组合、碱基,其中5’区域和3’区域中的一个或两个包含修饰的和/或具有修饰的主链的碱基,并且适用以下至少一项;
a)5’区域包含修饰的和/或具有修饰的主链的三个连续嘧啶碱基,
b)5’区域包含修饰的和/或具有修饰的主链的碱基,并且5’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
c)3’区域包含修饰的和/或具有修饰的主链的三个连续嘧啶碱基,
d)3’区域包含修饰的和/或具有修饰的主链的碱基,并且3’区域和中间区域之间的连接处包含三个连续嘧啶碱基,和
e)5’区域包含两个连续胞嘧啶碱基,它们被修饰和/或具有修饰的主链。
在一个实施方案中,中间区域的长度为约20、约15或约10个碱基。
在一个实施方案中,5’区域和/或3’区域的长度为约7、约5或约3个碱基。
在一个实施方案中,三个连续嘧啶碱基位于或靠近寡核苷酸的5’端和/或3’端。
本发明的5’三个连续嘧啶碱基的实例包括但不限于具有序列5’-CUU-3’、5’-CUT-3’、5’-CCU-3’、5’-UUC-3’、5’-UUU-3’或5’-CTT-3’的那些。在一个实施方案中,5’三个连续嘧啶碱基包含序列5’-CUU-3’。
本发明的3’三个连续嘧啶碱基的实例包括但不限于具有序列5’-UUC-3’、5’-TUC-3’、5’-UCC-3’、5’-CUU-3’、5’-UUU-3’或5’-TTC-3’的那些。在一个实施方案中,3’三个连续嘧啶碱基具有序列5’-UUC-3’。在另一个实施方案中,3’嘧啶碱基具有序列5’-CUUC-3’。
在一个实施方案中,嘧啶碱基中的一个、两个或全部三个是修饰的碱基和/或具有修饰的主链。
在一个实施方案中,连接处的三个连续嘧啶碱基具有序列5’-mCmUT-3’、5’-mCTT-3’、5’-TmUmC-3’或5’-TTmC-3’,其中m是修饰的碱基和/或具有修饰的主链。
可用于本发明的修饰的碱基的实例包括但不限于包含2’-O-甲基、2’-O-甲氧基乙氧基、2’-氟、2’-烯丙基、2’-O-[2-(甲氨基)-2-氧代乙基]、4’-硫代、4’-CH2-O-2’-桥、4’-(CH2)2-O-2’-桥、2’-LNA、2’-氨基、氟代阿糖核苷酸(fluoroarabinonucleotide)、苏糖核酸或2’-O-(N-甲基氨基甲酸酯)的那些。在一些实施方案中,修饰的碱基包含2’-O-甲基、2’-氟、2’-烯丙基、2’-O-[2-(甲氨基)-2-氧代乙基]、4’-硫代、4’-CH2-O-2’-桥、4’-(CH2)2-O-2’-桥、2’-氨基、氟代阿糖核苷酸、苏糖核酸或2’-O-(N-甲基氨基甲酸酯)。
可用于本发明的修饰的主链的实例包括但不限于包含硫代磷酸酯、取代硫原子的非桥接氧原子、膦酸酯诸如甲基膦酸酯、磷酸二酯、磷酸吗啉酸酯、磷酸哌嗪酸酯、酰胺、亚甲基(甲氨基)、甲缩醛(fromacetal)、硫代甲缩醛、肽核酸或磷酰胺诸如吗啉代磷二酰胺(PMO)、N3’-P5’亚磷酰胺或硫代亚磷酰胺的那些。
在一个实施方案中,寡核苷酸的至少一部分具有/是核糖核酸、脱氧核糖核酸、DNA硫代磷酸酯、RNA硫代磷酸酯、2’-O-甲基-寡核苷酸、2’-O-甲基-寡脱氧核糖核苷酸、2’-O-烃基核糖核酸、2’-O-烃基DNA、2’-O-烃基RNA硫代磷酸酯、2’-O-烃基DNA硫代磷酸酯、2’-F-硫代磷酸酯、2’-F-磷酸二酯、2’-甲氧基乙基硫代磷酸酯、2-甲氧基乙基磷酸二酯、脱氧亚甲基(甲基亚氨基)(脱氧MMI)、2’-O-烃基MMI、脱氧甲基膦酸酯、2’-O-烃基甲基膦酸酯、吗啉代、4’-硫代DNA、4’-硫代RNA、肽核酸、3’-酰胺、脱氧3’-酰胺、2’-O-烃基3’-酰胺、锁核酸、环己烷核酸、三环-DNA、2’氟-阿拉伯核酸、N3’-P5’磷酰胺、氨基甲酸酯连接、磷酸三酯连接、尼龙主链修饰及其任何组合。
在一个实施方案中,修饰的碱基包含2’O-甲基,寡核苷酸包含硫代磷酸酯主链。
在上述方面的一些实施方案中,两个连续胞嘧啶碱基包含2’-LNA和硫代磷酸酯主链。
在一个实施方案中,三个连续嘧啶碱基中的一个、两个或全部三个不与靶多核苷酸杂交。
在上述方面的实施方案中,两个连续胞嘧啶碱基中的一个或两个不与靶多核苷酸杂交。
在另一方面,本发明提供寡核苷酸,所述寡核苷酸包含:
i)5’端的5’-CUUGU-3’、5’-CCUAU-3’、5’-CAUUA-3’、5’-CGAAU-3’、5’-CUUAU-3’、5’-CUUUA-3’或5’ACUGU-3’,和
ii)3’端的5’-CUUCU-3’、5’-CAUAU-3’、5’-CUUCU-3’、5’-AAUUU-3’、5’-AAAUU-3’、5’-CCUUC-3’、5’-AAUCA-3’或5’-CGUCU-3’。
在上述方面的一个实施方案中,寡核苷酸包含末端5’U。在另一个实施方案中,寡核苷酸包含末端5’UC。
在一个实施方案中,将以下中的任一个修饰以包含如上所述的5’区域,优选末端,和/或3’区域,优选末端;
5’-AUGGAAUACUCUUGGUUACTT-3’和/或5’-GUAACCAAGAGUAUUCCAUTT-3’(用于治疗多神经病的siRNA链,称为Patisiran);
5’-GCGTTTGCTCTTCTTCTTGCG-3’(用于治疗巨细胞病毒性视网膜炎的反义寡核苷酸,称为福米韦森(fomivirsen));
5'-mG-mC*-mC*-mU*-mC*-dA-dG-dT-dC*-dT-dG-dC*-dT-dT-dC*-mG-mC*-mA-mC*-mC*-3',其中m为2’-O-(2-甲氧基乙基)核苷,d是2’-脱氧核苷,在C和U的5位具有甲基(*)(用于治疗纯合子家族性高胆固醇血症的反义寡核苷酸,称为米泊美生(Mipomersen));
其中下划线字母为2’-O-(2-甲氧基乙基)核糖核苷酸;非下划线字母为2’脱氧核糖核苷酸;所有嘧啶均为5-甲基化;所有连接均为硫代磷酸酯(用于治疗具有遗传性转甲状腺素介导的淀粉样变性的成人神经损伤的反义寡核苷酸,称为Inotersen);
5'-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3'(用于治疗杜氏肌营养不良症的反义寡核苷酸,称为依特立生);
5’-TCACTTTCATAATGCTGG-3’,它是在硫代磷酸酯主链上完全2'-O-甲氧基乙基(MOE)修饰的(用于治疗脊髓性肌萎缩症的反义寡核苷酸,称为诺西那生钠);
5'-XGTTGCCTCCGGTTCTGAAGGTGTTC-3',其中碱基通过合成的中性磷二酰胺吗啉寡聚物(PMO)主链连接,X是亲水性三甘醇链(用于治疗杜氏肌营养不良症的反义寡核苷酸,称为Golodirsen);或
5'-CAGAAAGAGUGUCUCAUCUUA-3'和/或5'-UAAGAUGAGACACUCUUUCUGGU-3'(用于治疗急性肝卟啉症的siRNA链,称为Givosiran)。此外,此类寡核苷酸可具有其他修饰,例如本领域中的那些标准修饰。
在一个实施方案中,任何上述方面的寡核苷酸在施用至动物时不抑制Toll样受体7(TLR7)活性。在一个实施方案中,动物是人。
在另一方面,本发明提供了包含一个或多个修饰的碱基和至少四个胸苷的寡核苷酸,其中所述寡核苷酸在施用至动物时增强Toll样受体8(TLR8)活性。
在一个实施方案中,寡核苷酸包含5'U。在另一个实施方案中,寡核苷酸包含5'UC。
在一个实施方案中,寡核苷酸包含:
a)长度为至少5个碱基的5’区域,所述碱基被修饰和/或具有修饰的主链,
b)包含10个碱基的区段的中间区域,其中至少四个碱基是胸苷,
c)长度为至少5个碱基的3’区域。
在进一步的实施方案中,至少四个胸苷碱基处于连续区段中。
在另一个实施方案中,至少四个胸苷碱基中的一个、两个、三个或四个不处于连续区段中。
在另一方面,本发明提供了一种寡核苷酸,其包含:
a)长度为至少5个碱基的5’区域,其中5'末端由末端5'-mUmC-3'或末端5'-mCmU-3'组成,其中m是修饰的碱基和/或具有修饰的主链,
b)包含10个碱基的区段的中间区域,其中至少两个碱基是胸苷,和
c)长度为至少5个碱基和/或具有修饰的主链的3’区域,
其中所述寡核苷酸在施用至动物(例如人)时增强Toll样受体8(TLR8)活性。
在一个实施方案中,上述两个方面的寡核苷酸也是其他方面所定义的寡核苷酸。
寡核苷酸可以是任何大小。合适大小的例子包括但不限于长度为至少约10、至少约18、至少约20、至少约21、至少约22、至少约23、至少约24、至少约25、至少约26、至少约27、至少约28、至少约29、至少约30、至少约40、约10至约50个核苷酸、约18至约50个核苷酸、约18至约30个核苷酸、约20至约30个核苷酸、约20至1,000个核苷酸、约20至5,000个核苷酸或约20个碱基。
本发明的寡核苷酸可用于多种目的。在一个实施方案中,寡核苷酸是反义寡核苷酸,例如用于与靶mRNA杂交以减少其翻译。在另一个实施方案中,寡核苷酸是用于基因沉默(例如RNA干扰)的链寡核苷酸或形成其一部分。在另一个实施方案中,寡核苷酸用于增强Toll样受体8(TLR8)活性但不与靶RNA杂交。
在一个实施方案中,寡核苷酸是gapmer反义寡核苷酸。在一个实施方案中,三个连续嘧啶碱基中的一个、两个或所有三个在体内被核酸内切酶去除。
在一个实施方案中,反义寡核苷酸下调基因的表达并增强Toll样受体8(TLR8)活性。
在一个实施方案中,用于基因沉默的双链寡核苷酸是siRNA或shRNA。
在一个实施方案中,寡核苷酸的长度为10到16个碱基,并且当施用至动物(例如人)时增强Toll样受体8(TLR8)活性。
在另一方面,本发明提供了一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少三个连续嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生包含三个连续嘧啶碱基的一种或多种候选寡核苷酸,其中适用以下一项或两项;
a)候选寡核苷酸在寡核苷酸5'端的七个碱基内包含三个连续嘧啶碱基,和
b)候选寡核苷酸在寡核苷酸3'端的七个碱基内包含三个连续嘧啶碱基,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
在一个实施方案中,候选寡核苷酸的三个连续嘧啶碱基具有修饰的碱基和/或修饰的主链。
在另一方面,本发明提供了一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少三个连续嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生一种或多种候选寡核苷酸,其包含5’区域、3’区域和包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,其中5’区域和3’区域中的一个或两个包含修饰的和/或具有修饰的主链的碱基,并且适用以下至少一项;
a)5’区域包含三个连续嘧啶碱基,它们被修饰和/或具有修饰的主链,
b)5’区域包含修饰的和/或具有修饰的主链的碱基,并且5’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
c)3’区域包含三个连续嘧啶碱基,它们被修饰和/或具有修饰的主链,和
d)3’区域包含修饰的和/或具有修饰的主链的碱基并且3’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
在另一方面,本发明提供了一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
(i)针对具有以下序列之一的区域扫描靶多核苷酸或其互补序列:5’-CUUGU-3’、5’-CCUAU-3’、5’-CAUUA-3’、5’-CGAAU-3’、5’-CUUAU-3’、5’-CUUUA-3’、5’ACUGU-3’、5’-CUUCU-3’、5’-CAUAU-3’、5’-CUUCU-3’、5’-AAUUU-3’、5’-AAAUU-3’、5’-CCUUC-3’、5’-AAUCA-3’或5’-CGUCU-3’,其中U可以是T,
ii)产生一种或多种候选寡核苷酸,包括:
a)5’端的5’-CUUGU-3’、5’-CCUAU-3’、5’-CAUUA-3’、5’-CGAAU-3’、5’-CUUAU-3’、5’-CUUUA-3’或5’ACUGU-3’和/或
b)3’端的5’-CUUCU-3’、5’-CAUAU-3’、5’-CUUCU-3’、5’-AAUUU-3’、5’-AAAUU-3’、5’-CCUUC-3’、5’-AAUCA-3’或5’-CGUCU-3’,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
在另一方面,本发明涉及一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少两个连续胞嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生包含两个连续胞嘧啶碱基的一种或多种候选寡核苷酸,其中候选寡核苷酸包含位于或靠近寡核苷酸5’端的两个连续胞嘧啶碱基,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
在一些实施方案中,寡核苷酸的两个连续胞嘧啶碱基具有修饰的碱基和/或修饰的主链。
在其他实施方案中,寡核苷酸包含:
a)包含修饰的和/或具有修饰的主链的碱基的5’区域,
b)包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,和
c)包含修饰的和/或具有修饰的主链的碱基的3’区域。
在一个实施方案中,5’区域和/或3’区域的长度为约3个碱基。
在另一个实施方案中,中间区域的长度为约10个碱基。
在相关方面,本发明涉及一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少两个连续胞嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生一种或多种候选寡核苷酸,其包含5’区域、3’区域和包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,其中5’区域和3’区域中的一个或两个包含修饰的和/或具有修饰的主链的碱基,并且5’区域包含两个连续胞嘧啶碱基,其被修饰和/或具有修饰的主链,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
在一个实施方案中,5’区域和/或3’区域的长度为约3个碱基。
在另一个实施方案中,中间区域的长度为约10个碱基。
在上述两个方面的某些实施方案中,两个连续胞嘧啶碱基中的一个或两个是修饰的碱基和/或具有修饰的主链。适当地,对于以上两个方面,两个连续胞嘧啶碱基包含2’-LNA和硫代磷酸酯主链。
在上述三个方面的一个实施方案中,所述方法还包括测试一种或多种候选寡核苷酸抑制Toll样受体7(TLR7)活性的能力,并选择不抑制TLR7活性的寡核苷酸。在这方面,上述方面的方法适用于降低寡核苷酸的TLR7抑制活性。
在设计和测试寡核苷酸时,发明人还观察到有助于增强Toll样受体8(TLR8)活性的新结构特征。
因此,在另一方面,本发明提供了一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对包含至少四个碱基是胸苷的区域扫描靶多核苷酸或其互补物;
ii)产生包含一个或多个修饰的碱基和至少四个胸苷的一种或多种候选寡核苷酸,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达和增强Toll样受体8(TLR8)活性的能力,以及
iv)选择降低靶基因的表达并增强TLR8活性的寡核苷酸。
在另一方面,本发明提供了一种选择增强Toll样受体8(TLR8)活性的寡核苷酸的方法,所述方法包括:
i)针对具有序列UC或CU和10个碱基的区段的区域扫描靶多核苷酸或其互补物,其中至少两个碱基是胸苷;
ii)产生一种或多种候选寡核苷酸,其包含;
a)长度为至少5个碱基的5’区域,其中5’末端由末端5’-mUmC-3’或末端5’-mCmU-3’组成,其中m是修饰的碱基和/或具有修饰的主链,
b)中间区域包含10个碱基的区段,其中至少两个碱基是胸苷,和
c)长度为至少5个碱基和/或具有修饰的主链的3’区域,
iii)测试一种或多种候选寡核苷酸增强TLR8活性的能力,以及
iv)选择增强TLR8活性的寡核苷酸。
在一些情况下,可能无法设计出具有所需嘧啶碱基的合适寡核苷酸。或者,在其他情况下,可能需要改进缺乏所需嘧啶碱基的预先存在的寡核苷酸的功能。因此,在另一方面,本发明提供了一种降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括通过将核苷酸序列添加到寡核苷酸的5’和/或3’端使得修饰的寡核苷酸在寡核苷酸的5’和/或3’端的七个碱基内包含三个连续嘧啶碱基。
在一个实施方案中,嘧啶碱基中一个、两个或所有三个是修饰的碱基和/或具有修饰的主链。
在另一方面,本发明提供了一种降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括修饰寡核苷酸,使得修饰的寡核苷酸包含以下至少一种;
a)5’区域包含三个连续嘧啶碱基,它们被修饰和/或具有修饰的主链,
b)5’区域包含修饰的和/或具有修饰的主链的碱基,并且5’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
c)3’区域包含三个连续嘧啶碱基,它们被修饰和/或具有修饰的主链,和
d)3’区域包含修饰的和/或具有修饰的主链的碱基并且3’区域和中间区域之间的连接处包含三个连续嘧啶碱基。
在以上两个方面的一个实施方案中,三个连续嘧啶碱基位于修饰的寡核苷酸的5’和/或3’端。
在另一方面,本发明提供了降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括通过将核苷酸序列添加到寡核苷酸的5’端来修饰寡核苷酸,使得修饰的寡核苷酸包含位于或靠近寡核苷酸的5’端的两个连续胞嘧啶碱基。
在某些实施方案中,两个连续胞嘧啶碱基中的一个或两个是修饰的碱基和/或具有修饰的主链。
在相关方面,本发明涉及一种降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括修饰寡核苷酸,使得修饰的寡核苷酸包含5’区域,其包含被修饰和/或具有修饰的主链的两个连续胞嘧啶碱基。
在上述两个方面的具体实施方案中,两个连续胞嘧啶碱基位于或靠近修饰的寡核苷酸的5’端。
在上述两个方面的其他实施方案中,两个连续胞嘧啶碱基包含2’-LNA和硫代磷酸酯主链。
在上述四个方面的另一个实施方案中,所述方法还包括测试修饰的寡核苷酸抑制TLR7活性的能力,并选择比未修饰的寡核苷酸更小程度地抑制(TLR7)活性的寡核苷酸。
还提供了使用本发明的方法选择的或使用本发明的方法修饰的寡核苷酸。
在另一方面,本发明涉及一种寡核苷酸,其包含表1至6中所列的核酸序列或其变体、由其组成或基本上由其组成。
