本申请要求于2021年9月1日提交的美国临时申请序列号63/239,645、于2022年12月22日提交的美国临时申请序列号63/292,685、于2022年2月25日提交的美国临时申请序列号63/268,580、于2022年3月31日提交的美国临时申请序列号63/362,294、于2022年4月4日提交的美国临时申请序列号63/362,423、于2022年5月2日提交的美国临时申请序列号63/337,560、于2022年6月22日提交的美国临时申请序列号63/354,456、于2021年9月1日提交的美国临时申请序列号63/239,671、于2021年12月17日提交的美国临时申请序列号63/290,960、于2022年1月11日提交的美国临时申请序列号63/298,565和于2022年2月25日提交的美国临时申请序列号63/268,577的优先权权益，所述美国临时申请的内容全文特别以引用方式并入本文。This application claims U.S. Provisional Application Serial No. 63/239,645 filed on September 1, 2021, U.S. Provisional Application Serial No. 63/292,685 filed on December 22, 2022, U.S. Provisional Application Serial No. 63/268,580 filed on February 25, 2022, U.S. Provisional Application Serial No. 63/362,294 filed on March 31, 2022, U.S. Provisional Application Serial No. 63/362,423 filed on April 4, 2022, and U.S. Provisional Application Serial No. 63/337,573 filed on May 2, 2022. 60. Priority benefit to U.S. Provisional Application Serial No. 63/354,456 filed on June 22, 2022, U.S. Provisional Application Serial No. 63/239,671 filed on September 1, 2021, U.S. Provisional Application Serial No. 63/290,960 filed on December 17, 2021, U.S. Provisional Application Serial No. 63/298,565 filed on January 11, 2022, and U.S. Provisional Application Serial No. 63/268,577 filed on February 25, 2022, the contents of which are specifically incorporated herein by reference in their entirety.
背景技术Background Art
杜兴氏肌营养不良(Duchenne Muscular Dystrophy，DMD)是一种遗传障碍，其特征在于由于蛋白质抗肌萎缩蛋白的改变导致进行性肌肉退化和无力。DMD中的遗传修饰，编码抗肌萎缩蛋白的基因会导致DMD。这些遗传修饰使DMD的阅读框移位导致非功能性截短的DMD蛋白。治疗DMD患者的一种方法需要向患者递送恢复DMD的阅读框的化合物。反义化合物可通过跳跃与DMD的阅读框移位相关的内部外显子来恢复DMD的阅读框，所述移位导致非功能性截短的DMD蛋白。外显子跳跃产生抗肌萎缩蛋白，所述蛋白保留了疾病状态下丧失的功能。Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to changes in the protein dystrophin. Genetic modifications in DMD, the gene encoding dystrophin, can cause DMD. These genetic modifications cause the reading frame shift of DMD to cause non-functional truncated DMD proteins. A method for treating DMD patients requires delivering compounds that restore the reading frame of DMD to the patient. Antisense compounds can restore the reading frame of DMD by skipping internal exons associated with the reading frame shift of DMD, which causes non-functional truncated DMD proteins. Exon skipping produces dystrophin, which retains the function lost in the disease state.
使用反义寡核苷酸治疗剂的一个显著问题是当全身施用时它们进入细胞内区室的能力有限。通过使用载剂系统诸如聚合物、阳离子脂质体或通过构建体的化学修饰，例如通过胆固醇分子的共价附接，可促进反义化合物的细胞内递送。然而，细胞内递送效率仍然很低，并且仍然需要改进的递送系统以增加这些反义化合物的效力。A significant problem with using antisense oligonucleotide therapeutic agents is that they have limited ability to enter intracellular compartments when systemically administered. By using carrier systems such as polymers, cationic liposomes or by chemical modification of constructs, for example, by the covalent attachment of cholesterol molecules, the intracellular delivery of antisense compounds can be promoted. However, intracellular delivery efficiency is still very low, and improved delivery systems are still needed to increase the effectiveness of these antisense compounds.
对于将反义化合物递送至细胞内区室以治疗由例如异常基因转录、剪接和/或翻译引起的疾病的有效组合物的需求尚未得到满足。There is an unmet need for effective compositions for delivering antisense compounds to intracellular compartments to treat diseases caused by, for example, aberrant gene transcription, splicing and/or translation.
发明内容Summary of the invention
本文描述了用于递送核酸的化合物。在实施方案中，核酸是反义化合物(AC)。在实施方案中，反义化合物靶向患有杜兴氏肌营养不良(DMD)的受试者中的外显子44。Compounds for delivering nucleic acids are described herein. In embodiments, the nucleic acid is an antisense compound (AC). In embodiments, the antisense compound targets exon 44 in a subject with Duchenne muscular dystrophy (DMD).