在另一方面,本发明提供包含本发明的寡核苷酸的组合物。
在一个实施方案中,该组合物还包含药学上可接受的载体。
在另一个实施方案中,该组合物还包含免疫应答调节剂。
在另一方面,本发明提供了一种降低细胞中靶基因的表达的方法,所述方法包括使细胞与本发明的寡核苷酸接触。
在另一方面,本发明提供治疗或预防受试者的疾病的方法,所述方法包括向受试者施用本发明的寡核苷酸,其中所述寡核苷酸降低与疾病有关的靶基因的表达。
在一个实施方案中,已经或将向动物施用免疫应答调节剂。
在一个实施方案中,免疫应答调节剂是Toll样受体(TLR)激动剂。合适的Toll样受体(TLR)激动剂的实例包括但不限于碱基类似物(包括:鸟苷类似物、脱氮-腺苷类似物、咪唑喹啉或衍生物、羟基腺嘌呤化合物或衍生物、噻唑喹诺酮化合物或衍生物,苯并氮(benzoazepine)化合物或衍生物),或RNA分子。
在一个实施方案中,TLR激动剂为鸟苷、尿苷、瑞喹莫德(R848)、洛索立宾、艾沙托立宾(Isatoribine)、咪喹莫德、CL075、CL097、CL264、CL307、852A或TL8-506。
还提供了本发明的寡核苷酸在制备用于治疗或预防受试者的疾病的药物中的用途,其中所述寡核苷酸降低与该疾病有关的靶基因的表达。
此外,提供了用于治疗或预防受试者的疾病的本发明的寡核苷酸,其中所述寡核苷酸降低与该疾病有关的靶基因的表达。
本文中的任何实施方案应被视为经必要的修改后适用于任何其他实施方案,除非另有说明。
本发明不限于本文描述的具体实施方案的范围,这些具体实施方案仅旨在用于示例的目的。如本文所述,功能等同的产品、组合物和方法显然在本发明的范围内。
贯穿本说明书,除非另有具体说明或上下文另有要求,提及单个步骤、物质组合物、步骤组或物质组合物组应被认为包括一个和多个(即一个或多个)那些步骤、物质组合物、步骤组或物质组合物组。
下文通过以下非限制性实施例并参考附图描述本发明。
附图说明
图1.TLR7/8对R848感测的ASO依赖性调节。
(A)用靶向cGAS的100nM所指示的ASO(表1)过夜预处理的野生型THP-1用1μg/mlR848刺激8.5小时或不刺激(未处理[NT]),然后通过ELISA测定上清液中的IP-10(左图)和TNF-α(右图)水平。显示的数据是三个生物学重复的三个(左)或两个(右)独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用与“无ASO的R848”条件的Dunnett多重比较检验)。对于任一细胞因子,ASO对NT细胞均无基础作用。
(B,C)表达NF-κB-萤光素酶报告因子的HEK-TLR7(B)和HEK-TLR8(C)细胞在用1μg/ml R848刺激之前用500nM所指示的ASO处理50分钟。过夜孵育后测量NF-κB-萤光素酶水平。相对于条件“无ASO的R848”条件的百分比(B)或倍数增加(C)是三个生物学重复的三个独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用与NT条件[B]或“无ASO的R848”条件[C]的Dunnett多重比较检验)。
(D)在用1μg/ml R848刺激24小时并通过ELISA测定上清液中的IP-10水平之前,UNC93B1缺陷型THP-1(KO)和具有拯救的UNC93B1表达(WT)的匹配对照用100nM ASO处理过夜。显示的数据是每个细胞系的三个生物学重复的两个独立实验的平均值(显示±s.e.m和未配对的t检验)。使用2.5μg/ml的cGAS配体ISD70作为阳性对照以诱导IP-10。(E,F)来自两名献血者的PBMC与100nM ASO孵育20-45分钟,并在TNF-αELISA(E)之前4小时或在IFN-αELISA(F)之前24小时用0.5μg/ml R848刺激。
(E)显示的数据是来自2名献血者的三个生物学重复的平均值(显示了±s.e.m和普通单向ANOVA,使用与“无ASO的R848”条件的Dunnett多重比较检验)。
(F)数据被标准化至条件“无ASO的R848”以限制患者之间的差异,并且是来自2名献血者的三个生物学重复的平均值(显示了±s.e.m和Brown-Forsythe和Welch ANOVA,使用与“无ASO的R848”条件的Dunnett’s T3多重比较检验)。
(G)使用的[cGAS]ASO2序列变体。中心DNA区域以浅灰色突出显示。对于3’和5’侧翼区域(以深灰色突出显示),DNA碱基为黑色,ASO2、ASOs-Cys3和ASO-PO的5个5’和3’碱基是2’OMe碱基,ASO2-LNA的3个5’和3’碱基是LNA碱基,ASO2-MOE的5个5’和3’碱基是2’MOE碱基。带下划线的碱基位于PS主链上。
(H,I)表达NF-κB-萤光素酶报告因子的HEKTLR7(H)和HEK-TLR8(I)细胞在用1μg/ml R848刺激前用500nM所指示的ASO处理20分钟。过夜孵育后测量NF-κB-萤光素酶水平。相对于条件“无ASO的R848”条件的百分比(H)或倍数增加(I)是三个生物学重复的三个(I)或两个(H)独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用与NT条件[H]或“无ASO的R848”条件[I]的Dunnett多重比较检验)。*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001,ns:不显著。
图2.鉴定具有低TLR7抑制和高TLR8增强作用的ASO。
(A,B)表达NF-κB-萤光素酶报告因子的HEK-TLR7(A)和HEK-TLR8(B)细胞在用1μg/ml R848刺激前用100nM或500nM所指示的ASO处理20-50分钟。过夜孵育后测量NF-κB-萤光素酶水平。相对于条件“无ASO的R848”的百分比(A)或倍数增加(B)是生物学重复的平均值(平均数据在表2中提供)。在独立实验中使用100nM和500nM ASO进行刺激(每个浓度显示的数据来自单个实验)。[CDKN2B-AS1]-852和[LINCPINT]-2504称为ASO852和ASO2504,并在图上标明。在500nM(A)时TLR7活性降低≥80%和在100nM(B)时TLR8增强≤2倍的ASO以灰色阴影突出显示。
(C,D)表达NF-κB-萤光素酶报告因子的HEK-TLR7(左图)和HEK-TLR8(右图)细胞用递增的ASO浓度(4、20、100和500nM)(C)或用500nM ASO(D)处理20分钟,随后用1μg/ml R848(C)或用递增的R848浓度(0.0156、0.031、0.062、0.125、0.250、0.5、1μg/ml)(D)刺激。过夜孵育后测量NF-κB-萤光素酶水平。相对于条件“无ASO的R848”(C)或NT条件(D)的百分比(左图)或倍数增加(右图)是三个生物学重复的两个独立实验的平均值(显示了±s.e.m和普通双向ANOVA,使用与ASO4条件[C]或仅R848条件[D]的Dunnett多重比较检验)。*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001,ns:不显著。
图3.对TLR7/8的ASO作用的分子决定因素的鉴定。
(A)上图:来自对HEK-TLR7细胞的筛选的ASO序列比对(图2A),其在100nM时显示出低TLR7抑制,并含有显著富集的基序(关于本分析中使用的17种ASO的详细信息,参见表2)。[CD.]为CDKN2B-AS1;[CT.]是CTNNB1。中心DNA区域以浅灰色突出显示,3’和5’2’OMe侧翼区域以深灰色突出显示。下图:构成非抑制基序的碱基的相对频率的MEME象形图。CUU基序为粗体浅灰色,而UUC基序带有下划线。
(B,E)表达NF-κB萤光素酶报告因子的HEK-TLR7细胞用100nM(B)或500nM(E)所指示的ASO处理20分钟,然后用1μg/ml R848刺激。过夜孵育后测量NF-κB-萤光素酶水平。相对于条件“无ASO的R848”的百分比是三个生物学重复的三个(B)或两个(E)独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件的Dunnett多重比较检验[B]或Mann-Whitney U检验[E])。
(C,D)来自PINT系列(C)和[cGAS]ASO11变体(D)的ASO的序列比对。(C)所有序列之间的保守区域以浅灰色突出显示。2’OMe侧翼区域以深灰色突出显示。(D)中心DNA区域以浅灰色突出显示,3’和5’2’OMe侧翼区域以深灰色突出显示。(C,D)CUU基序为粗体浅灰色,而UUC基序带有下划线。
(F,G)表达NF-κB-萤光素酶报告因子的HEK-TLR8细胞用100nM(F)或500nM(G)所指示的ASO处理20分钟,然后用1μg/ml R848刺激。过夜孵育后测量NF-κB-萤光素酶水平。相对于“无ASO的R848”条件的倍数增加是三个生物学重复的三个(F)或两个(G)独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件的Dunnett多重比较检验[F]或Mann-Whitney U检验[G])。
(H,I)根据末端5’mU(H)或5’mUmC(I)的存在或不存在,来自筛选的192个ASO被分为两组,并且将相对于仅R848(使用500nM ASO–表2)的NF-κB-萤光素酶水平的倍数增加显示为每个群体的小提琴图。显示了Mann-Whitney U检验。
(J)对来自筛选的192个ASO(表2)的顶部和底部20个TLR8增效剂的中心10个DNA碱基的碱基含量进行了分析。小提琴图显示了两个ASO群体的每个中心碱基的累积数量分布。显示了带有Sidak多重比较检验的普通双向ANOVA。
(K)ASO852和ASO2504变体。中心DNA区域以蓝色突出显示,3’和5’2’OMe侧翼区域以橙色突出显示。
(L)WT THP-1用100nM ASO预处理过夜,并用1μg/ml R848刺激7小时,并通过ELISA测定上清液中的IP-10水平。显示的数据是三个生物学重复的两个独立实验的平均值(显示了±s.e.m和未配对的t检验)。
(M)表达NF-κB-萤光素酶报告因子的HEK-TLR7和HEK-TLR8细胞在用1μg/ml R848刺激之前用500nM所指示的ASO处理20分钟。过夜孵育后测量NF-κB-萤光素酶水平。相对于条件“无ASO的R848”条件的百分比或倍数增加是三个生物学重复的三个独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件的Dunnett多重比较检验[TLR7]或Mann-Whitney U检验[TLR8])。*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001,ns:不显著。
图4.通过TLR8的R848感测的ASO增强的表征。
(A)MOLM13和OCIAML3细胞用500nM ASO孵育过夜,用1μg/ml R848刺激8小时(MOLM13)或24小时(OCI-AML3),并通过ELISA测定上清液中的IP-10水平。显示的数据是对所有条件的三个生物学重复的五次(MOML13)或四次(OCI-AML3)独立实验的平均值,除了仅ASO条件(在两个独立实验中进行)(显示了±s.e.m和普通单向ANOVA,使用相对于“无ASO的R848”条件的Dunnett多重比较检验)。
(B)MOLM13和THP-1用递增剂量的ASO(4、20、100、500nM)孵育过夜,用1μg/ml R848刺激8小时,上清液中的IP-10水平通过ELISA测定。显示的数据是三个生物学重复的两个独立实验的平均值(显示了±s.e.m和普通双向ANOVA,使用相对于ASO4条件的Dunnett多重比较检验)。
(C)在用1μg/ml R848刺激之前,用500nM所指示的ASO处理表达CCL5-萤光素酶报告因子(右侧)或IFN-β-萤光素酶报告因子(左侧)的HEK-TLR8细胞。孵育过夜后测量萤光素酶水平。数据显示为相对于NT条件的倍数增加,并且是三个生物学重复的两个独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件的Dunnett多重比较检验)。
(D)WT THP-1用500nM ASO852-dT(或NT)孵育过夜,并在RNA纯化前用1μg/ml R848刺激4小时或不刺激。通过RT-qPCR分析了4个IRF驱动基因的小组的表达。相对于18S表达报告所指示的基因的表达,并进一步标准化至“无ASO的R848”条件的平均值。显示的数据代表在生物学重复中进行的两个独立实验的平均值(显示了±s.e.m和MannWhitney U检验)。
(E)在用递增剂量的R848(1、5、10、15μg/ml)刺激之前,用500nM ASO852-dT(或NT)处理表达IFN-β-萤光素酶报告因子的HEK-TLR8细胞20分钟。孵育过夜后测量IFN-β-萤光素酶水平。数据显示为相对于NT条件的倍数增加,并且是三个生物学重复的两个独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件和选定的条件对的Tukey多重比较检验)。*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001,ns:不显著。
图5.展现TLR8增强作用的HPRT靶向ASO的合理选择。
(A)HeLa细胞用1nM(左侧)或10nM(右侧)HPRT靶向ASO进行反向转染,如实施例1和表3所述,并在孵育24小时后通过RT-qPCR测量HPRT水平。相对于SFRS9表达报告HPRT水平,并进一步标准化至非靶向ASONC1和ASONC5对照条件的平均值。显示的数据代表以三个生物学重复进行的三个独立实验的平均值(±s.e.m)。
(B,C)表达NF-κB-萤光素酶报告因子的HEKTLR7(B)和HEK-TLR8(C)细胞在用1μg/ml R848刺激之前用500nM ASO处理20分钟。过夜孵育后测量NF-κB-萤光素酶水平。相对于条件“无ASO的R848”(B)或NT条件(C)的百分比(B)或倍数增加(C)是三个生物学重复的三个独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件[B]或“无ASO的R848”条件[C]的Dunnett多重比较检验)。
(D)选定的具有低TLR7抑制作用的HPRT ASO序列。所有序列之间的保守区域以浅灰色突出显示。2’OMe侧翼区域以深灰色突出显示。CUU基序为粗体浅灰色,而UUC基序带有下划线。
(E,F)WT THP-1用100nM ASO孵育过夜。第二天,紧接在R848刺激(对于仅F,1μg/ml)之前,用lipofectamine 2000(2.5μl/ml,以增强ASO的细胞质递送)处理细胞。8小时后收集上清液用于IP-10ELISA(F),24小时后裂解细胞用于RNA纯化(E)。针对18S报告(E)HPRT水平,并标准化至NT条件。数据是两个重复的四个(E)或三个(F)独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于“无ASO的R848”条件[F]的Dunnett多重比较检验或Mann-Whitney U检验[E])。*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001,ns:不显著。
图6.HeLa细胞中靶向人HPRT基因的48个ASO的初步筛选。HeLa细胞用实施例1中详述的所指示的ASO量进行反向转染,并在孵育24小时后通过RT-qPCR测量HPRT水平。相对于SFRS9表达报告HPRT水平,并进一步标准化至非靶向ASONC1和ASONC5对照条件的平均值。显示的数据代表来自一个实验的三个生物学重复的平均值(±s.e.m)。
图7.TLR7和TLR8配体的ASO852-dT增强。在用洛索立宾(5mM)、CL075(1μg/ml)、嘎德莫特(1μg/ml)刺激之前,用100nM ASO852-dT(或NT)处理表达NF-κB-萤光素酶报告因子的HEK-TLR8细胞20分钟。过夜孵育后测量NF-κB-萤光素酶水平。数据显示为相对于NT条件的倍数增加,并是三个生物学重复的两个独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用Mann-Whitney U检验)。
图8.ASO对TLR7、8、9HEK细胞和THP-1细胞的基础活性。(A、B、C)表达NF-κB-萤光素酶报告因子的HEK-TLR7(A)、TLR8(B)和TLR9(C)细胞用500nM所指示的ASO处理。过夜孵育后测量NF-κB-萤光素酶水平。(A,B)细胞用1μg/ml R848处理作为阳性对照,单独地或在500nMASO2存在的情况下进行(仅针对图B)。(C)HEK-TLR9细胞用200nM ODN 2006刺激作为阳性对照。相对于NT条件的倍数增加是三个生物学重复的两个(A,B)或三个独立实验(C)的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件和选定的条件对的Dunnett多重比较检验[A,B]或Tukey多重比较检验[C])。(D)WT THP-1用100nM ASO预处理过夜(使用500nMASO的条件“852-dT 500nM”除外),并用1μg/ml R848刺激8小时,上清液中的IP-10水平通过ELISA测定。显示的数据是三个生物学重复的两个独立实验的平均值(显示了±s.e.m和普通单向ANOVA,使用相对于NT条件的Dunnett多重比较检验)。(A-D)除非另有说明,否则与NT条件的差异不显著。*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001。
图9.基序特异性TLR8增强。在R848刺激7小时(THP-1,IP-10ELISA)或对于NF-κB萤光素酶刺激过夜之前,用所指示浓度的ASO预处理THP-1细胞和表达NF-κB萤光素酶报告因子的HEK-TLR8细胞(对于THP-1过夜,对于HEK约30分钟)。显示的数据是三个生物学重复的3个独立实验的平均值。对于HEK-TLR8,相对于R848条件报告NF-κB-萤光素酶值。所有ASO条件使用R848共刺激。显示了SEM和单向ANOVA,使用相对于仅R848条件的Dunnett多重比较。
图10.通过ASO的尿苷感测的序列特异性增强。在尿苷刺激(20mM)7小时(THP-1、IP-10ELISA)或对于HEK-TLR8细胞刺激过夜之前,THP-1细胞和表达NF-κB-萤光素酶报告因子或RANTES-萤光素酶报告因子的HEK-TLR8细胞用100nM(THP-1)或500nM ASO(HEK)预处理(对于THP-1过夜,对于HEK约30分钟)。显示的数据是三个生物学重复的3个(THP-1和RANTES-Luc HEK)或2个(NF-κB-Luc HEK)独立实验的平均值。对于HEK-TLR8,相对于R848条件报告NF-κB-萤光素酶值;相对于NT条件报告RANTES-萤光素酶值。所有ASO条件使用尿苷共刺激(1ug/ml的R848条件除外)。显示了SEM和单向ANOVA,使用相对于“仅尿苷”条件的Dunnett多重比较。
图11.增强TLR8感测的LNA和2’MOE gapmer ASO的鉴定。在R848刺激(1μg/ml)过夜之前,表达NF-κB-萤光素酶报告因子的HEK-TLR8细胞用所指示浓度的ASO预处理约30分钟。相对于条件“无ASO的R848”的倍数增加是来自两个生物学重复的平均值(平均数据在表1中提供)。在独立实验中进行使用100nM和500nM ASO的刺激(每个浓度显示的数据来自单个实验)。
图12.增强TLR8的LNA ASO的验证。在R848刺激7小时(THP-1,IP-10ELISA)或对于NF-κB-萤光素酶刺激过夜之前,THP-1细胞和表达NF-κB-萤光素酶报告因子的HEK-TLR8细胞用所指示浓度的LNA ASO预处理(对于THP-1过夜,对于HEK约30分钟)。显示的数据是三个生物学重复(THP-1)或两个生物学重复(HEK)的3个独立实验的平均值。对于HEK-TLR8细胞,相对于“仅R848”条件报告NF-κB-萤光素酶值。所有ASO条件使用R848共刺激。显示了SEM和单向ANOVA,使用相对于仅R848条件的Dunnett多重比较。
图13.增强TLR8的2’MOE ASO的验证。在R848刺激(1μg/ml)过夜之前,表达NF-κB-萤光素酶报告因子的HEK-TLR8细胞用500nM所指示的2’MOE ASO预处理约30分钟。所有ASO条件使用R848共刺激。相对于条件“无ASO的R848”的倍数增加是来自生物学重复的平均值-显示的数据来自单个实验(±SEM)。
图14.dT20对TLR8感测的增强作用在洗掉寡核苷酸后不会持续存在。左:表达NF-κB-萤光素酶报告因子的HEK-TLR8细胞用500nM dT20预处理过夜,然后洗涤(或不洗涤)并用R848刺激(1μg/ml)刺激过夜。显示的数据是三个生物学重复的2个独立实验的平均值,并相对于仅R848条件报告。显示了SEM和单向ANOVA,使用相对于仅R848条件的Dunnett多重比较。右:THP-1细胞用100nM所指示的ASO孵育过夜(紫色)或2.5小时,然后R848刺激(1μg/ml)约7小时。IP-10水平通过ELISA测量。显示的数据来自三个生物学重复的单个实验(每个点代表一个生物学重复)。所有ASO条件使用R848共刺激。
图15.吞噬细胞与ASO转染细胞的共培养导致序列特异性TLR8增强。HEK WT细胞用500nM ASO3(非TLR8增强)或500nM 852dT(强增强TLR8)转染4小时,然后进行紫外线处理(254nm,120mJ/cm2),大量洗涤(2x5ml-以去除未转染的ASO),并与6天PMA分化的THP-1共培养过夜,随后进行24小时R848刺激(5μg/ml)。通过ELISA测量TNFα水平。显示的数据是相对于ASO3或ASO 852dT条件的,并且是使用生物学重复的2个独立实验的平均值。显示了SEM和未配对的双尾t检验。
图16.完全2’OMe修饰的ASO的TLR8增强。在R848刺激(1μg/ml)过夜之前,表达NF-κB-萤光素酶报告因子的HEK-TLR8细胞用500nM所指示的2’MOE ASO预处理约30分钟。所有ASO条件使用R848共刺激。相对于“无ASO的R848”条件的倍数增加是来自三个生物学重复的3个独立实验的平均值(显示了±SEM和单向ANOVA,使用相对于仅R848条件的Dunnett多重比较)。
图17.5’-末端CUU基序在2’OMe ASO而非LNA ASO的背景下调节TLR7感测。在R848刺激(1μg/ml)过夜之前,表达NF-κB-萤光素酶报告因子的HEK-TLR7细胞用500nM指示的2’MOE ASO预处理约30分钟。所有ASO条件使用R848共刺激。显示的数据是三个生物学重复的3个独立实验的平均值,并相对于仅R848条件报告。显示了SEM和单向ANOVA,使用相对于“660-Mut”条件的Dunnett多重比较。
图18.不抑制TLR7感测的LNA和2’MOE gapmer ASO的鉴定。在R848刺激(1μg/ml)过夜之前,表达NF-κB-萤光素酶报告因子的HEK-TLR7细胞用所指示浓度的ASO预处理约30分钟。相对于条件“无ASO的R848”的倍数增加是来自生物学重复的平均值(平均数据在表2中提供)。在独立实验中进行使用100nM和500nM ASO的刺激(对于每个浓度显示的数据来自单个实验)。
图19.具有有限TLR7抑制的LNA和2’MOE gapmer ASO的验证。在R848刺激(1μg/ml)过夜之前,表达NF-κB-萤光素酶报告因子的HEK-TLR7细胞用500nM指示的2’MOE ASO预处理约30分钟。所有ASO条件使用R848共刺激。显示的数据是三个生物学重复的3个独立实验(对于LNA)或单个实验(对于2’MOE)的平均值,并相对于仅R848条件报告。对于LNA ASO(左)显示了SEM和单向ANOVA,使用相对于“仅R848”条件的Dunnett多重比较。阴影条是指具有5’+C+C基序的ASO。对于2’MOE实验,发明人注意到E1和一些其他ASO(未显示)完全消融了TLR7感测,但G1-A2-C1-A9没有。
序列简述
SEQ ID NO:1和SEQ ID NO:2代表来自表1的阴性靶向对照ASO的核苷酸序列。SEQID NO:3到SEQ ID NO:20代表来自表1的靶向人cGAS mRNA及其修饰形式的ASO的核苷酸序列。SEQ ID NO:21和SEQ ID NO:22代表来自表1的ASO852和ASO852-DT的核苷酸序列。SEQID NO:23和SEQ ID NO:24代表来自表1的ASO2504和ASO2504-dT的核苷酸序列。SEQ ID NO:25代表来自表1的dT20的核苷酸序列。SEQ ID NO:26到SEQ ID NO:34代表来自表1的分别地Hs HPRT F517、Hs HPRT R591、Hs HPRT P554 FAM、Hs SFRS9 F594、Hs SFRS9 R690、HsSFRS9 P625 HEX、ODN 2006、ISD70-FWD和ISD70-REV的核苷酸序列。
SEQ ID NO:35至SEQ ID NO:82代表来自表2的CDKN2B-AS1 ASO的核苷酸序列。SEQID NO:83至SEQ ID NO:130代表来自表2的CTNNB1 ASO的核苷酸序列。SEQ ID NO:131至SEQID NO:178代表来自表2的EGFR ASO的核苷酸序列。SEQ ID NO:179至SEQ ID NO:226代表来自表2的LINC-PINT ASO的核苷酸序列。
SEQ ID NO:227至SEQ ID NO:273代表来自表3的HPRT ASO的核苷酸序列。SEQ IDNO:274至SEQ ID NO:282代表来自表4的分别地ASO1-UC、ASO2 LNA、ASO2-LNA Mut1、ASO2-LNA Mut2、ASO 660、ASO 660-Mut、C2Mut-1、C2Mut1-PS、C2Mut1-2OMe的核苷酸序列。SEQ IDNO:283至SEQ ID NO:373代表表5的LNA修饰的核苷酸序列。SEQ ID NO:374至SEQ ID NO:449代表表6的2’-MOE修饰的核苷酸序列。
发明详述
一般技术和定义
除非另有具体定义,否则本文使用的所有技术和科学术语应被视为具有与本领域普通技术人员通常理解的相同含义(例如,寡核苷酸设计、分子遗传学、反义寡核苷酸、基因沉默、基因表达和生物化学中的)。
除非另有说明,否则本发明中使用的重组蛋白、细胞培养和免疫学技术是标准程序,为本领域技术人员所熟知。这些技术在整个文献来源中都有描述和解释,例如J.Perbal,A Practical Guide to Molecular Cloning,John Wiley and Sons(1984),J.Sambrook等人,Molecular Cloning:A Laboratory Manual,Cold Spring HarbourLaboratory Press(1989),T.A.Brown(编辑),Essential Molecular Biology:APractical Approach,Volumes 1and 2,IRL Press(1991),D.M.Glover和B.D.Hames(编辑),DNA Cloning:A Practical Approach,Volumes 1-4,IRL Press(1995和1996),以及F.M.Ausubel等人(编辑),Current Protocols in Molecular Biology,GreenePub.Associates and Wiley-Interscience(1988,包括目前为止的所有更新),Ed Harlow和David Lane(编辑)Antibodies:A Laboratory Manual,Cold Spring HarbourLaboratory(1988),以及J.E.Coligan等人(编辑)Current Protocols in Immunology,John Wiley&Sons(包括目前为止的所有更新)。
术语“和/或”,例如“X和/或Y”应被理解为是指“X和Y”或“X或Y”,并且应被视为为两种含义或任一含义提供明确支持。
如本文所用,除非另有相反说明,术语约是指指定值的+/-10%,更优选+/-5%,更优选+/-1%。
贯穿本说明书,单词“包括”或诸如“包含”或“含有”的变型将被理解为暗示包括所陈述的元素、整数或步骤,或元素、整数或步骤的组,但不排除任何其他元素、整数或步骤,或元素、整数或步骤的组。
在寡核苷酸序列的上下文中,“基本上由...组成”是指所列举的寡核苷酸序列连同在其5’或3’端的另外一个、两个或三个核酸。
如本文所用,短语“不抑制Toll样受体7(TLR7)活性”或其变型是指在向动物施用本发明的寡核苷酸后,该动物仍然能够引发基于TLR7的免疫应答,比如针对病原体。在一个实施方案中,在寡核苷酸的存在下基于TLR7的免疫应答至少为在寡核苷酸不存在下的应答的约10%、20%、30%、40%、50%、60%、70%、80%、85%、90%、95%、97%、99%或100%。
类似地,短语“降低寡核苷酸的Toll样受体7(TLR7)抑制活性”或类似的意指在根据本发明进行修饰后,施用修饰的寡核苷酸的动物能够启动与起始(未修饰)寡核苷酸相比更强的基于TLR7的免疫应答。
如本文所用,短语“增强Toll样受体8(TLR8)活性”等意指在向动物施用本发明的寡核苷酸后,该动物具有增强的(增加的)基于TLR8的免疫应答。
如本文所用,“免疫应答调节剂”是指模拟、增强或需要宿主免疫细胞参与以获得最佳效果和/或具有激活、增强或加强特定免疫应答的已知能力的任何试剂。免疫应答调节剂的实例包括但不限于Toll样受体(TLR)激动剂,包括瑞喹莫德(R848)、洛索立宾、艾沙托立宾、咪喹莫德、CL075、CL097、CL264、CL307、852A和/或TL8-506。其他Toll样受体(TLR)激动剂可包括碱基类似物(包括:鸟苷类似物、脱氮腺苷类似物、咪唑喹啉或衍生物、羟基腺嘌呤化合物或衍生物、噻唑喹诺酮化合物或衍生物、苯并氮化合物或衍生物)和/或RNA分子。
本文使用的短语“药学上可接受的”是指在合理的医学判断范围内,适用于与人和动物的组织接触而无与合理的收益/风险比相称的过度毒性、刺激、过敏反应和/或其他问题或并发症的那些化合物、材料、组合物和/或剂型。
如本文所用,术语“治疗”、“医治”或“疗法”包括施用足以减轻或消除疾病的至少一种症状的治疗有效量的本文描述的化合物。
如本文所用,术语“预防”、“防止”或“防范”包括施用足以停止或阻碍疾病的至少一种症状的发展的治疗有效量的本文描述的化合物。
寡核苷酸
在本发明的上下文中,术语“寡核苷酸”是指核糖核酸(RNA)或脱氧核糖核酸(DNA)的寡聚体或多聚体,其中核苷酸单体的多聚体或寡聚体包含核碱基(本领域并且本文简称为“碱基”)的任意组合、修饰的核碱基、糖、修饰的糖、磷酸桥或修饰的磷原子桥(本文也称为“核苷酸间键”)。
寡核苷酸可以是单链的或双链的或它们的组合。单链寡核苷酸可以有双链区域,双链寡核苷酸可以有单链区域(例如microRNA或shRNA)。
“Gapmer”是指包含内部区域的寡核苷酸,该内部区域具有位于具有一个或多个核苷的外部区域之间的多个支持RNase H裂解的核苷,其中包含内部区域的核苷在化学上不同于包含外部区域的一种或多种核苷。内部区域可被称为“间隙”,外部区域可被称为“翼”。
如本文所用,诸如“靶基因”或“靶多核苷酸”的“靶”是指本发明的寡核苷酸直接或间接对其发挥作用的分子。通常,本发明的寡核苷酸或其部分与靶或靶的产物如由基因编码的mRNA或其部分能够在生理条件下杂交。
如本文所用,短语“降低靶基因的表达”或类似词是指本发明的寡核苷酸降低基因发挥生物学效应的能力。这可以通过减少由基因编码的RNA的量和/或减少从RNA翻译的蛋白质的量来直接或间接实现。
通常,本发明的寡核苷酸将在体外合成。然而,在一些不需要修饰的碱基和主链的情况下,它们可以在体外或体内在合适的系统中表达,例如通过重组病毒或细胞。
本发明的寡核苷酸可以缀合到增强寡核苷酸的活性、细胞分布或细胞摄取的一个或多个部分或基团。这些部分或基团可以共价结合到官能团如伯羟基或仲羟基。示例性的部分或基团包括嵌入剂、报告分子、多胺、聚酰胺、聚乙二醇、聚醚、增强寡聚物的药效学特性的基团和增强寡聚物的药代动力学特性的基团。典型的缀合基团包括胆固醇、脂质、磷脂、生物素、吩嗪、叶酸、菲啶、蒽醌、吖啶、荧光素、罗丹明、香豆素和染料。
在具体实施方案中,本文所述的寡核苷酸可包含合成寡核苷酸序列。如本文所用,“合成寡核苷酸序列”是指缺少天然存在的相应序列的寡核苷酸序列。举例来说,合成寡核苷酸序列不与特定的RNA分子例如编码内源多肽的RNA分子互补。因此,合成寡核苷酸序列适当地不能干扰转录后事件,例如RNA翻译。
如本文所用,寡核苷酸“变体”与参考核酸序列共有可定义的核苷酸序列关系。例如,参考核酸序列可以是表1至6中提供的序列(例如,SEQ ID NO.1-449)之一。“变体”寡核苷酸可以具有缺失或被不同核酸取代的参考核酸序列的一个或多个核酸。优选地,寡核苷酸变体与参考核酸序列共有至少70%或75%,优选至少80%或85%或更优选至少90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的序列同一性。
修饰的碱基
本发明的寡核苷酸可以具有核碱基(“碱基”)修饰或取代。
实例包括在2’位置包含以下之一的寡核苷酸:OH;F;O-,S-,或N-烷基;O-,S-,或N-烯基;O-,S-或N-炔基;或O-烷基-O-烷基,其中烷基、烯基和炔基可以是取代的或未取代的C1至C10烷基或C2至C10烯基和炔基。在一个实施方案中,寡核苷酸在2’位置包含以下之一:O[(CH2)nO]mCH3、O(CH2)nOCH3、O(CH2)nNH2、O(CH2)nCH3、O(CH2)nONH2和O(CH2)nON[(CH2)nCH3]2,其中n和m为1至约10。
修饰的寡核苷酸的进一步实例包括在2’位置包含以下之一的寡核苷酸:C1至C10低级烷基、取代的低级烷基、烯基、炔基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3、OCN、Cl、Br、CN、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2、杂环烷基、杂环烷芳基、氨基烷基氨基、聚烷基氨基、取代的甲硅烷基、RNA切割基团、报告基团、嵌入剂、改善寡核苷酸药代动力学性质的基团、或改善寡核苷酸药效学性质的基团,以及具有相似性质的其他取代基。
在一个实施方案中,修饰包括2’-甲氧基乙氧基(2’-O-CH2CH2OCH3(也称为2’-O-(2-甲氧基乙基)或2’-MOE)(Martin等人,1995),即,烷氧基烷氧基。在一些实施方案中,修饰不包含2’-MOE。在进一步的实施方案中,修饰包括2’-二甲基氨基氧基乙氧基,即,O(CH2)2ON(CH3)2基团(也称为2’-DMAOE),或2’-二甲基氨基乙氧基乙氧基(本领域也称为2’-O-二甲基-氨基-乙氧基-乙基或2’-DMAEOE),即2'-O-CH2-O-CH2-N(CH3)2。
其他修饰包括2’-甲氧基(2’-O-CH3)、2’-氨基丙氧基(2’-OCH2CH2CH2NH2)、2’-烯丙基(2'-CH2-CH=CH2)、2’-O-烯丙基(2'-O-CH2-CH=CH2)和2’-氟(2’-F)。2’-修饰可以在阿拉伯糖(上)位置或核糖(下)位置。在一个实施方案中,2’-阿拉伯糖修饰是2’-F。
也可以在寡核苷酸的其他位置(特别是3’端核苷酸上或2’-5’连接的寡核苷酸中糖的3’位置和5’端核苷酸的5’位置)进行类似的修饰。
寡核苷酸也可以具有糖模拟物,例如代替呋喃戊糖的环丁基部分。
教导制备此类修饰的糖结构的代表性美国专利包括但不限于US4,981,957、US5,118,800、US5,319,080、US5,359,044、US5,393,878、US5,446,137、US5,466,786、US5,514,785、US5,519,134、US5,567,811、US5,576,427、US5,591,722、US5,597,909、US5,610,300、US5,627,053、US5,639,873、US5,646,265、US5,658,873、US5,670,633、US5,792,747、和US5,700,920。
糖的进一步修饰包括锁核酸(LNA),其中2’-羟基连接到糖环的3’或4’碳原子,从而形成双环糖部分。在一个实施方案中,键联是桥接2’氧原子和4’碳原子的亚甲基(-CH2-)n基团,其中n是1或2。LNA及其制备描述于WO98/39352和WO99/14226中。然而,在一些实施方案中,修饰不包含LNA。
修饰的核碱基包括其他合成和天然核碱基,例如5-甲基胞嘧啶(5-me-C),5-羟甲基胞嘧啶,黄嘌呤,次黄嘌呤,2-氨基腺嘌呤,腺嘌呤和鸟嘌呤的6-甲基和其他烷基衍生物,腺嘌呤和鸟嘌呤的2-丙基和其他烷基衍生物,2-硫氧嘧啶,2-硫胸腺嘧啶和2-硫胞嘧啶,5-卤尿嘧啶和胞嘧啶,5-丙炔基(-CC-CH3)尿嘧啶和胞嘧啶以及嘧啶碱基的其他炔基衍生物,6-偶氮尿嘧啶,胞嘧啶和胸腺嘧啶,5-尿嘧啶(伪尿嘧啶),4-硫氧嘧啶,8-卤素,8-氨基,8-硫醇,8-硫代烷基,8-羟基和其他8-取代的腺嘌呤和鸟嘌呤,5-卤素特别是5-溴,5-三氟甲基和其他5-取代的尿嘧啶和胞嘧啶,7-甲基鸟嘌呤和7-甲基腺嘌呤,2-F-腺嘌呤,2-氨基-腺嘌呤,8-氮杂鸟嘌呤和8-氮杂腺嘌呤,7-脱氮鸟嘌呤和7-脱氮腺嘌呤和3-脱氮鸟嘌呤和3-脱氮腺嘌呤。
进一步修饰的核碱基包括三环嘧啶,例如吩噁嗪胞苷(1H-嘧啶并[5,4-b][1,4]苯并噁嗪-2(3H)-酮)、吩噻嗪胞苷(1H-嘧啶并[5,4-b][1,4]苯并噻嗪-2(3H)-酮)、G-钳,例如取代的吩噁嗪胞苷(例如,9-(2-氨基乙氧基)-H-嘧啶并[5,4-b][1,4]苯并噁嗪-2(3H)-酮)、咔唑胞苷(2H-嘧啶并[4,5-b]吲哚-2-酮)、吡啶吲哚胞苷(H-吡啶并[3',2':4,5]吡咯并[2,3-d]嘧啶-2-酮)。