本公开涉及包含以下的化合物：The present disclosure relates to compounds comprising:
(a)细胞穿透肽(CPP)序列(例如，环肽)；和(a) a cell penetrating peptide (CPP) sequence (e.g., a cyclic peptide); and
(b)与包含前mRNA序列中的DMD基因的外显子44的至少一部分的靶序列互补的反义化合物(AC)。在实施方案中，AC与包含以下的靶序列互补：前mRNA序列中的DMD基因的外显子44的至少一部分、侧接前mRNA序列中的DMD基因的外显子44的内含子序列的至少一部分或两者。在实施方案中，AC与靶序列的杂交改变DMD前mRNA的剪接模式，以恢复阅读框并能够产生功能性抗肌萎缩蛋白。(b) an antisense compound (AC) complementary to a target sequence comprising at least a portion of exon 44 of a DMD gene in a pre-mRNA sequence. In an embodiment, AC is complementary to a target sequence comprising: at least a portion of exon 44 of a DMD gene in a pre-mRNA sequence, at least a portion of an intron sequence flanking exon 44 of a DMD gene in a pre-mRNA sequence, or both. In an embodiment, hybridization of AC to the target sequence changes the splicing pattern of DMD pre-mRNA to restore the reading frame and enable the production of functional dystrophin.
在实施方案中，AC包含选自硫代磷酸酯(PS)核苷酸、二氨基磷酸酯吗啉代(phosphorodiamidate morpholino，PMO)核苷酸、锁核酸(LNA)、肽核酸(PNA)、包含2’-O-甲基(2’-OMe)修饰的主链的核苷酸、2’O-甲氧基-乙基(2’-MOE)核苷酸、2’，4’约束乙基(cEt)核苷酸和2’-脱氧-2’-氟-β-D-阿糖核酸(2’F-ANA)的至少一种修饰的核苷酸或核酸。在实施方案中，AC包含至少一个PMO(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个PMO，包括其中的所有范围)。在实施方案中，AC中的每个核苷酸均为PMO。In an embodiment, AC comprises at least one modified nucleotide or nucleic acid selected from phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino (PMO) nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), nucleotides comprising a 2'-O-methyl (2'-OMe) modified backbone, 2'O-methoxy-ethyl (2'-MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides, and 2'-deoxy-2'-fluoro-β-D-arabino nucleic acid (2'F-ANA). In embodiments, AC comprises at least one PMO (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 PMOs, including all ranges therein). In embodiments, every nucleotide in AC is a PMO.
在实施方案中，AC包含以下序列：In an embodiment, AC comprises the following sequence:
5’-AAA CGC CGC CAT TTC TCA ACA GAT C-3’。5’-AAA CGC CGC CAT TTC TCA ACA GAT C-3’.
在实施方案中，环肽是FGFGRGRQ。在实施方案中，环肽是GfFGrGrQ。在实施方案中，环肽是FfΦGRGRQ。In an embodiment, the cyclic peptide is FGFGRGRQ. In an embodiment, the cyclic peptide is GfFGrGrQ. In an embodiment, the cyclic peptide is FfΦGRGRQ.
在实施方案中，EEV是：Ac-PKKKRKV-AEEA-Lys-(环[FGFGRGRQ])-PEG12-OH。In an embodiment, the EEV is: Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH.
本公开涉及包含本文所述的化合物的药物组合物。The present disclosure relates to pharmaceutical compositions comprising the compounds described herein.
本公开涉及包含本文所述的化合物的细胞。The present disclosure relates to cells comprising the compounds described herein.
本公开涉及一种治疗DMD的方法，所述方法包括向患者施用本文所述的化合物。The present disclosure relates to a method of treating DMD comprising administering to a patient a compound described herein.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1A和图1B示出了用于将治疗部分，例如反义化合物(AC)连接到细胞穿透肽(CPP)的缀合化学。CPP可与AC的5’端、3’端或主链缀合。Figures 1A and 1B show the conjugation chemistry used to link a therapeutic moiety, such as an antisense compound (AC), to a cell penetrating peptide (CPP). The CPP can be conjugated to the 5' end, 3' end or the backbone of the AC.
图2A和图2B示出了用于连接细胞穿透肽(CPP)(如所示)与反义化合物(AC)的缀合化学，其中CPP包括PEG4接头并且示出了不具有含有聚乙二醇(PEG2或miniPeG)部分的接头的AC(图2A)和具有含有聚乙二醇(PEG2或miniPeG)部分的接头的AC(图2B)。图中的“R”代表棕榈酰基。2A and 2B show a method for attaching a cell penetrating peptide (CPP) (e.g. 2A) and an antisense compound (AC), wherein the CPP includes a PEG4 linker and AC without a linker containing a polyethylene glycol (PEG2 or miniPeG) moiety (FIG. 2B) and AC with a linker containing a polyethylene glycol (PEG2 or miniPeG) moiety are shown. "R" in the figure represents palmitoyl.