修饰的核碱基还可以包括其中嘌呤或嘧啶碱基被其他杂环取代的碱基,例如,7-脱氮-腺嘌呤、7-脱氮鸟苷、2-氨基吡啶和2-吡啶酮。其他核碱基包括在US3,687,808中公开的那些,在J.I.Kroschwitz(编辑),The Concise Encyclopedia of Polymer Science andEngineering,第858-859页,John Wiley and Sons(1990)中公开的那些,Englisch等人(1991)公开的那些,以及Y.S.Sanghvi,Chapter 15:Antisense Research andApplications,第289-302页,S.T.Crooke,B.Lebleu(编辑),CRC Press,1993公开的那些。
这些核碱基中的某些对于增加寡核苷酸的结合亲和力特别有用。这些包括5-取代的嘧啶、6-氮杂嘧啶和N-2、N-6和O-6取代的嘌呤,包括2-氨基丙基腺嘌呤、5-丙炔基尿嘧啶和5-丙炔基胞嘧啶。5-甲基胞嘧啶取代已显示将核酸双链体稳定性提高0.6-1.2oC。在一个实施方案中,这些核碱基取代与2’-O-甲氧基乙基糖修饰组合。
教导制备某些上述修饰的核碱基以及其他修饰的核碱基的代表性美国专利包括但不限于US3,687,808、US4,845,205、US5,130,302、US5,134,066、US5,175,273、US5,367,066、US5,432,272、US5,457,187、US5,459,255、US5,484,908、US5,502,177、US5,525,711、US5,552,540、US5,587,469、US5,594,121、US5,596,091、US5,614,617、US5,645,985、US5,830,653、US5,763,588、US6,005,096、US5,681,941和US5,750,692。
除非另有说明,提及A、T、G、U或C可以指天然存在的碱基或其修饰形式。
在特定的实施方案中,本文所述的寡核苷酸的两个或更多个碱基(例如,2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个碱基,包括其中的任何范围)被修饰。在一些实施方案中,本文描述的寡核苷酸的所有碱基都被修饰。在备选实施方案中,本文描述的寡核苷酸的碱基没有被修饰。
主链
本公开内容的寡核苷酸包含具有修饰的主链或非天然核苷间键联的那些。具有修饰的主链的寡核苷酸包含在主链中保留磷原子的寡核苷酸和在主链中不具有磷原子的寡核苷酸。
其中含有磷原子的修饰的寡核苷酸主链包括,例如,硫代磷酸酯、手性硫代磷酸酯,二硫代磷酸酯,磷酸三酯,氨烷基磷酸三酯,甲基和其他烷基膦酸酯,包括3’-亚烷基膦酸酯,5’-亚烷基膦酸酯和手性膦酸酯,次膦酸酯,氨基磷酸酯,包括3’-氨基氨基磷酸酯和氨基烷基氨基磷酸酯,硫代氨基磷酸酯,硫代烷基膦酸酯,硫代烷基磷酸三酯,硒代磷酸酯和具有正常3’-5’键联的硼磷酸酯,这些的2’-5’连接的类似物,以及具有反转极性的那些,其中一个或多个核苷酸间键联是3’至3’、5’至5’或2’至2’键联。具有反转极性的寡核苷酸在最3’的核苷酸间键联处包含单个3’至3’键联,即,其可以是无碱基(核碱基缺失或在其位置具有羟基)的单个反转核苷残基。还包括各种盐、混合盐和游离酸形式。
教导制备上述含磷键联的代表性美国专利包括但不限于US3,687,808、US4,469,863、US4,476,301、US5,023,243、US5,177,196、US5,188,897、US5,264,423、US5,276,019、US5,278,302、US5,286,717、US5,321,131、US5,399,676、US5,405,939、US5,453,496、US5,455,233、US5,466,677、US5,476,925、US5,519,126、US5,536,821、US5,541,306、US5,550,111、US5,563,253、US5,571,799、US5,587,361、US5,194,599、US5,565,555、US5,527,899、US5,721,218、US5,672,697和US5,625,050。
其中不包括磷原子的修饰的寡核苷酸主链包括例如由短链烷基或环烷基核苷间键联、混合的杂原子和烷基或环烷基核苷间键联、或一个或多个短链杂原子或杂环核苷间键联形成的主链。这些包括具有以下的那些:吗啉代键联(部分由核苷的糖部分形成);硅氧烷主链;硫化物、亚砜和砜主链;甲酰基和硫代甲酰基主链;亚甲基甲酰基和硫代甲酰基主链;核糖乙酰主链;含有烯烃的主链;氨基磺酸酯主链;亚甲基亚氨基和亚甲基肼主链;磺酸酯和磺酰胺主链;酰胺主链;以及具有混合的N、O、S和CH2组成部分的其他主链。
教导制备上述寡核苷酸的代表性美国专利包括但不限于:US5,034,506、US5,166,315、US5,185,444、US5,214,134、US5,216,141、US5,235,033、US5,264,562、US5,264,564、US5,405,938、US5,434,257、US5,466,677、US5,470,967、US5,489,677、US5,541,307、US5,561,225、US5,596,086、US5,602,240、US5,610,289、US5,602,240、US5,608,046、US5,610,289、US5,618,704、US5,623,070、US5,663,312、US5,633,360、US5,677,437、US5,792,608、US5,646,269和US5,677,439。
反义寡核苷酸
术语“反义寡核苷酸”应理解为与特定mRNA分子(例如编码内源性多肽)的至少一部分互补并能够干扰转录后事件例如mRNA翻译的寡核苷酸。反义方法的使用是本领域众所周知的(参见例如,G.Hartmann和S.Endres,Manual of Antisense Methodology,Kluwer(1999))。
在一个实施方案中,反义寡核苷酸在生理条件下杂交,即反义寡核苷酸(其是完全或部分单链的)在细胞中的正常条件下至少能够与mRNA(例如编码内源多肽)形成双链多核苷酸。
反义寡核苷酸可包括对应于结构基因的序列或影响对基因表达或剪接事件的控制的序列。例如,反义序列可以对应于内源基因的靶向编码区,或5’-非翻译区(UTR)或3’-UTR,或它们的组合。它可以与可以在转录期间或之后被剪除的内含子序列部分互补,优选仅与靶基因的外显子序列互补。鉴于UTR的差异通常更大,靶向这些区域提供更大的基因抑制特异性。
反义寡核苷酸可以与整个基因转录物或其部分互补。反义序列与靶向的转录物的同一性程度应为至少90%,更优选为95-100%。反义RNA或DNA分子当然可以包含不相关的序列,这些序列可以起到稳定分子的作用,例如本文所述。
基因沉默
寡核苷酸分子,特别是RNA,可用于调节基因表达。术语“RNA干扰”、“RNAi”或“基因沉默”一般指这样的过程,其中dsRNA分子降低与双链RNA分子共有实质或完全的同源性的核酸序列的表达。然而,已表明可以使用非RNA双链分子实现RNA干扰(参见,例如,US20070004667)。
双链区应该为至少19个连续的核苷酸,例如约19至23个核苷酸,或可以更长,例如30个或50个核苷酸,或100个核苷酸或更多。可以使用对应于整个基因转录物的全长序列。优选地,它们的长度为约19至约23个核苷酸。
核酸分子的双链区域与靶转录物的同一性程度应为至少90%,更优选为95-100%。核酸分子当然可以包含不相关的序列,其可以起到稳定分子的作用。
如本文所用,术语“短干扰RNA”或“siRNA”是指包含能够抑制或下调基因表达的核糖核苷酸的多核苷酸,例如通过以序列特异性方式介导RNAi,其中双链部分的长度少于50个核苷酸,优选长度为约19至约23个核苷酸。例如,siRNA可以是包含自身互补的有义和反义区的核酸分子,其中反义区包含与靶核酸分子中的核苷酸序列或其一部分互补的核苷酸序列和有义区具有对应于靶核酸序列或其一部分的核苷酸序列。siRNA可以由两条独立的寡核苷酸组装而成,其中一条链是有义链,另一条是反义链,其中反义链和有义链是自身互补的。两条链可以具有不同的长度。
如本文所用,术语siRNA意在等同于用于描述能够介导序列特异性RNAi的多核苷酸的其他术语,例如微小RNA(miRNA)、短发夹RNA(shRNA)、短干扰寡核苷酸、短干扰核酸(siNA)、短干扰修饰寡核苷酸、化学修饰的siRNA、转录后基因沉默RNA(ptgsRNA)等。此外,如本文所用,术语RNAi意在等同于用于描述序列特异性RNA干扰的其他术语,例如转录后基因沉默、翻译抑制或表观遗传学。例如,siRNA分子可用于在转录后水平或转录前水平对基因进行表观遗传沉默。在一个非限制性实例中,siRNA分子对基因表达的表观遗传调控可由改变基因表达的染色质结构的siRNA介导的修饰引起。
“shRNA”或“短发夹RNA”是指一种RNA分子,其中少于约50个核苷酸,优选约19至约23个核苷酸与位于同一RNA分子上的互补序列碱基配对,并且其中所述序列和互补序列被至少约4至约15个核苷酸的未配对区域隔开,该区域在由碱基互补的两个区域产生的茎结构上方形成单链环。单链环序列的一个示例包括:5’UUCAAGAGA3’。
包括的shRNA是双指或二指和多指发夹dsRNA,其中RNA分子包含由单链间隔区分隔的两个或多个这种茎环结构。
候选寡核苷酸的设计和测试
正如本领域技术人员所知,除了本发明的设计要素外,在生产寡核苷酸时还有许多已知因素需要考虑。这具体取决于寡核苷酸的用途,但包括特征诸如寡核苷酸-靶核酸相互作用的强度和稳定性,例如mRNA二级结构、热力学稳定性、杂交位点的位置和/或功能基序。
本发明的一些方法涉及针对特定特征扫描靶多核苷酸或其互补物。这可以通过眼睛或使用本领域已知的计算机程序来完成。可用于设计、分析和预测反义寡核苷酸的功能特征的软件程序包括Mfold、Sfold、NUPACK、Nanofolder、Hyperfold和/或RNA设计器。可用于设计、分析和预测用于基因沉默的寡核苷酸的功能特征的软件程序包括dsCheck、E-RNAi和/或siRNA-Finder。
在一个实施方案中,可用的软件用于选择可能有用的寡核苷酸,然后针对如本文所述的所需特征扫描这些寡核苷酸。可替代地,可以容易地开发软件来针对如本文所述的所需特征扫描靶多核苷酸或其互补物。
一旦合成,就可以使用本领域的标准程序针对所需活性测试候选寡核苷酸。这可能涉及将候选物施用至体外表达感兴趣的基因的细胞,并分析基因产物(例如RNA和/或蛋白质)的量。在另一个实例中,将候选物施用至动物,并且针对靶RNA和/或蛋白质的量和/或使用功能测定来筛选动物。在另一个实施方案中,针对与靶多核苷酸(例如mRNA)杂交的能力测试寡核苷酸。
在一些例子中,表达和寡核苷酸活性可以通过mRNA逆转录定量实时PCR(RT-qPCR)来确定。例如,可以从已经与候选寡核苷酸一起孵育的细胞中提取和纯化RNA。然后从分离的RNA合成cDNA并且可以使用本领域已知的方法和试剂进行RT-qPCR。在一个示例中,RNA可以使用ISOLATE II RNA Mini试剂盒(Bioline)从细胞中纯化,cDNA可以根据制造商的说明使用High-Capacity cDNA Archive试剂盒(Thermo Fisher Scientific)从分离的RNA合成。根据制造商的说明,RT-qPCR可以在HT7900和QuantStudio 6RT-PCR系统(ThermoFisher Scientific)上使用Power SYBR Green Master Mix(Thermo Fisher Scientific)进行。
测试TLR7活性的抑制
本发明的一些方面涉及测试TLR7活性的抑制,这可以使用本领域已知的任何方法来确定。在一些实施方案中,细胞中的TLR7活性可以通过一种或多种促炎细胞因子(例如TNFα、IP-10)的表达和/或分泌和/或转录因子(例如NF-κB)的激活或表达来测量。
寡核苷酸抑制TLR7活性的能力可以例如通过将表达TLR7的细胞与寡核苷酸一起孵育,然后用TLR7激动剂刺激所述细胞,和分析细胞群中的总体TLR7应答,或分析在规定的时间段后具有TLR7阳性活性的细胞的比例来分析。
在此类实例中,TLR7活性的抑制可通过观察与其中在没有寡核苷酸的情况下(或在适当的对照抑制剂的存在下)用TLR7激动剂处理细胞的阳性对照条件相比细胞群的总体降低的TLR7应答或较低比例的具有TLR7阳性活性的细胞来鉴定。在一个示例中,293XLhTLR7(称为HEK-TLR7)细胞用pNF-κB-Luc4报告因子转染,与寡核苷酸一起孵育,然后用R848刺激。TLR7活性可以通过萤光素酶测定来确定,该测定通过发光测量活化的NF-κB。TLR7活性也可以通过测量细胞因子水平来分析,例如通过ELISA。
增强TLR8活性的测试
本发明的一些方面涉及测试TLR8活性的增强,这可以使用本领域已知的任何方法来确定。在一些实施方案中,细胞中的TLR8活性可以通过一种或多种促炎细胞因子(例如TNFα、IP-10)的表达和/或分泌,和/或转录因子(例如NF-κB)的激活或表达来测量。
寡核苷酸增强TLR8活性的能力可以例如通过将表达TLR8的细胞与寡核苷酸一起孵育,然后用TLR8激动剂刺激所述细胞,并分析细胞群中的总体TLR8应答,或分析在规定的时间段后具有TLR8阳性活性的细胞的比例来分析。
在此类实例中,TLR8活性的增强可通过观察与在寡核苷酸不存在的情况下(或在适当的对照非增效剂存在的情况下)用TLR8激动剂处理细胞的阴性对照条件相比细胞群的总体降低的TLR8应答或更高比例的具有TLR8阳性活性的细胞的比例来鉴定。在一个示例中,293XLhTLR8(称为HEK-TLR8)细胞用pNF-κB-Luc4报告因子转染,与寡核苷酸一起孵育,然后用R848刺激。TLR8活性可以通过萤光素酶测定来确定,该测定通过发光测量活化的NF-κB。TLR8活性也可以通过测量细胞因子水平来分析,例如通过ELISA。
“增强”是指功能特征相对于对照条件的增加。TLR8活性的增强可能大于约100%,例如约2倍,约3倍,约4倍,约5倍,约6倍,约7倍,约8倍,约9倍,约10倍,约11倍,约12倍,约13倍,约14倍,约15倍,约20倍或约50倍。优选地,TLR8增强的水平为约2倍至50倍、约2倍至20倍和/或约5倍至20倍以上。
用途
本发明的寡核苷酸被设计用于施用至动物。在一个示例中,动物是脊椎动物。例如,动物可以是哺乳动物、鸟类、脊索动物、两栖动物或爬行动物。示例性受试者包括但不限于人、灵长类动物、家畜(例如绵羊、牛、鸡、马、驴、猪)、伴侣动物(例如狗、猫)、实验室试验动物(例如小鼠、兔、大鼠、豚鼠、仓鼠)、圈养野生动物(例如狐狸、鹿)。在一个示例中,哺乳动物是人。
本发明的寡核苷酸可用于靶向任何感兴趣的基因/多核苷酸/功能。通常,寡核苷酸用于修饰动物的性状,更通常用于治疗或预防疾病。在一个优选的实施方案中,疾病将受益于动物在施用寡核苷酸后能够产生TLR7和/或TLR8应答,特别是在TLR7应答未被抑制和/或TLR8应答增强的情况下。
可以使用本发明的寡核苷酸治疗或预防的疾病包括但不限于癌症(例如乳腺癌、卵巢癌、中枢神经系统癌症、胃肠癌、膀胱癌、皮肤癌、肺癌、头颈癌、血液癌和淋巴癌、骨癌)罕见遗传病、神经肌肉和神经系统疾病(例如,脊髓性肌萎缩症、肌萎缩性侧索硬化症、杜兴氏肌营养不良症、亨廷顿病、巴滕病、帕金森病、肌萎缩性侧索硬化症、共济失调-毛细血管扩张症、脑瘫)病毒(例如巨细胞病毒、丙型肝炎病毒、埃博拉出血热病毒、人免疫缺陷病毒、冠状病毒)、心血管疾病(例如家族性高胆固醇血症、高甘油三酯血症)、自身免疫和炎症性疾病(例如关节炎、狼疮、结肠袋炎、银屑病、哮喘),以及非酒精性和酒精性脂肪肝疾病。
本发明的寡核苷酸的靶基因(多核苷酸)的例子包括但不限于PLK1ERBB2、PIK3CA、ERBB3、HDAC1、MET、EGFR、TYMS、TUBB4B、FGFR2、ESR1、FASN、CDK4、CDK6、NDUFB4、PPAT、NDUFB7、DNMT1、BCL2、ATP1A1、HDAC3、FGFR1、NDUFS2、HDAC2、NDUFS3、HMGCR、IGF1R、AKT1、BCL2L1、CDK2、MTOR、PDPK1、CSNK2A1、PIK3CB、CDK12、MCL1、ATR、PLK4、MEN1、PTK2、FZD5、KRAS、WRN、CREBBP、NRAS、MAT2A、RHOA、TPX2、PPP2CA、ALDOA、RAE1、SKP1、ATP5A1、EIF4G1、CTNNB1、TFRC、CDH1、CCNE1、CLTC、METAP2、GRB2、MDM4、SLC16A1、FERMT2、ENO1、STX4、SF3B1、RBBP4、FEN1、MRPL28、CCNA2、PTPN11、SAE1、KMT2D、APC、CAD、NAMPT、OGT、HSPA8、USP5、CSNK1A1、PGD、VRK1、SEPSECS、SUPT4H1、DNAJC9、TRIAP1、DLD、PTPN7、VDAC1、STAT3、TCEB2、ADSL、GMPS、DHPS、METAP1、TAF13、CFL1、SCD、RBM39、PGAM1、FNTB、PPP2R1A、ARF1、UBE2T、UMPS、MYC、PRMT5、EIF4G2、SKP2、STAG2、ATF4、WDR77、ILK、METTL16、SOD1、DDX6、FURIN、AARS、FNTA、PABPC1、RANBP2、CDC25B、SLC2A1、CENPE、ADAR、CDC42、RNF31、CCNC、PRIM1、SLC38A2、SNUPN、PDCD6IP、RTN4IP1、VMP1、TGFBR1、TXN、UBE2N、UAP1、RAC1、GGPS1、RAB10、RAB6A、TPI1、RPE、THG1L、UBE2D3、RHEB、PKM、GMNN、HGS、NCKAP1、NUP98、SMARCA2、RNF4、DDX39B、ACLY、XPO1、PPP1R8、YAP1、MTHFD1、LPAR1、TAF1、UROD、STXBP3、HSP90B1、VHL、EFR3A、FECH、MRPL44、AIFM1、MAGOH、MRPL17、SUZ12、RNMT、RAB1B、PNPT1、RAD1、WDR48、PITRM1、MRPL47、AP2M1、EIF4A1、UBE2C、LONP1、VPS4A、SNRNP25、TUBGCP6、DNM2、UBE2M、EXOSC9、TAF1B、CDC37、ATP6V1G1、POP1、JUP、PRPS1、GPX4、CFLAR、CHMP4B、ACTB、ACTR1A、PTPN23、SHC1、TRPM7、SLC4A7、HSPD1、XRN1、WDR1、ITGB5、UBR4、ATP5B、CPD、TUFM、MYH9、ATP5F1、ATP6V1C1、SOD2、PFAS、NFE2L2、ARF4、ITGAV、DHX36、KIF18A、DDX5、XRCC5、DNAJC11、ZBTB8OS、NCL、SDHB、ATP5C1、NDC1、SNF8、CUL3、SLC7A1、ASNA1、EDF1、TMED10、CHMP6、ARIH1、DDOST、RPL28、DIMT1、CMPK1、PPIL1、PPA2、SMAD7、CEP55、MVD、MVK、PDS5A、KNTC1、CAPZB、GMPPB、TPT1、ACIN1、SAR1A、TAF6L、PTBP1、PAK2、CRKL、NHLRC2、INO80、SLC25A3、ACTR3、DDX3X、HUWE1、TBCA、IK、SSBP1、ARPC4、SLC7A5、OSGEP、PDCD2、TRAF2、SNAP23、RPN1、EIF5A、GEMIN4、BMPR1A、AHCYL1、CHMP5、TRAPPC1、LRP8、ARID2、UBE2L3、STAMBP、KDSR、UQCRC2、PNN、USP7、TBCD、ATP6V0E1、PCYT1A、TAZ、POLRMT、CELSR2、TERF1、BUB1、YRDC、SMG6、TBX3、SLC39A10、IPO13、CDIPT、UBA5、EMC7、FERMT1、VEZT、CCND1、CCND2、FPGS、JUN、PPM1D、PGGT1B、NPM1、GTF2A1、MBTPS1、HMGCS1、LRR1、HSD17B12、LCE2A、NUP153、FOSL1、IRS2、CYB5R4、PMPCB、ARHGEF7、TRRAP、NRBP1、ARMC7、MOCS3、TIPARP、SEC61A1、PFDN5、MYB、IRF4、STX5、MYCN、FOXA1、SOX10、GATA3、ZEB2、MYBL2、MFN2、TBCB、KLF4、TRIM37、CEBPA、STAG1、POU2AF1、HYPK、FLI1、NCAPD2、MAF、NUP93、RBBP8、HJURP、SMARCB1、SOCS3、GRWD1、NKX2-1、FDXR、SPDEF、SBDS、SH3GL1、KLF5、CNOT3、ZNF407、CPSF1、RPTOR、EXT1、SMC1A、GUK1、TIMM23、FAU、ACO2、ALG1、CCNL1、SCAP、SRSF6、SPAG5、SOX9、LDB1、ASPM、LIG1、TFDP1、RPAIN、CENPA、MIS12、ILF3、HSCB、ERCC2、SOX2、ARFRP1、PMF1、POLR3E、MAD2L2、PELP1、NXT1、WDHD1、ZWINT、E2F3、FZR1、JUNB、OGDH、NOB1、SKA3、TACC3、UTP14A、XRN2、SMG5、IDH3A、CIAO1、COQ4、ZFP36L1、CDCA5、PRKRA、PFDN6、PAK1IP1、PSTK、EDC4、UTP18、TOMM22、CASC5、PTTG1、RBBP5、PPP1R12A、FARS2、FOXM1、SIN3A、BUB1B、GNB1L、SMC5、SARS2、SYNCRIP、IPPK、FANCD2、WDR46、FANCI、DCP2、RFC2、RNF20、DMAP1、MED23、MBNL1、CTPS1、TBP、MMS19、RAD51C、CDS2、NONO、USP18、PARS2、FBXW11、SUMO2、RRP12、FAM50A、URB2、MCM4、SLC25A28、IPO7、MAX、SFSWAP、SBNO1、DPAGT1、TINF2、BRCA2、NUP50、RPIA、EP400、IKBKAP、KIF14、RTTN、CCDC115、GEMIN6、WWTR1、BCS1L、GTF3A、SCYL1、NELFB、DDX39A、TRA2B、SYVN1、ISL1、CYB5B、ACSL3、DPH3、E2F1、IREB2、SREBF1、SMC6、IRF8、ID1、PDCD11、SNAPC2、TIMM17A、ANAPC10、NUP85、SEH1L、VBP1、NUDC、MTX2、RPP25L、ISY1、LEMD2、ATP5D、EXOSC2、TAF1C、PPIL4、SEPHS2、HNRNPH1、CTR9、CDC26、TIMM13、FAM96B、CEBPZ、UFL1、ZNF236、COPG1、TPR、MIOS、UBE2G2、MED12、GTF3C1、PPP2R2A、UBIAD1、WTAP、MYBBP1A、NUP88、NELFCD、WDR73、RTCB、CEP192、GTF3C5、LENG1、RINT1、MED24、COX6B1、DCTN6、SLC25A38、LYRM4、STRAP、TTF2、DDX27、GTF2F1、ZNHIT2、BCLAF1、WDR18、GTF2H2C、NDE1、TIMM9、CHMP7、IPO11、TGIF1、NOC4L、EXOSC6、WDR24、INTS6、DDX41、UBE2S、ARGLU1、SHOC2、ATP5J、CSTF2、RPP30、NHP2、GRHL2、RPL22L1、WDR74、UTP23、CCDC174、RPP21、UBE2J2、GEMIN8、ATP6V0B、KIAA1429、PNO1、MED22、ENY2、THOC7、DDX19A、SUGP1、PELO、ELAC2、CHCHD4、RNPC3、INTS3、PSMG4、UQCRC1、TAF1A、TSR1、UTP6、TRMT5、EIF1AD、GTF3C2、DCTN3、GPS1、WDR7、EXOSC8、KANSL1、SPRTN、KANSL3、EXOSC5、PRCC、TRNAU1AP、EIF3J、TAMM41、HAUS6、OIP5、HAUS5、TAF6、MRPS22、MRPS34、WBP11、COG8、DHX38、DNLZ、LAGE3、FUBP1、MED26、SLC7A6OS、MARS2、RBM28、ASCC3、PSMG3、TUBGCP5、PCF11或LAS1L。
在一个实施方案中,待靶向的基因包括PKN3、VEGFA、KIF11、MYC、EPHA2、KRAS(G12)、ERBB3、BIRC5、HIF1A、BCL2、STAT3、AR、EPAS1、BRCA2或CLU。
可以如本文所述进行修饰的商业寡核苷酸的实例包括但不限于inclisiran、米泊美生(Kynamro)、诺西那生钠(Spinraza)、依特立生(Exondys51)、miravirsen(SPC3649)、RG6042(IONIS-HTTRx)、inotersen、volanesorsen、golodirsen(Vyondys53)、福米韦森(Vitravene)、patisiran、givosiran、inclisiran、danvatirsen和IONIS-AR-2.5Rx。
组合物
本公开内容的寡核苷酸可以与其他分子、分子结构或化合物的混合物混合、封装、缀合(例如融合)或以其他方式缔合,产生例如脂质体、受体靶向分子、口服的、直肠的、局部的或其他制剂,用于帮助摄取、分布和/或吸收。教导制备此类吸收、分布和/或吸收辅助制剂的代表性美国专利包括但不限于US5,108,921、US5,354,844、US5,416,016、US5,459,127、US5,521,291、US5,543,158、US5,547,932、US5,583,020、US5,591,721、US4,426,330、US4,534,899、US5,013,556、US5,108,921、US5,213,804、US5,227,170、US5,264,221、US5,356,633、US5,395,619、US5,416,016、US5,417,978、US5,462,854、US5,469,854、US5,512,295、US5,527,528、US5,534,259、US5,543,152、US5,556,948、US5,580,575和US5,595,756。
本公开内容的寡核苷酸可以在药学上可接受的载体中施用。药学上可接受的载体可以是固体或液体。药学上可接受的载体的有用实例包括但不限于不影响本公开内容的活性剂的活性的稀释剂、溶剂、表面活性剂、赋形剂、悬浮剂、缓冲剂、润滑剂、佐剂、媒介物、乳化剂、吸收剂、分散介质、包衣、稳定剂、保护胶体、粘合剂、增稠剂、触变剂、渗透剂、螯合剂、等渗剂和吸收延迟剂。
在一个实施方案中,药物载体是注射用水(WFI),并且药物组合物被调节至pH7.4、7.2-7.6。在一个实施方案中,盐是钠盐或钾盐。
寡核苷酸可能包含手性(不对称)中心或整个分子可能是手性的。单独的立体异构体(对映异构体和非对映异构体)和它们的混合物在本公开内容的范围内。
本公开内容的寡核苷酸可以是药学上可接受的盐、酯或酯的盐或施用后能够(直接或间接)提供生物活性代谢物的任何其他化合物。如本文所用的术语“药学上可接受的盐”是指寡核苷酸的生理学上和药学上可接受的盐,其保留母体化合物的所需的生物活性并且在施用时不产生不想要的毒理学作用。在US6,287,860中进一步描述了药学上可接受的盐的例子及其用途。
本公开内容的寡核苷酸可以是前药或前药的药学上可接受的盐,或其他生物等效物。如本文所用,术语“前药”是指以非活性形式制备的治疗剂,其在施用时通过内源性酶或其他化学物质和/或条件的作用转化为活性形式(即,药物)。特别地,根据WO93/24510、WO94/26764和US5,770,713中公开的方法,将本公开内容的寡核苷酸的前药形式制备为SATE[(S乙酰基-2-硫代乙基)磷酸酯]衍生物。
例如,前药可以在体内转化(例如通过在血液中水解)为其具有医疗作用的活性形式。药学上可接受的前药描述于T.Higuchi和V.Stella,Prodrugs as Novel DeliverySystems,Vol.14of the A.C.S.Symposium Series(1976);"Design of Prodrugs"ed.H.Bundgaard,Elsevier,1985;以及在Edward B.Roche,ed.,Bioreversible Carriersin Drug Design,American Pharmaceutical Association and Pergamon Press,1987。有机化学领域的技术人员将理解,许多有机化合物可以与它们在其中反应或它们从其沉淀或结晶的溶剂形成络合物。这些络合物被称为“溶剂化物”。例如,与水的络合物被称为“水合物”。
在一个实施方案中,本发明的寡核苷酸可以与络合剂络合以增加寡核苷酸的细胞摄取。络合剂的例子包括阳离子脂质。阳离子脂质可用于将寡核苷酸递送至细胞。
术语“阳离子脂质”包括具有极性和非极性结构域的脂质和合成脂质,它们能够在生理pH值处或在生理pH值附近带正电荷,并且它们结合聚阴离子,例如核酸,并促进核酸递送到细胞中。通常阳离子脂质包括饱和和不饱和的烷基和脂环族醚以及胺、酰胺或其衍生物的酯。阳离子脂质的直链和支链烷基和烯基可包含例如1至约25个碳原子。优选的直链或支链烷基或烯烃基团具有六个或更多个碳原子。脂环族基团包括胆固醇和其他类固醇基团。阳离子脂质可以用多种抗衡离子(阴离子)制备,包括例如Cl-、Br-、I-、F-、乙酸盐、三氟乙酸盐、硫酸盐、亚硝酸盐和硝酸盐。
阳离子脂质的实例包括聚乙烯亚胺、聚酰胺胺(PAMAM)星爆树枝状大分子、Lipofectin(DOTMA和DOPE的组合)、Lipofectase、LIPOFECTAMINETM(例如,LIPOFECTAMINETM2000)、DOPE、细胞转染素(Gilead Sciences,Foster City,Calif.)和Eufectins(JBL,SanLuis Obispo,Calif.)。示例性阳离子脂质体可由以下制成:N-[1-(2,3-二油醇氧基)-丙基]-N,N,N-三甲基氯化铵(DOTMA)、N-[1-(2,3-二油醇氧基)-丙基]-N,N,N-甲基硫酸三甲基铵(DOTAP)、3.β.-[N--(N’,N’-二甲基氨基乙烷)氨基甲酰基]胆固醇(DC-Chol)、2,3,-二油烯氧基-N-[2(精胺甲酰氨基)乙基]-N,N-二甲基-1-丙胺三氟乙酸盐(DOSPA)、1,2-二肉豆蔻氧基丙基-3-二甲基-羟乙基溴化铵;和二甲基双十八烷基溴化铵(DDAB)。寡核苷酸也可以与例如聚(L-赖氨酸)或抗生物素蛋白复合,并且脂质可以或可以不包含在该混合物中,例如甾醇-聚(L-赖氨酸)。
阳离子脂质已在本领域中用于将寡核苷酸递送至细胞(参见,例如,US5,855,910;5,851,548;5,830,430;5,780,053;5,767,099;Lewis等人,1996;Hope等人,1998)。可用于促进本发明的寡核苷酸摄取的其他脂质组合物可与本发明的方法结合使用。除了上面列出的那些之外,其他脂质组合物也是本领域已知的并且包括例如在US4,235,871;US4,501,728;4,837,028;4,737,323中教导的那些。
在一个实施方案中,脂质组合物还可以包含试剂,例如病毒蛋白,以增强寡核苷酸的脂质介导的转染。在另一个实施方案中,N-取代的甘氨酸寡核苷酸(拟肽)可用于优化寡核苷酸的摄取。
在另一个实施方案中,用于递送本发明的寡核苷酸的组合物包含具有约一个至约四个碱性残基的肽。这些碱性残基可位于例如肽的氨基末端、C末端或内部区域。具有相似侧链的氨基酸残基家族已在本领域中定义。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸(也可以认为是非极性的)、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如,酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。除碱性氨基酸外,肽的大部分或所有其他残基可选自非碱性氨基酸,例如除赖氨酸、精氨酸或组氨酸以外的氨基酸。优选使用具有长中性侧链的中性氨基酸的优势。
在一个实施方案中,通过连接将寡核苷酸转运到细胞中的肽序列(在本文中称为“转运肽”)来修饰寡核苷酸。在一个实施方案中,组合物包括与编码蛋白质的靶核酸分子互补的寡核苷酸,和共价连接的转运肽。
在另一个实施方案中,寡核苷酸连接到靶向部分,例如N-乙酰半乳糖胺(GalNAc)、抗体、抗体样分子或适配体(参见,例如,Toloue和Ford(2011)以及Esposito等人(2018))。
施用
在一个实施方案中,本公开内容的寡核苷酸被全身施用。如本文所用,“全身施用”是肠内或肠胃外的施用途径。
如本文所用,“肠内”是指涉及胃肠道的任何部分的施用形式,其包括例如片剂、胶囊或滴剂形式的寡核苷酸的口服施用;胃饲管、十二指肠饲管或胃造口术;和例如栓剂或灌肠剂形式的寡核苷酸的直肠施用。
如本文所用,“肠胃外”包括通过注射或输注施用。例子包括静脉内(进入静脉)、动脉内(进入动脉)、肌肉内(进入肌肉)、心内(进入心脏)、皮下(在皮肤下)、骨内输注(进入骨髓)、皮内(进入皮肤本身)、鞘内(进入椎管)、腹膜内(输注或注射入腹膜)、膀胱内(输注入膀胱)、透皮(通过完整皮肤扩散)、跨粘膜(通过粘膜扩散)、吸入。
在一个实施方案中,药物组合物的施用是皮下的。
寡核苷酸可以以单次剂量或以周期为基础的重复剂量施用,例如每天一次、每两天一次、每三、四、五、六、七、八、九、十、十一、十二、十三或十四天一次、每周一次、每周两次、每周三次、每两周一次、每三周一次、每月一次、每两个月一次、每三个月至六个月一次或每十二个月一次。
在一个实施方案中,施用是每周1至3次,或每周、每两周、三周、四周一次,或每两个月一次。
在一个实施方案中,施用是每周一次。
在一个实施方案中,低剂量施用持续3至6个月,例如约25-50mg/周持续至少三至六个月和然后长达12个月并长期施用。
示例性剂量为约10至5,000mg。示例性剂量包括25、50、100、150、200、1,000、2,000mg。示例性剂量包括1.5mg/kg(约50至100mg)和3mg/kg(100-200mg)、4.5mg/kg(150-300mg)、10mg/kg、20mg/kg或30mg/kg。在一个实施方案中,剂量每周施用一次。因此,在一个实施方案中,约10至30或20至40或20至28mg的低剂量可施用至通常体重为约25至65kg的受试者。在一个实施方案中,寡核苷酸以每剂量小于50mg或小于30mg或约25mg的剂量施用以产生治疗效果。
实施例
实施例1-方法
伦理学声明
从健康捐献者收集外周血单核细胞(PBMC)已根据HREC参考编号02052A获得Monash Health的批准。
细胞分离、培养和刺激
如前所述(Gantier等人,2010),使用Histopaque-1770(Sigma-Aldrich)通过密度离心从全血捐赠中分离出PBMC,并接种在补充有1x抗生素/抗真菌剂和10%热灭活胎牛血清的RPMI 1640加L-谷氨酰胺培养基(Thermo Fisher Scientific)(称为完全RPMI)中。
分别稳定表达TLR8、TLR7和TLR9的293XL-hTLR8-HA(称为HEK-TLR8)和293XL-hTLR7-HA(称为HEK-TLR7)和293XL-hTLR9-HA购自Invivogen,并维持在补充有10%热灭活胎牛血清(Thermo Fisher Scientific)和补充有10μg/ml杀稻瘟菌素(Invivogen)的1×抗生素/抗真菌剂(Thermo Fisher Scientific)的Dulbecco改良的Eagle培养基(ThermoFisher Scientific)(称为完全DMEM)中。亲代野生型(WT)THP-1、UNC93B1缺陷型THP-1(Schmid-Burgk等人,2014)和用荧光野生型UNC93B1重构的匹配克隆(Pelka等人,2014)在完全RPMI中生长。OCI-AML3和MOLM13在补充有20%热灭活胎牛血清和1×抗生素/抗真菌剂的RPMI中生长(它们的身份通过依靠PowerPlex HS16系统试剂盒,Promega的内部细胞系鉴定服务确认)。所有细胞均在37℃、5%CO2条件下培养。细胞系每周传代2-3次,并通过PCR常规检测支原体污染。
对于刺激,在用1μg/ml R848(Invivogen)刺激之前,用ASO处理THP-1、MOLM13和OCI-AML3过夜。在用R848、CL075、嘎德莫特(均来自Invivogen)或7-烯丙基-7,8-二氢-8-氧代鸟苷(洛索立宾–SigmaAldrich)刺激之前,HEK-TLR7和HEKTLR8用所指示的浓度的ASO处理20-50分钟。所有ASO均由Integrated DNA Technologies(IDT)合成,并重新悬浮在pH8.0的无RNase的TE缓冲液(Thermo Fisher Scientific)中。表1、2和3中提供了ASO序列和修饰。cGAS配体ISD70(表1)如前所述(Pepin等人,2020)制备,并用lipofectamine 2000以2.5μg/ml的最终浓度进行转染。B类CpG寡核苷酸人TLR9配体ODN 2006由IDT合成并重悬于无RNase的TE缓冲液中。
萤光素酶测定
稳定表达TLR7、8或9的HEK293细胞用pNF-κB-Luc4报告因子(Clontech)、pLuc-IFN-β(来自K.Fitzgerald,University of Massachusetts的馈赠)或pCCL5[RANTES]-Luc(来自G.Scholz,University of Melbourne的馈赠)使用lipofectamine 2000(ThermoFisher Scientific)根据制造商的方案转染。简而言之,500,000-700,000个细胞用400ng报告因子和1.2μl Lipofectamine 2000反向转染到6孔板的每孔中,并在37℃和5%CO2条件下孵育3-24小时。转染后,恰好在ASO和过夜TLR刺激(如上所述)之前,从6孔中收集细胞并等分到96孔中。第二天,将细胞在40μl(对于96孔板)的1X Glo裂解缓冲液(Promega)中在室温下裂解10分钟。然后使用40μl萤光素酶测定试剂(Promega)对15μl裂解物进行萤火虫萤光素酶测定。用Fluostar OPTIMA(BMG LABTECH)发光计对发光进行量化。