图3示出了使用代表性CPP进行内体逃逸载体(EEV)设计的实例。应当理解，CPP可包括本文公开的CPP中的任一种。An example of endosomal escape vehicle (EEV) design using representative CPPs is shown in Figure 3. It should be understood that the CPP may include any of the CPPs disclosed herein.
图4A示出了EEV-PMO-MDX-23-1的制备示意图。图4B是RT-PCR分析，表明与用PMO-MDX-23-1治疗的小鼠相比，用EEV-PMO-MDX-23-1治疗的小鼠产生了缺乏内部外显子(外显子23)的抗肌萎缩蛋白。图4C示出了施用PMO-MDX-23-1和EEV-PMO-MDX-23-1后，不同治疗肌肉组中抗肌萎缩蛋白外显子跳跃产物。Figure 4A shows a schematic diagram of the preparation of EEV-PMO-MDX-23-1. Figure 4B is an RT-PCR analysis showing that mice treated with EEV-PMO-MDX-23-1 produced dystrophin lacking an internal exon (exon 23) compared to mice treated with PMO-MDX-23-1. Figure 4C shows the dystrophin exon skipping products in different treated muscle groups after administration of PMO-MDX-23-1 and EEV-PMO-MDX-23-1.
图5A至图5D示出了递送PMO-MDX-23-1或EEV-PMO-MDX-23-1后MDX小鼠四头肌(图5A)、胫骨前肌(TA)(图5B)、横膈膜(图5C)和心脏(图5D)中外显子跳跃的百分比。Figures 5A to 5D show the percentage of exon skipping in the quadriceps (Figure 5A), tibialis anterior (TA) (Figure 5B), diaphragm (Figure 5C), and heart (Figure 5D) of MDX mice after delivery of PMO-MDX-23-1 or EEV-PMO-MDX-23-1.
图6A至图6D示出了递送EEV-PMO-MDX-23-1后MDX小鼠胫骨前肌(TA)(图6A)、四头肌(图6B)、横膈膜(图6C)和心脏(图6D)中外显子23剪接的百分比。Figures 6A-6D show the percentage of exon 23 splicing in the tibialis anterior (TA) (Figure 6A), quadriceps (Figure 6B), diaphragm (Figure 6C), and heart (Figure 6D) of MDX mice after delivery of EEV-PMO-MDX-23-1.
图7A至图7D示出了递送PMO-MDX-23-1或EEV-PMO-MDX-23-1后通过蛋白印迹法检测的四头肌(图7A)、胫骨前肌(TA)(图7B)、横膈膜(图7C)和心脏(图7D)中外显子23校正抗肌萎缩蛋白的量。Figures 7A to 7D show the amount of exon 23 corrected dystrophin detected by Western blotting in quadriceps (Figure 7A), tibialis anterior (TA) (Figure 7B), diaphragm (Figure 7C), and heart (Figure 7D) after delivery of PMO-MDX-23-1 or EEV-PMO-MDX-23-1.
图8A至图8D示出了静脉内递送10mpk或30mpkEEV-PMO-MDX-23-1后横膈膜(图8A)、心脏(图8B)、四头肌(图8C)和胫骨前肌(图8D)中外显子23校正抗肌萎缩蛋白和α-肌动蛋白的蛋白印迹。8A-8D show Western blots of exon 23 corrected dystrophin and α-actin in the diaphragm ( FIG. 8A ), heart ( FIG. 8B ), quadriceps ( FIG. 8C ), and tibialis anterior ( FIG. 8D ) muscles following intravenous delivery of 10 mpk or 30 mpk EEV-PMO-MDX-23-1.
图9A至图9B示出了用30mpk EEV-PMO-MDX-23-1或30mpkPMO-MDX-23-1治疗后两周(图9A)和四周(图9B)MDX小鼠中的抗肌萎缩蛋白水平。9A-9B show dystrophin levels in MDX mice two weeks ( FIG. 9A ) and four weeks ( FIG. 9B ) after treatment with 30 mpk EEV-PMO-MDX-23-1 or 30 mpk PMO-MDX-23-1.
图10A至图10D示出了施用30mpk PMO-MDX-23-1或30mpkEEV-PMO-MDX-23-1的MDX小鼠胫骨前肌(图10A)、四头肌(图10B)、横膈膜(图10C)和心脏(图10D)中外显子23校正的百分比。与单独施用PMO-MDX-23-1的小鼠相比，施用EEV-PMO-MDX-23-1的小鼠表现出增强的剪接校正。Figures 10A to 10D show the percentage of exon 23 correction in the tibialis anterior muscle (Figure 10A), quadriceps (Figure 10B), diaphragm (Figure 10C), and heart (Figure 10D) of MDX mice administered 30 mpk PMO-MDX-23-1 or 30 mpk EEV-PMO-MDX-23-1. Mice administered EEV-PMO-MDX-23-1 showed enhanced splicing correction compared to mice administered PMO-MDX-23-1 alone.