在HeLa细胞中用ASO下调HPRT
对于每个孔中50μl的总体积,通过在OptiMEM I(Thermo Fisher Scientific)中将各种ASO剂量与0.5μl Lipofectamine 2000(Thermo Fisher Scientific)复合,对每个ASO在96孔板中以三次生物学重复进行反向转染。将HeLa细胞(20,000)悬浮在添加了10%胎牛血清(FCS)的100μl DMEM中,添加到脂质-ASO复合物中,然后在37℃和5%CO2条件下孵育24小时。使用SV总RNA分离试剂盒(Promega)和DNase1处理收集RNA。根据制造商的说明,使用SuperScript II逆转录酶(Thermo Fisher Scientific)从约200ng总RNA使用锚定寡核苷酸dT和随机六聚体引物(Integrated DNA Technologies)合成cDNA。在384孔板形式的10μl反应中,使用~10ng cDNA和Immolase DNA聚合酶(Bioline)、500nM每种引物和250nM探针进行qPCR反应。扩增反应在Applied Biosystems 7900HT(Thermo FisherScientific)上进行。对于每个样品,所有qPCR反应一式三份进行并取平均值。
线性化的克隆扩增子用作拷贝数标准,以建立每个测定的绝对定量测量。HPRT(NM000194)和SFRS9(NM 003769)表达水平通过多重5’-核酸酶测定进行量化,并且针对SFRS9标准化的HPRT水平用作内部参考对照。表1中提供了所用引物和探针测定的序列。相对于NC1和NC5阴性对照ASO计算了敲低效率。
细胞因子检测
根据制造商的说明,使用BD OptEIA ELISA装置(BD Biosciences,分别为#555212和#550926)测量人TNF-α和IP-10。人IFN-α检测如先前报道的那样进行(Gantier,2013)。四甲基联苯胺底物(Thermo Fisher Scientific)用于在Fluostar OPTIMA(BMG LABTECH)酶标仪上量化细胞因子。
mRNA逆转录定量实时PCR(RT-qPCR)
使用ISOLATE II RNA Mini试剂盒(Bioline)从细胞中纯化总RNA。根据制造商的说明,使用High-Capacity cDNA Archive试剂盒(Thermo Fisher Scientific)从分离的RNA合成随机六聚体cDNA。RT-qPCR在HT7900和QuantStudio 6RT-PCR系统(Thermo FisherScientific)上使用Power SYBR Green Master Mix(Thermo Fisher Scientific)进行。每个PCR以技术重复进行,并将人18S用作参考基因。每个扩增子都经过凝胶纯化并用于生成用于基因表达定量的标准曲线(用于每次运行)。每次运行都使用熔解曲线来确认扩增的特异性。
使用的引物如下:人RSAD2:hRSAD2-RT-FWDTGGTGAGGTTCTGCAAAGTAG;hRSAD2-RT-REVGTCACAGGAGATAGCGAGAATG;hIFIT1:hIFIT1-FWDTCACCAGATAGGGCTTTGCT;hIFIT1-REVCACCTCAAATGTGGGCTTTT;h18S:h18S-FWDCGGCTACCACATCCAAGGAA;h18S-REVGCTGGAATTACCGCGGCT;hIFI44:hIFI44-FWDATGGCAGTGACAACTCGTTTG;hIFI44:TCCTGGTAACTCTCTTCTGCATA;hIFNB:hIFNB-FWDGCTTGGATTCCTACAAAGAAGCA;hIFNBREV:ATAGATGGTCAATGCGGCGTC;hHPRT-FWD:GACTTTGCTTTCCTTGGTCAG;hHPRT-REVGGCTTATATCCAACACTTCGTGGG;来自RSAD2、IFIT1和18SPCR的扩增子通过Sanger测序验证。IFI44和IFNB引物来自PrimerBank(Wang等人,2012),HPRT引物由IDT设计。
统计分析
使用Prism 8(GraphPad Software Inc.)进行统计分析。每个实验都以三个生物学重复进行(除了以两个生物学重复进行的图2A、2B、4D和5E以外)并重复独立的至少两次。在比较条件组时使用单向和双向方差分析(ANOVA),而在比较条件对时使用双尾未配对非参数Mann-Whitney U检验或未配对双尾t检验。使用的符号:*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001,“ns”为不显著。
实施例2:ASO对TLR7/8感测的序列和主链依赖性影响
发明人最初研究了靶向先天免疫传感器cGAS的mRNA的一组11 2’Ome gapmer ASO对未分化THP-1细胞的免疫应答的活性。令人惊讶的是,对于选定的ASO(例如ASO2、ASO9、ASO11,但不是ASO4-图1A),在细胞中TLR7/8的R848刺激后,使用ASO预处理过夜导致IP-10和TNF-α产生的强增强。先前的研究报告了富含T的PS寡核苷酸可以促进TLR8感测,同时抑制TLR7(Gorden等人,2006年;Jurk等人,2006年)。由于THP-1可以响应TLR7和TLR8配体(Gantier等人,2008),发明人推测他们观察到的ASO对R848感测的序列特异性影响可能是由于它们对TLR7和TLR8的不同活性。为了定义这一点,发明人接下来在稳定表达TLR7或TLR8(此后称为HEK-TLR7和HEKTLR8)以及NF-κB-萤光素酶报告因子的HEK 293细胞中测试了我们的序列组(图1B、1C、8A和8B)。
有趣的是,发明人发现大多数ASO强烈抑制R848的TLR7感测,但ASO8和ASO11除外,它们是不太有效的抑制剂(图1B)。相反,与发明人在THP-1细胞中的观察结果直接比对,几种ASO强烈增强了R848的TLR8感测(例如ASO2、ASO9和ASO11-图1C)。ASO自身不刺激TLR7或TLR8(图8A、8B)。着眼于同样增强TLR8但对TLR7具有不同活性的ASO2和ASO11,发明人进一步验证了它们在缺乏UNC93B1的THP-1中的TLR7/8依赖性活性,这对TLR7/8信号传导至关重要(Pelka等人,2014)。ASO2和ASO11对R848感测的增强作用在缺乏UNC93B1的THP-1中未被发现,但可以在重建UNC93B1-Citrine表达后恢复(Pelka等人,2014)(图1D),从而支持TLR7/8在这个作用中的参与。此外,用ASO2和ASO11刺激人外周血单核细胞(PBMC)强烈增强R848诱导的TNF-α,但不影响IFN-α水平,表明对R848的TLR8感测的优先影响(Gantier等人,2008)(图1E、1F)。这种对IFN-α和TNF-α的作用与通过ASO的PBMC的TLR9激活无关,因为ASO2不激活HEK-TLR9细胞中的TLR9信号传导,而ASO11则激活(图8C)。
为了确定ASO对TLR7/8的这种作用是否依赖于它们的主链或碱基修饰,发明人接下来研究了一系列基于ASO2序列的ASO变体(图1G、1H、1I)。用R848刺激的HEK-TLR7细胞中的这些变体的分析表明,含有PS主链的所有ASO2变体抑制TLR7,与使用的碱基修饰的类型无关(仅DNA,2’OMe,LNA或2’MOE)(图1G,1H,表1)。相反,通过TLR8的R848感测的增强直接取决于5’和3’端碱基修饰,其中2’OMe在此序列上下文中提供最佳增强(图1G、1I)。添加3’端Cy3接头降低TLR8感测的这种增强作用,而用2’MOE或LNA碱基取代2’OMe碱基消除这种增强作用。
增强作用不局限于双重TLR7/8激动剂R848,并且对于CL075(TLR8激动剂)、洛索立宾(TLR7激动剂)也见到增强作用,和对于嘎德莫特(TLR7激动剂)也见到一定程度的增强作用(图7)。
类似于在TLR7上观察到的作用,PS主链对于TLR8增强也是必需的;然而,它本身对于此作用是不足够的,因为2’OME和LNA ASO2变体也在PS主链上合成,并且对于特征仅在于PS修饰的变体(ASO2-PS)观察到仅有限的增强作用。总的来说,这些结果表明PS ASO可以以序列依赖的方式显示有效的TLR7/8免疫调节。
表1:本研究中使用的各种寡核苷酸(全部为5’-3’)。DNA单独大写,‘m’表示2’OMe碱基,‘/i2MOEr’表示2’MOE碱基,‘+’表示LNA碱基,*表示硫代磷酸酯主链。Cy3Sp表示Cy3标签。FAM、HEX、ZEN、3IABkFQ是qPCR探针修饰。
实施例3:用于鉴定具有低TLR7抑制和高TLR8增强作用的ASO的筛选
通过选择PS ASO观察到的TLR7抑制和TLR8增强与富含T的PS寡核苷酸可以促进类似的活性的之前的报道(Gorden等人,2006;Jurk等人,2006)一致。然而,一些2’OMe ASO序列(例如ASO8和ASO11)对TLR7具有较低抑制活性的发现表明,PS主链促进的TLR7抑制可能在选定的2’Ome gapmer ASO中被抵消。发明人推断,定义该活性的模式可以帮助设计对TLR7具有降低的免疫抑制活性的ASO。此外,观察到ASO11能够增强R848的TLR8感测,同时保留TLR7活性,这表明对TLR7和8的活性不受相同序列决定因素的控制。
为了进一步表征这些观察结果,发明人筛选了一个包含192个2’OMe ASO的文库。值得注意的是,这些ASO旨在针对4个不同的转录物(每个转录物48个ASO),其中ASO之间具有最小的单碱基增量(表2)。针对每个TLR在两个不同的ASO浓度下进行筛选,并测量它们对HEK-TLR7和HEK-TLR8细胞中通过R848的NF-κB萤光素酶诱导的影响(图2A、2B和表2)。
与最初的ASO小组一致,发明人发现大多数以500nM使用的ASO强烈抑制TLR7感测。因此,78%的ASO将TLR7上的R848活性降低了超过80%,只有2个ASO在两种浓度下将TLR7感测降低了不到40%(图2A和表2–ASOs‘[CDKN2B-AS1]-852’和‘[LINC-PINT]-2504’,以下称为ASO852和ASO2504)。相反,ASO对TLR8增强的影响在ASO中是不同的,51%的ASO在100nM时将R848感测增强至少2倍(图2B)。重要的是,尽管ASO852和ASO2504都显示出低TLR7抑制作用,但它们对TLR8具有不同的活性(图2A、2B和表2)。在HEK293-TLR7和HEK293-TLR8细胞中进行的ASO剂量反应研究证实,与ASO4相比,ASO2504和ASO852对通过TLR7的R848感测几乎没有影响,但ASO852对TLR8感测的增强程度明显高于ASO4(图2C)。分析ASO852对不同剂量R848的影响后发现,它使TLR7对R848的敏感性降低了约2.5倍(图2D)。然而,ASO852处理使R848对TLR8的活性增加了约13倍(图2D)。
表2:196个ASO筛选数据。靶向的基因名称在括号中提供(例如[EGFR])。ASO的合成具有以下修改:DNA单独大写,‘m’表示2’OMe碱基修饰,*表示硫代磷酸酯主链。给出了指定ASO浓度下每个筛选的平均NF-κB-萤光素酶数据(使用1μg/ml R848共刺激)。图3B和3F中进一步研究了来自PINT家族的带下划线的序列。黑色粗体序列用作17个低TLR7抑制剂并使用MEME分析基序富集(与[cGAS]ASO8和ASO11一起),如图3A所示。
实施例4:2’OMe ASO对TLR7的抑制可以通过CUU末端基序恢复
基于上述筛选对19种具有最低TLR7抑制活性的ASO进行MEME基序发现分析(Bailey和Elkan,1994)导致观察到显示末端5’和3’“C”碱基以及尿苷残基的ASO的2’OMe区域在8个序列中过度呈现(图3A,表2)。此外,来自使用单碱基增量的筛选的序列家族(称为PINT家族)的分析表明,密切相关的序列对TLR7具有不同的抑制活性(图3B、3C和表2-从ASO‘[LINC-PINT]-108’到[LINC-PINT]-116’,称为ASO108-ASO116)。
对HEK293-TLR7细胞中ASO的PINT家族的验证证实,ASO111仅能够阻断该家族中R848对TLR7的激活(图3B、3C)。至关重要的是,序列比对分析显示ASO111是唯一在其5’或3’端区域缺少CUU/CUT/CTT基序的序列(图3C)。由于[cGAS]ASO11在其5’和3’端也包含此类末端2’OMe CUU基序(以及图3A中包含富集基序的其他4个序列),发明人接下来测试了[cGAS]ASO11变体(ASO11-Mut1和ASO11-Mut2),其中5’和3’端2’OMe区域与ASO2的这些区域交换,该ASO2缺乏此类“CUU”基序并且是TLR7的有效阻遏物(图3D、3E)。与5’和3’端区域的关键作用一致,与亲本ASO11相比,两个ASO11突变体显著增加了TLR7抑制(图3D、3E)。这些发现共同表明,5’和3’CUU基序是2’OMe-PS ASO对TLR7抑制的重要调节剂。
实施例5:TLR8增强由2’OMe PS ASO的中心DNA区域和5’端驱动
发明人还分析了PINT家族(ASO108-116)和[cGAS]ASO11突变体的TLR8增强。虽然两个序列更有效(例如ASO108和ASO110),但大多数序列显示出相似的TLR8增强作用,表明这些分子的中心区域主要参与TLR8调节(图3C、3F)。类似地,ASO11突变仅轻微影响TLR8增强,尽管添加ASO2 3’端(ASO11-Mut1)显著降低,而ASO2 5’端(ASO11-Mut2)显著增加TLR8感测(图3D、3G)。这些结果表明,2’OMe-PS ASO对TLR8增强的控制主要受ASO的中心10-merDNA区域控制,但2’OMe末端也发挥了作用。
为了进一步支持ASO的5’-末端区域的贡献,发明人注意到在我们的筛选中TLR8感测的前20个增效剂中的大多数具有末端5’-U(20个中有14个),而这种末端5’-U的出现在最弱的20个增效剂中的频率要低得多(20个中有3个)(表2)。比较有或没有末端5’-U的序列的192个ASO的TLR8增强的分析证实了包含末端5’-U的序列的显著增加的TLR8感测增强(图3H和表2)。对于末端5’-UC基序(出现在最强ASO的9/20中-图3I和表2),观察到类似的趋势。至关重要的是,ASO108和ASO110是PINT家族中仅有的两个具有这种末端5’-UC基序的ASO,该基序也存在于ASO2和ASO11-Mut2中。
发明人还注意到,TLR8增强ASO852的中心10聚体DNA区域包含富含T的中心区域(TTTCTGTGGT),而ASO2504的该区域富含A(TAAAAAAATT)。比较最强和最弱20个TLR8感测增效剂的中心DNA区域,证实ASO中胸苷残基的显著增加的比例最能增强TLR8感测-中心胸苷的中位数为4个(图3J和表2)。由于这与之前关于富含T的区域对TLR8增强很重要的报道直接一致(Gorden等人,2006;Jurk等人,2006),发明人将ASO852突变为ASO852-dT,其中包含10个dT的中心区段(图3K)。此外,发明人将ASO2504的中心10聚体DNA区域与ASO852的中心DNA区域交换,产生ASO2504-Mut(图3K),因为ASO2504没有像ASO852那样增强TLR8。在THP-1细胞、HEK-TLR7和HEK-TLR8细胞中比较这些寡核苷酸对R848感测的活性(图3L、3M、8A、8B和8D)。2504-Mut ASO在驱动THP-1细胞产生IP-10和驱动HEK-TLR8细胞产生NF-κB萤光素酶方面明显更有效,这一观察结果支持ASO的中心富含T的10聚体DNA区域在其对TLR8的作用方面的关键作用。此外,ASO852-dT在诱导IP-10方面也比ASO852更有效,其刺激水平与在THP-1细胞中使用20聚体dT PS寡核苷酸(dT20)获得的刺激水平相似。相反,对于ASO852-dT和ASO2504-Mut的中心区域的替换不会显著影响R848的TLR7感测,而dT20阻止TLR7激活。在HEK-TLR7、HEK-TLR8或THP-1细胞中单独使用的这些ASO均不影响HEK细胞中的NF-κB萤光素酶和THP-1中的IP-10产生(图8A、8B、8D)。然而,增加ASO852的中心富含T的区域进一步增强了其对TLR9驱动的NF-κB萤光素酶的基础活性-这与dT20单独也在HEK-TLR9细胞中充当温和的TLR9配体的观察结果一致(图8C)。
实施例6:ASO对R848的TLR8增强导致IRF激活
ASO852-dT在R848感测时强烈增强IP-10产生的能力在另外两个表达TLR8的AML细胞系(MOLM13和OCI-AML3;图4A)中得到证实,并且很容易用低至4至20nM ASO852-dT观察到(图4B)。鉴于所见到的IP-10的高产量(表明IRF激活),发明人测试了ASO852-dT系列在由IRF驱动的表达CCL5-萤光素酶或IFN-β-萤光素酶报告因子的HEKTLR8细胞(Chow等人,2018;Schafer等人,1998)中的活性。虽然R848单独没有激活任何一个报告因子,但与寡核苷酸的共同刺激增强了两个启动子,其中ASO852-dT和dT20是最有效的,其次是ASO852/2504-Mut,ASO2504是效果最不好的(图4C);因此,这一发现镜像反映了在THP-1细胞中对于IP-10产生所获得的结果(图3L)。
为了进一步支持IRF驱动的应答的诱导,发明人在R848刺激THP-1细胞后4小时对包括IFNB1在内的几种IRF驱动基因进行了RT-qPCR分析。虽然仅用R848几乎没有观察到IFIT1、RSAD2、IFI44和IFNB1的诱导,但通过与ASO852-dT的共刺激,所有这些基因都显著增加(图4D)。发明人已经观察到ASO共刺激强烈增加了HEK-TLR8细胞中TLR8对R848的敏感性(图2D),表明对IFN-β诱导所见的影响可能是由于TLR8对R848的敏感性增加。HEK-TLR8细胞中IFN-β-萤光素酶报告因子对R848的剂量反应(范围从1到15μg/ml)表明,高剂量的R848在这些细胞中参与IFN-β反应的程度与低剂量R848+ASO852-dT相似。
总的来说,这些结果表明ASO(例如ASO852-dT)促进了IRF驱动的应答的激活,否则只能通过非常高剂量的R848才能实现。
实施例7:增强TLR8感测的基因靶向ASO的鉴定
发明人接下来试图建立原理证明,即可以实现结合基因靶向和TLR8增强(同时避免TLR7抑制)的双功能ASO。为此,发明人测试了针对人HPRT基因的mRNA设计的一组48个2’OMe ASO。