图11A至图11C示出了单一IV剂量(40mg/kg)的EEV-PMO-MDX-23-1后至多8周在mdx小鼠中观测到的心脏(图11A)、胫骨前肌(图11B)和横膈膜(图11C)中的外显子23跳跃和抗肌萎缩蛋白校正。Figures 11A-11C show exon 23 skipping and dystrophin correction observed in the heart (Figure 11A), tibialis anterior muscle (Figure 11B), and diaphragm (Figure 11C) in mdx mice up to 8 weeks after a single IV dose (40 mg/kg) of EEV-PMO-MDX-23-1.
图12A至图12D示出了重复剂量(20mg/kg)的EEV-PMO-MDX-23-2后D2-mdx模型中的外显子23跳跃。图12A(心脏)；图12B(横膈膜)；图12C(胫骨前肌)；图12D(三头肌)Figures 12A-12D show exon 23 skipping in the D2-mdx model following repeated doses (20 mg/kg) of EEV-PMO-MDX-23-2. Figure 12A (heart); Figure 12B (diaphragm); Figure 12C (tibialis anterior); Figure 12D (triceps)
图13A至图13C与单独PMO-MDX-23相比，每月用20mg/kgEEV-PMO-MDX-23-2治疗后，D2-mdx小鼠示出了正常化的血清肌酸激酶(CK)水平(图12A)和肌肉功能显著改善(图12B至图12C)。(ns不显著、*p＜0.05、**p＜0.01、***p＜0.001、****p＜0.0001)。Figures 13A-13C D2-mdx mice showed normalized serum creatine kinase (CK) levels (Figure 12A) and significant improvement in muscle function (Figures 12B-12C) after monthly treatment with 20 mg/kg EEV-PMO-MDX-23-2 compared to PMO-MDX-23 alone (ns not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
图14A至图14D示出了通过两步RT-PCR评估的EEV-PMO-MDX-23-2注射后1周的剂量依赖性外显子跳跃。图14A(三头肌)；图14B(胫骨前肌)；图14C(隔膜)；图15D(心脏)。Figures 14A to 14D show dose-dependent exon skipping of EEV-PMO-MDX-23-2 one week after injection as assessed by two-step RT-PCR. Figure 14A (triceps); Figure 14B (tibialis anterior); Figure 14C (diaphragm); Figure 15D (heart).
图15A至图15D示出了施用80mpk EEV-PMO-MDX-23-2后效果的持续时间。图15A(三头肌)；图15B(胫骨前肌)；图15C(横膈膜)；图15D(心脏)。Figures 15A-15D show the duration of effect following administration of 80 mpk of EEV-PMO-MDX-23-2. Figure 15A (triceps); Figure 15B (tibialis anterior); Figure 15C (diaphragm); Figure 15D (heart).
图16示出了所有4个组织(三头肌、胫骨前肌、横膈膜和心脏)中的累积外显子跳跃。FIG. 16 shows cumulative exon skipping in all four tissues (triceps, tibialis anterior, diaphragm, and heart).
图17示出了D2.mdx线挂数据(wire hang data)。治疗12周后，用EEV-PMO-MDX23-180mpk Q2W治疗的动物的线挂时间与WT动物在统计上无法区分。DBA WT媒介物(盐水)；D2.mdx媒介物(盐水)；D2.mdx EEV-PMO-MDX23-1；D2.mdx EEV-PMO-MDX23-2；D2.mdx PMO-MDX23(5’-GGCCAAACCTCGGCTTACCTGAAAT-3’)Figure 17 shows D2.mdx wire hang data. After 12 weeks of treatment, the wire hang time of animals treated with EEV-PMO-MDX23-180mpk Q2W was statistically indistinguishable from WT animals. DBA WT Vehicle (saline); D2.mdx Vehicle (saline); D2.mdx EEV-PMO-MDX23-1; D2.mdx EEV-PMO-MDX23-2; D2.mdx PMO-MDX23 (5'-GGCCAAACCTCGGCTTACCTGAAAT-3')
图18A至图18D示出了给药前(图18A)以及给药后4周(图18B)、8周(图18C)和12周(图18D)D2MDX小鼠中的肌酸激酶活性。18A to 18D show creatine kinase activity in D2MDX mice before dosing ( FIG. 18A ) and 4 weeks ( FIG. 18B ), 8 weeks ( FIG. 18C ), and 12 weeks ( FIG. 18D ) after dosing.
图19A至图19B示出了给药前(图19A)和给药后12周(图19B)D2MDX小鼠的握力。19A-19B show the grip strength of D2MDX mice before dosing ( FIG. 19A ) and 12 weeks after dosing ( FIG. 19B ).
图20A至图20D示出了EEV-PMO-DMD44-1(图5A)、EEV-PMO-DMD44-2(图5B)、EEV-PMO-DMD44-3(图5C)和EEV-PMO-DMD44-4(图5D)的合成方案。20A to 20D show the synthesis schemes of EEV-PMO-DMD44-1 ( FIG. 5A ), EEV-PMO-DMD44-2 ( FIG. 5B ), EEV-PMO-DMD44-3 ( FIG. 5C ), and EEV-PMO-DMD44-4 ( FIG. 5D ).
图21示出了用1、3或10μM EEV-PMO-DMD44-1；EEV-PMO-DMD44-2或EEV-PMO-DMD-3处理后DMDΔ45肌细胞中的抗肌萎缩蛋白恢复。FIG. 21 shows dystrophin restoration in DMDΔ45 myocytes after treatment with 1, 3, or 10 μM EEV-PMO-DMD44-1; EEV-PMO-DMD44-2, or EEV-PMO-DMD-3.
图22A和图22B示出了经由IV注射用EEV-PMO-DMD44-1(图7A)和EEV-PMO-DMD44-2(图7B)治疗的hDMD小鼠组织中的外显子跳跃和药物浓度。22A and 22B show exon skipping and drug concentrations in tissues of hDMD mice treated with EEV-PMO-DMD44-1 ( FIG. 7A ) and EEV-PMO-DMD44-2 ( FIG. 7B ) via IV injection.
图23A至图23B描绘了NHP模型中EEV-PMO-DMD44-1的外显子跳跃(图23A)和药物暴露(图23B)。23A-23B depict exon skipping ( FIG. 23A ) and drug exposure ( FIG. 23B ) of EEV-PMO-DMD44-1 in the NHP model.
图24A至图24B描绘了NHP模型中EEV-PMO-DMD44-2的外显子跳跃(图24A)和药物暴露(图24B)。24A-24B depict exon skipping ( FIG. 24A ) and drug exposure ( FIG. 24B ) of EEV-PMO-DMD44-2 in the NHP model.
图25A至图25B示出了用EEV-PMO-DMD44-1处理的DMD患者来源的肌细胞中的外显子跳跃(图25A)和抗肌萎缩蛋白的恢复(图25B)。25A-25B show exon skipping ( FIG. 25A ) and restoration of dystrophin ( FIG. 25B ) in DMD patient-derived myocytes treated with EEV-PMO-DMD44-1.
图26A至图26C以10、20、40和80mg/kg静脉内(IV)施用EEV-PMO-DMD44-1后在hDMD转基因小鼠的心脏(图26A)和骨骼肌(图26B和图26C)中观察到剂量依赖性组织暴露和外显子跳跃。26A-26C Dose-dependent tissue exposure and exon skipping were observed in the heart ( FIG. 26A ) and skeletal muscle ( FIG. 26B and FIG. 26C ) of hDMD transgenic mice following intravenous (IV) administration of EEV-PMO-DMD44-1 at 10, 20, 40, and 80 mg/kg.
图27示出了当将EEV-PMO-DMD44-1施用于非人灵长类动物(NHP)后，其具有延长的循环半衰期。FIG. 27 shows that EEV-PMO-DMD44-1 has a prolonged circulation half-life when administered to non-human primates (NHPs).
图28示出了在以30mg/kg IV输注1小时后7天，单剂量EEV-PMO-DMD44-1在NHP的骨骼肌和心脏中产生了有意义的外显子跳跃水平。Figure 28 shows that a single dose of EEV-PMO-DMD44-1 produced significant levels of exon skipping in skeletal muscle and heart of NHPs 7 days after 1 hour IV infusion at 30 mg/kg.
图29A至图29C描绘了单一IV剂量(15mg/kg)EEV-PMO-DMD44-1或R6(聚精氨酸)缀合的外显子44跳跃PMO后，hDystrophin小鼠心脏(图29A)、横膈膜(图29B)和三头肌(图29C)中的外显子跳跃。Figures 29A-29C depict exon skipping in the heart (Figure 29A), diaphragm (Figure 29B), and triceps (Figure 29C) of hDystrophin mice following a single IV dose (15 mg/kg) of EEV-PMO-DMD44-1 or R6 (polyarginine)-conjugated exon 44-skipping PMO.