对HeLa细胞的初步研究表明,48个ASO中的29个在10nM时显著降低HPRT mRNA水平至少50%(图6和表3)。因此,发明人将注意力集中在这个29个序列的子组上,用于比较R848感测的基因靶向、TLR7抑制和TLR8增强的以下实验(分别为图5A、5B和5C)。与之前的发现一致,这些实验证实大多数ASO阻断了R848对TLR7的激活,[HPRT]ASO551、ASO660-663和ASO665 ASO明显除外(图5B)。至关重要的是,这些序列中的每一个都在其5’或3’端至少包含一个CUU/CUT基序,进一步表明这些基序在保留TLR7感测方面的重要作用(图5D)。有趣的是,ASO551和ASO662 ASO都包含一个5’-CUU和一个3’-UUC基序,但ASO551 ASO是唯一完全保留TLR7感测的ASO。由于这些5’-CUU和3’-UUC基序的位置在两个ASO之间不同,因此CUU基序的最佳定位可能在ASO的末端5’端,表明末端3’端UUC基序也可能重要。在这一点上,发明人再次注意到[LINC-PINT]ASO109和[cGAS]ASO8也具有此类末端3’-UUC基序。这些数据与显示末端5’和3’C在非抑制序列中普遍存在的MEME基序一致(图3A)。
表3:HPRT靶向ASO序列。ASO的合成具有以下修改:DNA单独大写,‘m’表示2’OMe碱基修饰,*表示硫代磷酸酯主链。图5中使用的29个序列以粗体显示。
此外,除4个HPRT ASO(ASO329、ASO321、ASO333、ASO666)外,大多数ASO都在不同程度上显著增强了TLR8感测(图5C)。基于这些分析,发明人选择ASO662作为具有良好HPRT靶向性(在10nM时>70%)、保留TLR7活性(~80%)并增强R848的TLR8感测~5倍的ASO。此外,发明人选择ASO847作为具有接近ASO662的高基因靶向活性(>93%)、强TLR7抑制和TLR8增强活性的ASO。两种ASO被转染到THP-1细胞中并导致显著的HPRT下调,这对于ASO847更为明显-与来自HeLa细胞的数据一致(图5A和5E)。此外,按照相同的转染方案,ASO662强烈增强了R848诱导的IP-10产生(达到与对照dT20寡核苷酸相似的水平)(图5F)。出乎意料的是,ASO847在R848共刺激后未能增加IP-10的产生,这可能是由于其对TLR7的抑制作用,其在THP-1细胞中也有功能(Gantier等人,2008)。
实施例8:TLR8增强-通过5’端基序进行调节
之前已经观察到2’OMe ASO可以通过中心10个DNA碱基的感测来增强R848的TLR8感测,并且富含“T”的区域是更好的增强剂(如对于ASO-852及其变体ASO-852dT所见)(图3-Alharbi等人,2020)。
此外,发明人表明,改变ASO11的5’端2’OMe区域以包括ASO2的5’UCCGG区域导致TLR8增强的显著增加(图3-Alharbi等人,2020)。比较以5’U或5’UC基序终止的ASO的TLR8增强表明,基于192个ASO,5’U或5’UC ASO是显著更强的TLR8增效剂(Alharbi等人,2020)。
为了直接提示5’U/UC在TLR8感测调节中的作用,发明人接下来测试了将末端2’OMe UC基序添加到非TLR8增强ASO的效果。发明人测试的第一个分子是ASO1-UC,它是一个22nt的分子,带有附加的5’UC基序(在5’端有7个2’OMe碱基)。虽然ASO1没有增强HEKTLR8或THP-1细胞中的TLR8,但其5’端变体显著促进了两种模型中的TLR8增强(图9)。这证明了添加5’端UC基序可用作将TLR8增强赋予其他非增强2’OMe gapmer ASO的策略。
发明人还测试了ASO HPRT-660的5’端修饰的效果,发明人之前发现它是TLR8的强增效剂(图5-Alharbi等人,2020)。ASO 660的天然序列包含一个5’CUU区域,发明人将其突变为5’GAA区域(给出ASO660-Mut)。该突变完全消除了HEK-TLR8和THP-1细胞中的TLR8增强,证实了5’端尿苷残基在2’OMe ASO增强TLR8中的重要性(图9)。
为了进一步推动这些结果,发明人测试了相同策略对ASO2 LNA的影响,发明人之前发现ASO2 LNA不会增强TLR8(图1-Alharbi等人,2020)。在此序列上下文中,添加5’端2’OMe UC基序(导致LNA ASO2-Mut1)未能促进TLR8增强。类似地,具有5’CUU和3’UUC 2’OMe基序的ASO2的5’和3’延伸(产生LNA ASO2-Mut2)不影响TLR8增强。这表明5’端修饰策略虽然能够赋予2’OMe ASO的TLR8增强,但与gapmer LNA ASO不兼容。发明人将此归因于LNA修饰可能会改变核酸酶对5’端的处理(参见下文-关于TLR8的LNA ASO增强的部分)(图9)。
表4:以下实施例中使用的各种寡核苷酸(全部为5’-3’)。DNA单独大写,‘m’表示2’OMe碱基,‘/i2MOEr’表示2’MOE碱基,‘+’表示LNA碱基,*表示硫代磷酸酯主链。
实施例9:TLR8增强不限于R848样分子
先前的报告表明,TLR8的R848依赖性激活依赖于其与尿苷通常结合的位点的结合(Tanji等人,2015)(称为位点1)。在另一方面,含尿苷RNA的降解产物与TLR8二聚体的第二个位点(位点2)结合,通常为短双核苷酸(例如UG或UUG或CG)(Tanji等人,2015)。
结合位点2的短RNA中的尿苷残基对于尿苷/R848结合位点1激活TLR8不是必需的,因为使用尿苷增强TLR8对PS-ssRNA41(缺乏尿苷)和TLR13配体Sa19(具有单个尿苷残基)的感测(Shibata等人,2016)。有趣的是,PS-多聚A、多聚C或多聚G未能增强TLR8对尿苷的感测-与结构数据比对,而是提示与选择性RNA基序的结合(Shibata等人,2016年)。
推测本发明的2’OMe ASO(或其降解产物)通过结合TLR8的位点2增强了R848感测,发明人接下来测试了2’OMe ASO是否可以增强TLR8对游离尿苷的感测。与此一致,发明人在HEK-TLR8(具有NF-κB-萤光素酶)和THP-1细胞(具有IP-10)中观察到使用选定的2’OMe(ASO660,852dT)和dT20对于尿苷感测的序列特异性TLR8增强(图10)。然而,ASO660的5’端突变体未能增强TLR8感测-镜像反映了对于R848所见的效果,并证实了ASO的序列依赖性效应。
发明人之前已经表明,R848感测的ASO增强导致IRF激活增加(推测是通过TASL募集的IRF5)。与此一致,TLR8对尿苷感测的ASO增强导致RANTES-萤光素酶诱导(以序列特异性方式-比较ASO660和ASO660-Mut)(图10)。
总的来说,这些结果证实了2’OMe ASO对TLR8的增强作用不仅限于合成的咪唑喹啉化合物,而且对于天然尿苷(与TLR8的位点1结合)也是可见的。
重要的是,由于ASO660对R848和尿苷增强的影响完全被ASO 660Mut中的其5’CUU基序替换为GAA基序所消除,目前的结果表明,增强TLR8所需的短双核苷酸需要起源于该ASO的5’端(对于ASO1和ASO1-UC有相似的发现)。CUU在ASO 660中增强作用的重要性似乎不与RNase T2的作用一致,该RNase T2优先切割ssRNA中的GU或AU基序(在PS和磷酸二酯主链两者上)(Greulich等人,2019)。至关重要的是,ASO 660中的CUU基序位于5’mCmUmU/mCmG背景(所有2’OMe)中。
LNA ASO2 Mut2还包含5’mCmUmU/+C+G背景(其中+是LNA碱基)。由于这不会导致TLR8增强,发明人推测释放CUU片段所必需的核酸内切酶在LNA碱基的情况下无效-注意到如果切割在5’mCmU/mU位置进行,则都具有此序列的LNA ASO2 Mut2和ASO 660之间将不存在TLR8增强的差异。
实施例10:LNA和2MOE gapmer ASO对TLR8的增强作用
发明人之前证明,具有LNA或2’MOE修饰的gapmer ASO2 ASO不会显著增强R848的TLR8感测(图1-Alharbi等人,2020)。基于这些寡核苷酸被加工成与TLR8的位点2结合以增强尿苷和R848活性的片段的概念,这些发现表明LNA和2’MOE修饰阻碍了ASO2的加工(还注意到LNA ASO2只有16nt并且在5’和3’端缺少2nt)。
为了更广泛地了解LNA和2’MOE ASO对TLR8感测的影响,发明人在用R848处理的HEK-TLR8细胞中筛选了100和500nM的一组91个LNA ASO和76个2’MOE ASO(图11、表5和表6)。对于LNA ASO,发明人发现TLR8增强是有限的,只有27%(25/91)的ASO在500nM时导致NF-κB萤光素酶增加>2倍,没有ASO在100nM时增强超过2倍。这与2’OMe ASO形成鲜明对比,其中>50%的分子在100nM时将TLR8感测增强>2倍(图2-Alharbi等人,2020年)。
对于2’MOE ASO,发明人发现TLR8增强大于LNA,但小于2’OMe,其中50%(38/76)的ASO在500nM导致NF-κB萤光素酶增加>2倍,34%(26/76)ASO在100nM时增强超过2倍(图11)。
发明人接下来试图使用来自筛选的一些分子在HEK TLR8细胞的重复实验中验证这些结果。对于LNA ASO,发明人证实A7和H11显著增强了R848的TLR8感测(图12)。然而,当在THP-1细胞中测试相同的序列时,与dT20相比,增强作用非常有限(减少约10倍),A7是唯一持续增加IP-10产生的序列(H11在这些细胞中未能这样做)(图12)。虽然这些结果与LNAASO增强TLR8感测的能力有限一致,但它们也表明LNA ASO的降解模式可能是细胞依赖性的(即THP-1细胞中可能不存在HEK细胞中降解ASO H11的核酸内切酶)。
发明人还在HEK-TLR8细胞的初步实验中验证了2’MOE筛选的结果(图13)。虽然此实验仅导致R848对TLR8进行适度激活(这并未最佳激活此处的细胞),但它验证了最强增强性ASO(G9、D2、D7、B5、A9)。重要的是,发明人在筛选数据中指出,来自HPRT家族的具有单核苷酸增量的序列对TLR8表现出不同的影响。此初步实验验证了这一点,表明ASO 663而不是664/665/666ASO可以增强TLR8感测。尽管需要进一步确认,但这是特别令人感兴趣的,因为用2’OMe化学制成的相同ASO系列显示出类似的趋势,其中2’OMe HPRT 663是最强的TLR8增效剂(663>664>665)(图5-Alharbi等人,2020)。
由于发明人使用的2’OMe和2’MOE gapmer ASO具有相同的长度/序列并且仅在5’和3’5nt区域中使用的碱基的性质不同,这些结果表明核酸内切酶切割的模式是保守的(不阻止核酸酶的序列靶向),尽管与2’OMe ASO相比,2’MOE ASO的效率可能较低。
表5–通过LNA ASO的TLR7和TLR8调节
表6-2’MOE ASO的TLR7和TLR8调节
实施例11:TLR8增强需要位点1和位点2配体的共同施用
在迄今为止的所有实验中,发明人在R848刺激之前使用其ASO对细胞进行预处理(在HEK中约30分钟-在THP-1细胞中过夜)。至关重要的是,发明人在尿苷或R848刺激期间始终将ASO保留在上清液中。为了更好地确定对R848的影响是否需要寡核苷酸的从头降解,发明人将PS-dT20与HEK-TLR8细胞预孵育,然后在R848刺激之前将它们洗掉(图14)。令人惊讶的是,在R848刺激时从上清液中去除dT20会强烈降低所述寡核苷酸对TLR8的增强作用。这表明dT20一旦进入内体就会迅速降解,并且其降解产物(可能与TLR8的位点2结合)的寿命非常短,并且对于增强R848活性至关重要。
还值得注意的是,发明人注意到与过夜孵育相比,他们的2’OMe ASO在THP-1中的短暂孵育(~2.5小时)大大降低了它们的增强能力,但这并没有改变dT20的效果(图14)。基于dT20的作用在HEK中洗掉后不持续的观察结果,发明人推测其2’OMe ASO的降解可能比dT20慢(从而解释了为什么它们的作用在使用2.5h预孵育时与dT20相比降低)。
总的来说,这些结果表明ASO稳定性与TLR8增强之间存在负相关,这需要进一步研究。由于HPRT 663-665系列的2’MOE增强似乎与2’OMe相似,这表明2’MOE ASO仍以与2’OMe相似的方式加工(可能通过相同的核酸酶),但可能不那么快。这与2’MOE ASO通常比2’OMeASO更有效地促进mRNA下调的概念一致-可能部分与更高的细胞内稳定性相关。
这可以为选择和设计更长时间地增强尿苷或R848的TLR8感测的ASO提供机会,当ASO和R848/尿苷/位点1激动剂未同时施用时,这将尤为重要。
实施例12:共培养细胞的TLR8增强-以及在癌症中的作用
接下来,发明人有兴趣测试用他们的增强TLR8的2’OMe ASO之一转染的细胞(使用852dT作为模型)在与吞噬细胞共培养时是否可以增强吞噬细胞中的TLR8感测。发明人推断,未加工的ASO或它们的降解产物可能有利于TLR8的R848感测,这是基于最近的一篇出版物,该出版物表明用合成的PS修饰的DNA分子转染的凋亡细胞的吞噬作用导致DNA在吞噬细胞中的吞噬溶酶体递送(Ahn等人,2018)。
在此,发明人用2’OMe ASO3(非TLR8增强)或852dT(强增强TLR8)转染HEK细胞,然后进行紫外线处理并与PMA分化的THP-1共培养过夜,然后进行24小时R848刺激(图15)。在此设置中,与转染ASO3的HEK细胞的共培养物的R848刺激仅略微上调TNFα的产生(2.8倍),而与转染852dT的HEK细胞的共培养物的R848刺激强烈上调TNFα的产生(>13倍)(图15)。
这些结果表明,HEK中的细胞内ASO(或其降解产物)找到了通往THP-1细胞内体的途径,从而导致TLR8感测的增强。
这一观察结果可能在肿瘤靶向ASO的背景中产生影响。因此,具有癌细胞杀伤活性和TLR8增强活性的双功能ASO也可以被紧密包围的吞噬细胞吸收(或其降解产物)。当用尿苷/R848位点1配体刺激时,这些肿瘤吞噬细胞可能最终被强烈激活以促进更多免疫细胞的募集,同时也有利于它们本应吞噬的癌细胞肽的MHC呈递。至关重要的是,不直接参与垂死癌细胞吞噬作用(并且没有内体ASO降解产物和癌症表位呈现)的肿瘤吞噬细胞不会被R848强烈激活。
实施例13:使用完全2’OMe ASO的TLR8增强
发明人还测试了完全2’OMe修饰的ASO(没有中心DNA“间隙”)增强TLR8的能力。为此,发明人比较了ASO C2Mut1及其完全缺乏2’OMe(称为C2Mut1-PS)或完全2’OMe修饰(C2Mut1-2OMe)的变体的作用。这些实验表明,即使没有中心DNA区域,该ASO仍显著增强TLR8(注意,与其他序列相比,该ASO家族不是强增效剂)(图16)。至关重要的是,这些结果表明完全2’OMe寡核苷酸可以被细胞自发吸收以激活内体TLR8,而无需转染。
实施例14:TLR7抑制-通过5’端基序进行调节
发明人还对R848的TLR7感测测试了突变序列(ASO1-UC、LNA ASO2-mut1和mut2,以及ASO 660/ASO 660-Mut)。根据他们之前的发现,即5’端CUU基序对于限制2’OMe ASO对TLR7的抑制很重要,ASO 660-Mut(缺少其5’端mCmUmU基序)比ASO-660具有显著更强的抑制作用(图17)。相反,LNA ASO2 Mut2(也含有5’mCmUmU基序)保留了TLR7抑制作用。在ASO1-UC中添加5’UC基序不会改变TLR7抑制(但它确实会显著增加TLR8增强)。
实施例15:TLR7抑制-通过碱基修饰进行调节
发明人之前已经观察到ASO2 LNA或2’MOE变体强烈抑制TLR7感测(图1,Alharbi等人,2020)。为了确定LNA或2’MOE ASO中的选定基序是否会阻止TLR7抑制,发明人在用R848处理的HEK-TLR7细胞中测试了他们的100和500nM的91个LNA ASO和76个2’MOE ASO组(图18)。
对于LNA ASO,发明人发现TLR7抑制是主要的,在500nM时只有85%(78/91)的ASO导致NF-κB萤光素酶降低50%,而在该剂量下52%(48/91)的ASO导致NF-κB萤光素酶减少至80%。然而,这种TLR7抑制比发明人对于2’OMe观察到的频率要低一些,其中>78%的分子在500nM时将TLR7感测抑制了80%(图2-Alharbi等人,2020)。
对于2’MOE,发明人发现95%(72/76)的ASO在500nM导致NF-κB萤光素酶减少50%,54%(41/76)的ASO在该剂量下导致NF-κB萤光素酶减少80%(图18)。然而,重要的是,与发明人对2’OMe的观察相似,一些选择的ASO不抑制TLR7(例如,LNA的A9、H11、D1、H1,以及2’MOE的G1、C2、C1、A9、A2)。
发明人接下来试图在重复实验中验证这些结果,重点关注不抑制TLR7的ASO。具体而言,发明人注意到LNA ASO A9、H11、D1、H1均具有5’+C+C基序(+表示LNA修饰)-并且此类基序在所有强TLR7抑制剂(即在500nM时具有>75%的抑制)中均不存在。发明人还包括了抑制TLR7的A11、B1和C11。重复实验证实了筛选的趋势,验证了LNA D1和H11没有显著降低NF-κB萤光素酶活性(注意到A9略微增加了NF-κB萤光素酶,而H1仅略微降低了NF-κB萤光素酶)(图19)。
对于2’MOE ASO,发明人仅进行了一个初步实验,该实验证实了对于G1、A2、C1和A9的降低的TLR7抑制(与测试的其他ASO相比,后者在该实验中完全消除了TLR7信号)。重要的是,发明人注意到具有单核苷酸增量的序列E1(665)、A2(666)和G1(667)对TLR7表现出不同的影响。因此,只有A2和G1 2’MOE ASO具有降低的TLR7活性(表明存在调节TLR7抑制的基序,类似于发明人对2’OMe ASO的发现)。虽然需要进一步确认,但这是特别令人感兴趣的,因为用2’OMe化学制成的相同ASO系列不同地抑制TLR7–666和6672’OMe ASO比665更多地抑制TLR7(图5-Alharbi等人,2020)(图19)。
总的来说,这些结果证实,一些序列对TLR7的免疫抑制能力低于具有所有三种gapmer ASO化学的其他序列。
本领域的技术人员将理解,在不脱离广泛描述的本发明的精神或范围的情况下,可以对如具体实施方案中所示的本发明进行多种变化和/或修饰。因此,本发明的实施方案在所有方面都被认为是说明性的而不是限制性的。
本文讨论和/或引用的所有出版物均以其整体并入本文。
已经包括在本说明书中的文件、行为、材料、装置、文章或类似物的任何讨论仅用于为本发明提供上下文的目的。不应视为承认任何或所有这些事项构成现有技术基础的一部分或是与本发明相关领域的公知常识,因为它存在于本申请的每项权利要求的优先权日之前。
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Claims (80)