图30A至图30E描绘了通过1-STEP RT-PCR检测至多12周，hDMD小鼠心脏(图30A)、横膈膜(图30B)、胫骨前肌(图30C)、腓肠肌(图30D)和三头肌(图30E)中的外显子跳跃。Figures 30A to 30E depict exon skipping in the heart (Figure 30A), diaphragm (Figure 30B), tibialis anterior (Figure 30C), gastrocnemius (Figure 30D), and triceps (Figure 30E) of hDMD mice detected by 1-STEP RT-PCR up to 12 weeks.
图31示出了单一IV剂量后至多12周NHP中的外显子跳跃。FIG. 31 shows exon skipping in NHPs up to 12 weeks after a single IV dose.
图32A至图32C示出了通过LC-MS/MS确定的PMO与EEV-PMO与EEV-NLS-PMO在THP细胞中的定位：全细胞摄取(图32A)；亚细胞定位(图32B)；和核摄取(图32C)。32A-32C show the localization of PMO versus EEV-PMO versus EEV-NLS-PMO in THP cells as determined by LC-MS/MS: whole cell uptake ( FIG. 32A ); subcellular localization ( FIG. 32B ); and nuclear uptake ( FIG. 32C ).
具体实施方式DETAILED DESCRIPTION
化合物Compound
本文公开了用于治疗杜兴氏肌营养不良(DMD)的化合物。在实施方案中，DMD是由外显子44中的突变引起的。在实施方案中，化合物被设计成递送与前mRNA序列中的DMD基因的靶序列互补的反义化合物(AC)，其中靶序列包含外显子44的5’侧接内含子的至少一部分、外显子44的至少一部分、外显子44的3’侧接内含子的至少一部分或它们的组合。在实施方案中，化合物被设计成将反义化合物(AC)细胞内递送至有需要的受试者。Disclosed herein are compounds for treating Duchenne muscular dystrophy (DMD). In embodiments, DMD is caused by a mutation in exon 44. In embodiments, compounds are designed to deliver antisense compounds (AC) complementary to the target sequence of the DMD gene in the pre-mRNA sequence, wherein the target sequence comprises at least a portion of the 5' flanking intron of exon 44, at least a portion of exon 44, at least a portion of the 3' flanking intron of exon 44, or a combination thereof. In embodiments, compounds are designed to deliver antisense compounds (AC) intracellularly to subjects in need thereof.
在实施方案中，化合物改变与AC结合的靶前mRNA的剪接模式，导致再剪接靶蛋白的形成。在一个实施方案中，再剪接靶蛋白比在不存在AC的情况下通过剪接靶前mRNA产生的靶蛋白更具功能性。在实施方案中，与通过剪接产生的靶蛋白的功能相比，再剪接靶蛋白使靶蛋白的功能提高至少约1％、至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约99％、至少约100％、至少约150％、至少约200％、至少约250％、至少约300％、至少约350％、至少约400％、至少约450％、至少约500％或更多。在实施方案中，与通过剪接产生的靶蛋白的功能相比，再剪接靶蛋白使靶蛋白的功能提高约1％、约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％、约99％、约100％、约150％、约200％、约250％、约300％、约350％、约400％、约450％、约500％或更多，包括其间的所有值和范围。在实施方案中，再剪接靶蛋白将功能恢复至野生型靶蛋白功能的至少约1％、至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％或至少约99％，以及至多约100％。在实施方案中，再剪接靶蛋白将功能恢复至野生型靶蛋白功能的约1％、约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％、约99％或约100％，包括其间的所有值和范围。In an embodiment, the compound changes the splicing pattern of the target pre-mRNA bound to the AC, resulting in the formation of a re-spliced target protein. In one embodiment, the re-spliced target protein is more functional than the target protein produced by splicing the target pre-mRNA in the absence of the AC. In embodiments, re-splicing of the target protein improves the function of the target protein by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, or more, compared to the function of the target protein produced by splicing. In embodiments, re-splicing of the target protein improves the function of the target protein by about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500% or more, including all values and ranges therebetween, compared to the function of the target protein produced by splicing. In embodiments, the re-splicing target protein restores function to at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, and up to about 100% of the function of the wild-type target protein. In embodiments, the re-splicing target protein restores function to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the function of the wild-type target protein, including all values and ranges therebetween.
在各种实施方案中，本文公开的化合物具有AC部分和细胞穿透肽(CPP)部分。在一些实施方案中，CPP部分是环状的(在本文中称为环肽)。在实施方案中，化合物能够穿过细胞膜并体内结合到靶前mRNA。在实施方案中，化合物包含：a)至少一个CPP部分；和b)至少一种AC，其中CPP直接或间接(例如，经由接头)偶联到AC。在实施方案中，化合物包含1、2、3、4、5、6、7、8、9、10或更多个AC部分。在实施方案中，化合物包含1、2、3、4、5、6、7、8、9、10或更多个CPP部分。在实施方案中，化合物包含一个AC部分。在实施方案中，化合物包含两个AC部分。如本文所用，“偶联”可指CPP与AC之间的共价或非共价缔合，包括CPP与AC的融合以及CPP与AC的化学缀合。将CPP非共价附接到AC的方法的非限制性实例是通过链霉亲和素/生物素相互作用，例如通过将生物素缀合到CPP并将AC融合到链霉亲和素。在所得化合物中，CPP经由生物素与链霉亲和素之间的非共价缔合来与AC偶联。In various embodiments, the compounds disclosed herein have an AC portion and a cell penetrating peptide (CPP) portion. In some embodiments, the CPP portion is cyclic (referred to herein as a cyclic peptide). In embodiments, the compound is able to pass through the cell membrane and bind to the target pre-mRNA in vivo. In embodiments, the compound comprises: a) at least one CPP portion; and b) at least one AC, wherein the CPP is directly or indirectly (e.g., via a linker) coupled to the AC. In embodiments, the compound comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AC portions. In embodiments, the compound comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more CPP portions. In embodiments, the compound comprises an AC portion. In embodiments, the compound comprises two AC portions. As used herein, "coupling" may refer to a covalent or non-covalent association between a CPP and an AC, including the fusion of a CPP with an AC and the chemical conjugation of a CPP with an AC. A non-limiting example of a method of non-covalently attaching a CPP to an AC is through a streptavidin/biotin interaction, such as by conjugating biotin to the CPP and fusing the AC to the streptavidin. In the resulting compound, the CPP is coupled to the AC via a non-covalent association between biotin and streptavidin.
在实施方案中，CPP直接或经由接头间接缀合到AC，从而形成CPP-AC缀合物。AC与CPP的缀合可发生在这些部分上的任何适当的位点处。在实施方案中，AC的5’或3’端可与CPP中的氨基酸的C-末端、N-末端或侧链缀合。在实施方案中，CPP是环肽。In an embodiment, the CPP is conjugated to the AC directly or indirectly via a linker, thereby forming a CPP-AC conjugate. Conjugation of AC to the CPP may occur at any suitable site on these parts. In an embodiment, the 5' or 3' end of the AC may be conjugated to the C-terminus, N-terminus or side chain of an amino acid in the CPP. In an embodiment, the CPP is a cyclic peptide.
在实施方案中，AC可通过AC的5’或3’端上的部分化学缀合到CPP。在实施方案中，AC可通过CPP上氨基酸的侧链缀合到CPP。CPP上能够形成共价键或可如此修饰的任何氨基酸侧链可用于将AC连接到CPP。CPP上的氨基酸可以是天然或非天然氨基酸。在实施方案中，CPP上用于缀合AC的氨基酸是天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺、赖氨酸、鸟氨酸、2，3-二氨基丙酸或它们的类似物。在实施方案中，侧链被连接到AC或接头的键取代。在实施方案中，氨基酸是赖氨酸或其类似物。在实施方案中，氨基酸是谷氨酸或其类似物。在实施方案中，氨基酸是天冬氨酸或其类似物。在实施方案中，CPP是环肽。In an embodiment, AC can be chemically conjugated to CPP through a portion on the 5' or 3' end of AC. In an embodiment, AC can be conjugated to CPP through the side chain of an amino acid on CPP. Any amino acid side chain on CPP that can form a covalent bond or can be so modified can be used to connect AC to CPP. The amino acid on CPP can be a natural or non-natural amino acid. In an embodiment, the amino acid used to conjugate AC on CPP is aspartic acid, glutamic acid, glutamine, asparagine, lysine, ornithine, 2,3-diaminopropionic acid or their analogs. In an embodiment, the side chain is replaced by a bond connected to AC or a linker. In an embodiment, the amino acid is lysine or an analog thereof. In an embodiment, the amino acid is glutamic acid or an analog thereof. In an embodiment, the amino acid is aspartic acid or an analog thereof. In an embodiment, CPP is a cyclic peptide.