1.一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少三个连续嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生包含所述三个连续嘧啶碱基的一种或多种候选寡核苷酸,其中适用以下一项或两项;
a)候选寡核苷酸包含寡核苷酸5’端的7个碱基内的三个连续嘧啶碱基,和
b)候选寡核苷酸包含寡核苷酸3’端的7个碱基内的三个连续嘧啶碱基,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
2.权利要求1的方法,其中候选寡核苷酸的三个连续嘧啶碱基具有修饰的碱基和/或修饰的主链。
3.权利要求1或权利要求2的方法,其中所述寡核苷酸包含:
a)包含修饰的和/或具有修饰的主链的碱基的5’区域,
b)包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,和
c)包含修饰的和/或具有修饰的主链的碱基的3’区域。
4.一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少三个连续嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生一种或多种候选寡核苷酸,所述候选寡核苷酸包含5’区域、3’区域和包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,其中所述5’区域和所述3’区域中的一个或两个包含修饰的和/或具有修饰的主链的碱基,并且适用以下至少一项;
a)5’区域包含修饰的和/或具有修饰的主链的三个连续嘧啶碱基,
b)5’区域包含修饰的和/或具有修饰的主链的碱基,并且5’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
c)3’区域包含修饰的和/或具有修饰的主链的三个连续嘧啶碱基,和
d)3’区域包含修饰的和/或具有修饰的主链的碱基,并且3’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
5.权利要求3或权利要求4的方法,其中所述中间区域长度为约10个碱基。
6.权利要求3至5中任一项的方法,其中所述5’区域和/或所述3’区域的长度为约5个碱基。
7.权利要求1至6中任一项的方法,其中所述三个连续嘧啶碱基在所述寡核苷酸的5’端和/或3’端的5个碱基内。
8.权利要求1至7中任一项的方法,其中所述三个连续嘧啶碱基在所述寡核苷酸的5’端和/或3’端的3个碱基内。
9.权利要求1至8中任一项的方法,其中所述三个连续嘧啶碱基位于所述寡核苷酸的5’端和/或3’端。
10.权利要求1至9中任一项的方法,其中5’三个连续嘧啶碱基具有序列5’-CUU-3’、5’-CUT-3’、5’-CCU-3’、5’-UUC-3’、5’-UUU-3’或5’-CTT-3’。
11.权利要求10的方法,其中5’三个连续嘧啶碱基包含序列5’-CUU-3’。
12.权利要求1至11中任一项的方法,其中所述3’三个连续嘧啶碱基具有序列5’-UUC-3’、5’-TUC-3’、5’-UCC-3’、5’-CUU-3’、5’-UUU-3’或5’-TTC-3’。
13.权利要求12的方法,其中3’三个连续嘧啶碱基具有序列5’-UUC-3’。
14.权利要求13的方法,其中所述3’嘧啶碱基具有序列5’-CUUC-3’。
15.权利要求1至14中任一项的方法,其中所述嘧啶碱基中的一个、两个或全部三个是修饰的碱基和/或具有修饰的主链。
16.权利要求4至15中任一项的方法,其中连接处的三个连续嘧啶碱基具有序列5’-mCmUT-3’、5’-mCTT-3’、5’-TmUmC-3’或5’-TTmC-3’,其中m是修饰的碱基和/或具有修饰的主链。
17.权利要求1至16中任一项的方法,其中所述修饰的碱基包含2’-O-甲基、2’-O-甲氧基乙氧基、2’-氟、2’-烯丙基、2’-O-[2-(甲氨基)-2-氧代乙基]、4’-硫代、4’-CH2-O-2’-桥、4’-(CH2)2-O-2’-桥、2’-LNA、2’-氨基、氟代阿糖核苷酸、苏糖核酸或2’-O-(N-甲基氨基甲酸酯)。
18.权利要求2至17中任一项的方法,其中所述修饰的主链包含硫代磷酸酯、取代硫原子的非桥接氧原子、膦酸酯诸如甲基膦酸酯、磷酸二酯、磷酸吗啉酸酯、磷酸哌嗪酸酯、酰胺、亚甲基(甲氨基)、甲缩醛、硫代甲缩醛、肽核酸或磷酰胺诸如吗啉代磷二酰胺(PMO)、N3’-P5’亚磷酰胺或硫代亚磷酰胺。
19.权利要求1至18中任一项的方法,其中所述寡核苷酸的至少一部分具有/是核糖核酸、脱氧核糖核酸、DNA硫代磷酸酯、RNA硫代磷酸酯、2’-O-甲基-寡核苷酸、2’-O-甲基-寡脱氧核糖核苷酸、2’-O-烃基核糖核酸、2’-O-烃基DNA、2’-O-烃基RNA硫代磷酸酯、2’-O-烃基DNA硫代磷酸酯、2’-F-硫代磷酸酯、2’-F-磷酸二酯、2’-甲氧基乙基硫代磷酸酯、2-甲氧基乙基磷酸二酯、脱氧亚甲基(甲基亚氨基)(脱氧MMI)、2’-O-烃基MMI、脱氧甲基膦酸酯、2’-O-烃基甲基膦酸酯、吗啉代、4’-硫代DNA、4’-硫代RNA、肽核酸、3’-酰胺、脱氧3’-酰胺、2’-O-烃基3’-酰胺、锁核酸、环己烷核酸、三环-DNA、2’氟-阿拉伯核酸、N3’-P5’磷酰胺、氨基甲酸酯连接、磷酸三酯连接、尼龙主链修饰及其任何组合。
20.权利要求1至19中任一项的方法,其中所述修饰的碱基包含2’O-甲基和硫代磷酸酯主链。
21.权利要求1至20中任一项的方法,其中所述寡核苷酸的长度为至少10、至少约18、至少约20、至少约25、约10至约50个核苷酸、约18至约50个核苷酸、约18至约30个核苷酸、约20至约30个核苷酸、约20至约5000个核苷酸或约20个碱基。
22.权利要求1至21中任一项的方法,其中所述寡核苷酸是用于基因沉默的反义寡核苷酸或双链寡核苷酸。
23.权利要求21或权利要求22的方法,其中所述寡核苷酸是gapmer反义寡核苷酸。
24.权利要求22或权利要求23的方法,其中所述三个连续嘧啶碱基中的一个、两个或全部三个在体内通过核酸内切酶去除。
25.权利要求22的方法,其中用于基因沉默的双链寡核苷酸是siRNA或shRNA。
26.权利要求21的方法,其中所述寡核苷酸的长度为10至16个碱基,并且当施用至动物时增强Toll样受体8(TLR8)活性。
27.权利要求1至26中任一项的方法,其中当施用至动物时,所述寡核苷酸不抑制Toll样受体7(TLR7)活性。
28.权利要求27的方法,其中所述动物是人。
29.一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有以下序列之一的区域扫描靶多核苷酸或其互补物:5’-CUUGU-3’、5’-CCUAU-3’、5’-CAUUA-3’、5’-CGAAU-3’、5’-CUUAU-3’、5’-CUUUA-3’、5’ACUGU-3’、5’-CUUCU-3’、5’-CAUAU-3’、5’-CUUCU-3’、5’-AAUUU-3’、5’-AAAUU-3’、5’-CCUUC-3’、5’-AAUCA-3’或5’-CGUCU-3’,其中U可以是T,
ii)产生一种或多种候选寡核苷酸,所述候选寡核苷酸包含:
a)5’端的5’-CUUGU-3’、5’-CCUAU-3’、5’-CAUUA-3’、5’-CGAAU-3’、5’-CUUAU-3’、5’-CUUUA-3’或5’ACUGU-3’,和/或
b)3’端的5’-CUUCU-3’、5’-CAUAU-3’、5’-CUUCU-3’、5’-AAUUU-3’、5’-AAAUU-3’、5’-CCUUC-3’、5’-AAUCA-3’或5’-CGUCU-3’,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
30.一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少两个连续胞嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生包含两个连续胞嘧啶碱基的一种或多种候选寡核苷酸,其中候选寡核苷酸在寡核苷酸的5’端或靠近寡核苷酸的5’端包含两个连续胞嘧啶碱基,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
31.权利要求30的方法,其中所述寡核苷酸的两个连续胞嘧啶碱基具有修饰的碱基和/或修饰的主链。
32.权利要求30或权利要求31的方法,其中所述寡核苷酸包含:
a)包含修饰的和/或具有修饰的主链的碱基的5’区域,
b)包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,和
c)包含修饰的和/或具有修饰的主链的碱基的3’区域。
33.一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对具有至少两个连续胞嘧啶碱基的区域扫描靶多核苷酸或其互补物;
ii)产生一种或多种候选寡核苷酸,所述候选寡核苷酸包含5’区域、3’区域和包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,其中5’区域和3’区域中的一个或两个包含修饰的和/或具有修饰的主链的碱基,5’区域包含修饰的和/或具有修饰的主链的两个连续胞嘧啶碱基,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达的能力,和
iv)选择降低靶基因的表达的寡核苷酸。
34.权利要求32或权利要求33的方法,其中所述5’区域和/或所述3’区域的长度为约3个碱基。
35.权利要求32至34中任一项的方法,其中所述中间区域长度为约10个碱基。
36.权利要求30至35中任一项的方法,其中所述两个连续胞嘧啶碱基包含2’-LNA和硫代磷酸酯主链。
37.权利要求1至36中任一项的方法,其还包括检测所述一种或多种候选寡核苷酸抑制Toll样受体7(TLR7)活性的能力,以及选择不抑制TLR7活性的寡核苷酸。
38.权利要求29至37中任一项的方法,其中所述寡核苷酸还包含权利要求2、3或5至28中任一项的一个或多个特征。
39.一种选择用于降低靶基因的表达的寡核苷酸的方法,所述方法包括:
i)针对包含至少四个碱基是胸苷的区域扫描靶多核苷酸或其互补物;
ii)产生一种或多种候选寡核苷酸,所述候选寡核苷酸包含一个或多个修饰的碱基和至少四个胸苷,
iii)测试一种或多种候选寡核苷酸降低靶基因的表达和增强Toll样受体8(TLR8)活性的能力,和
iv)选择降低靶基因的表达并增强TLR8活性的寡核苷酸。
40.权利要求39的方法,其中所述寡核苷酸包含:
a)长度为至少5个碱基的5’区域,所述碱基被修饰和/或具有修饰的主链,
b)包含10个碱基的区段的中间区域,其中所述碱基中的至少四个是胸苷,
c)长度为至少5个碱基的3’区域。
41.权利要求39或权利要求40的方法,其中
i)至少四个胸苷碱基处于连续区段中,和/或
ii)至少四个胸苷碱基中的一个、两个、三个或四个不处于连续区段中。
42.一种选择增强Toll样受体8(TLR8)活性的寡核苷酸的方法,所述方法包括:
i)针对具有序列UC或CU和10个碱基的区段的区域扫描靶多核苷酸或其互补物,其中所述碱基中的至少两个是胸苷;
ii)产生一种或多种候选寡核苷酸,所述候选寡核苷酸包含:
a)长度为至少5个碱基的5’区域,其中5’端由末端5’-mUmC-3’或末端5’-mCmU-3’组成,其中m是修饰的碱基和/或具有修饰的主链,
b)包含10个碱基的区段的中间区域,其中所述碱基中的至少两个是胸苷,和
c)长度为至少5个碱基和/或具有修饰的主链的3’区域,
iii)测试一种或多种候选寡核苷酸增强TLR8活性的能力,和
iv)选择增强TLR8活性的寡核苷酸。
43.权利要求39至42中任一项的方法,其中所述寡核苷酸还包含权利要求2、3或5至28中任一项的一个或多个特征。
44.一种降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括通过向寡核苷酸的5’和/或3’端添加核苷酸序列来修饰寡核苷酸,使得修饰的寡核苷酸包含寡核苷酸的5’和/或3’端的7个碱基内的三个连续嘧啶碱基。
45.权利要求44的方法,其中所述嘧啶碱基中的一个、两个或全部三个是修饰的碱基和/或具有修饰的主链。
46.一种降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括修饰寡核苷酸,使得修饰的寡核苷酸包含以下至少一种;
a)包含三个连续嘧啶碱基的5’区域,所述三个连续嘧啶碱基被修饰和/或具有修饰的主链,
b)包含修饰的和/或具有修饰的主链的碱基的5’区域,并且5’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
c)包含三个连续嘧啶碱基的3’区域,所述三个连续嘧啶碱基被修饰和/或具有修饰的主链,和
d)包含修饰的和/或具有修饰的主链的碱基的3’区域,并且3’区域和中间区域之间的连接处包含三个连续嘧啶碱基。
47.权利要求44至46中任一项的方法,其中所述三个连续嘧啶碱基位于修饰的寡核苷酸的5’端和/或3’端。
48.一种降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括通过向寡核苷酸的5’端添加核苷酸序列来修饰寡核苷酸,使得修饰的寡核苷酸在寡核苷酸的5’端或靠近寡核苷酸的5’端包含两个连续胞嘧啶碱基。
49.权利要求48的方法,其中两个连续胞嘧啶碱基中的一个或两个是修饰的碱基和/或具有修饰的主链。
50.一种降低寡核苷酸的Toll样受体7(TLR7)抑制活性的方法,所述方法包括修饰寡核苷酸,使得修饰的寡核苷酸包含5’区域,所述5’区域包含两个连续胞嘧啶碱基,所述两个连续胞嘧啶碱基被修饰和/或具有修饰的主链。
51.权利要求48至50中任一项的方法,其中所述两个连续胞嘧啶碱基位于或靠近所述修饰的寡核苷酸的5’端。
52.权利要求48至51中任一项的方法,其中所述两个连续胞嘧啶碱基包含2’-LNA和硫代磷酸酯主链。
53.权利要求44至52中任一项的方法,其中所述修饰的寡核苷酸还包含权利要求2、3、5至28、31、32或34至36中任一项的一个或多个特征。
54.权利要求44至53中任一项的方法,其还包括测试所述修饰的寡核苷酸抑制TLR7活性的能力,以及选择比未修饰的寡核苷酸更小程度地抑制(TLR7)活性的寡核苷酸。
55.一种寡核苷酸,其使用权利要求1至43中任一项的方法选择,或使用权利要求44至54中任一项的方法修饰。
56.一种寡核苷酸,其包含寡核苷酸5’端和/或3’端的7个碱基内的三个连续嘧啶碱基。
57.一种寡核苷酸,其包含两个连续胞嘧啶碱基,其在寡核苷酸的5’端或靠近5’端被修饰和/或具有修饰的主链。
58.一种寡核苷酸,其包含5’区域、3’区域和包含核糖核酸、脱氧核糖核酸或其组合、碱基的中间区域,其中5’区域和3’区域中的一个或两个包含修饰的和/或具有修饰的主链的碱基,并且适用以下至少一项;
a)5’区域包含修饰的和/或具有修饰的主链的三个连续嘧啶碱基,
b)5’区域包含修饰的和/或具有修饰的主链的碱基,并且5’区域和中间区域之间的连接处包含三个连续嘧啶碱基,
c)3’区域包含修饰的和/或具有修饰的主链的三个连续嘧啶碱基,
d)3’区域包含修饰的和/或具有修饰的主链的碱基,并且3’区域和中间区域之间的连接处包含三个连续嘧啶碱基,和
e)5’区域包含两个连续胞嘧啶碱基,其被修饰和/或具有修饰的主链。
59.权利要求57或权利要求58的寡核苷酸,其中所述两个连续胞嘧啶碱基包含2’-LNA和硫代磷酸酯主链。
60.权利要求56至59中任一项的寡核苷酸,其中所述三个连续嘧啶碱基和/或所述两个连续胞嘧啶碱基中的至少一个不与靶多核苷酸杂交。
61.权利要求60的寡核苷酸,其中连续嘧啶碱基中的所有三个不与靶多核苷酸杂交,和/或两个连续胞嘧啶残基中的两个不与靶多核苷酸杂交。
62.一种寡核苷酸,包含:
i)5’端的5’-CUUGU-3’、5’-CCUAU-3’、5’-CAUUA-3’、5’-CGAAU-3’、5’-CUUAU-3’、5’-CUUUA-3’或5’ACUGU-3’,和
ii)3’端的5’-CUUCU-3’、5’-CAUAU-3’、5’-CUUCU-3’、5’-AAUUU-3’、5’-AAAUU-3’、5’-CCUUC-3’、5’-AAUCA-3’或5’-CGUCU-3’。
63.一种寡核苷酸,所述寡核苷酸包含一个或多个修饰的碱基和至少四个胸苷,其中当施用至动物时,所述寡核苷酸增强Toll样受体8(TLR8)活性。
64.权利要求63的寡核苷酸,其包含:
a)长度为至少5个碱基的5’区域,所述碱基被修饰和/或具有修饰的主链,
b)包含10个碱基的区段的中间区域,其中所述碱基中的至少四个是胸苷,
c)长度为至少5个碱基的3’区域。
65.权利要求63或权利要求64的寡核苷酸,其中
i)至少四个胸苷碱基处于连续区段中,和/或
ii)至少四个胸苷碱基中的一个、两个、三个或四个不处于连续区段中。
66.一种寡核苷酸,其包含:
a)长度为至少5个碱基的5’区域,其中5’端由末端5’-mUmC-3’或末端5’-mCmU-3’组成,其中m是修饰的碱基和/或具有修饰的主链,
b)包含10个碱基的区段的中间区域,其中所述碱基中的至少两个是胸苷,和
c)长度为至少5个碱基和/或具有修饰的主链的3’区域,
其中当施用至动物时,所述寡核苷酸增强Toll样受体8(TLR8)活性。
67.权利要求63至66中任一项的寡核苷酸,其也是如权利要求55至62中任一项定义的寡核苷酸。
68.权利要求55至67中任一项的寡核苷酸,其还包含权利要求2、3、5至28、31、32、34至36、40或41中任一项的一个或多个特征。
69.一种寡核苷酸,其包含表1至表6中列出的核酸序列或其变体或由其组成或基本上由其组成。
70.一种组合物,其包含权利要求55至69中任一项的寡核苷酸。
71.权利要求70的组合物,其还包含药学上可接受的载体。
72.权利要求71的组合物,其还包含免疫应答调节剂。
73.一种降低细胞中靶基因的表达的方法,所述方法包括使所述细胞与权利要求55至69中任一项的寡核苷酸接触。
74.一种治疗或预防受试者的疾病的方法,所述方法包括向所述受试者施用权利要求55至69中任一项的寡核苷酸,其中所述寡核苷酸降低与所述疾病相关的靶基因的表达。
75.权利要求74的方法,其中动物已经或将被施用免疫应答调节剂。
77.权利要求76的方法,其中TLR激动剂是瑞喹莫德(R848)、洛索立宾、艾沙托立宾、咪喹莫德、CL075、CL097、CL264、CL307、852A或TL8-506。
78.权利要求55至69中任一项的寡核苷酸在制备用于治疗或预防受试者的疾病的药物中的用途,其中所述寡核苷酸降低与所述疾病相关的靶基因的表达。
79.权利要求55至69中任一项的寡核苷酸,用于治疗或预防受试者的疾病,其中所述寡核苷酸降低与所述疾病相关的靶基因的表达。
80.本文公开和本申请说明书中单独或总体指示的步骤、特征、整数、组合物和/或化合物,以及所述步骤或特征中的两个或更多个的任何和所有组合。
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