内体逃逸载体(EEV)Endosomal escape vector (EEV)
本文提供了内体逃逸载体(EEV)，其可用于转运AC穿过细胞膜，例如，将AC递送至细胞的胞质或细胞核。EEV可包含细胞穿透肽(CPP)，例如，与环外肽(EP)缀合的环状细胞穿透肽(cCPP)。EP可互换地称为调节肽(MP)。EP可包含核定位信号(NLS)的序列。EP可与AC偶联。EP可与cCPP偶联。EP可与AC和cCPP偶联。EP、AC、cCPP或它们的组合之间的偶联可以是非共价的或共价的。EP可通过肽键附接到cCPP的N-末端。EP可通过肽键附接到cCPP的C-末端。EP可通过cCPP中氨基酸的侧链附接到cCPP。EP可通过赖氨酸的侧链附接到cCPP，赖氨酸可与cCPP中的谷氨酰胺的侧链缀合。EP可与AC的5’或3’端缀合。EP可与接头偶联。环外肽可与接头的氨基基团缀合。EP可经由EP和cCPP的C-末端通过cCPP和/或EP上的侧链与接头偶联。例如，EP可包含末端赖氨酸，其然后可通过酰胺键与含有谷氨酰胺的cCPP偶联。当EP含有末端赖氨酸并且赖氨酸的侧链可用于附接cCPP时，C-末端或N-末端可附接到AC上的接头。Provided herein is an endosomal escape vector (EEV), which can be used to transport AC across the cell membrane, for example, to deliver AC to the cytoplasm or nucleus of a cell. EEV may include a cell penetrating peptide (CPP), for example, a cyclic cell penetrating peptide (cCPP) conjugated to an extracyclic peptide (EP). EP is interchangeably referred to as a regulatory peptide (MP). EP may include a sequence of a nuclear localization signal (NLS). EP may be coupled to AC. EP may be coupled to cCPP. EP may be coupled to AC and cCPP. The coupling between EP, AC, cCPP or a combination thereof may be non-covalent or covalent. EP may be attached to the N-terminus of cCPP by a peptide bond. EP may be attached to the C-terminus of cCPP by a peptide bond. EP may be attached to cCPP by the side chain of an amino acid in cCPP. EP may be attached to cCPP by the side chain of lysine, and lysine may be conjugated to the side chain of glutamine in cCPP. EP may be conjugated to the 5' or 3' end of AC. The EP can be coupled to a linker. The exocyclic peptide can be conjugated to the amino group of the linker. The EP can be coupled to the linker via the C-terminus of the EP and the cCPP through side chains on the cCPP and/or the EP. For example, the EP can contain a terminal lysine, which can then be coupled to a cCPP containing glutamine through an amide bond. When the EP contains a terminal lysine and the side chain of the lysine can be used to attach the cCPP, the C-terminus or the N-terminus can be attached to the linker on the AC.
环外肽Exocyclic peptide
环外肽(EP)可包含2至10个氨基酸残基，例如2、3、4、5、6、7、8、9或10个氨基酸残基，包括其间的所有范围和值。EP可包含6至9个氨基酸残基。EP可包含4至8个氨基酸残基。The exocyclic peptide (EP) may comprise 2 to 10 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues, including all ranges and values therebetween. The EP may comprise 6 to 9 amino acid residues. The EP may comprise 4 to 8 amino acid residues.
环外肽中的每个氨基酸可以是天然的或非天然的氨基酸。术语“非天然氨基酸”是指一种有机化合物，它是天然氨基酸的同源物，因为它具有与天然氨基酸类似的结构，从而模拟天然氨基酸的结构和反应性。非天然氨基酸可以是修饰的氨基酸和/或氨基酸类似物，其不是20种常见天然存在的氨基酸中的一种，也不是稀有天然氨基酸硒代半胱氨酸或吡咯赖氨酸。非天然氨基酸也可以是天然氨基酸的D-异构体。合适的氨基酸的实例包括但不限于丙氨酸、别亮氨酸(allosoleucine)、精氨酸、瓜氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、萘基丙氨酸、苯丙氨酸、脯氨酸、焦谷氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、它们的衍生物或它们的组合。这些和其他氨基酸连同它们在本文中使用的缩写列于表1中。例如，氨基酸可以是A、G、P、K、R、V、F、H、Nal或瓜氨酸。Each amino acid in the exocyclic peptide can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a homologue of a natural amino acid because it has a structure similar to that of a natural amino acid, thereby simulating the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and/or amino acid analogs that are not one of the 20 common naturally occurring amino acids, nor are they rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives or combinations thereof. These and other amino acids are listed in Table 1 together with the abbreviations they use herein. For example, the amino acid can be A, G, P, K, R, V, F, H, NaI, or citrulline.
EP可包含至少一个带正电荷的氨基酸残基，例如至少一个赖氨酸残基和/或至少一个包含含有胍基或其质子化形式的侧链的胺酸残基。EP可包含1或2个氨基酸残基，其包含含有胍基或其质子化形式的侧链。包含含有胍基的侧链的氨基酸残基可以是精氨酸残基。质子化形式在整个公开内容中可意指其盐。The EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one amino acid residue comprising a side chain containing a guanidine group or a protonated form thereof. The EP may comprise 1 or 2 amino acid residues comprising a side chain containing a guanidine group or a protonated form thereof. The amino acid residue comprising a side chain containing a guanidine group may be an arginine residue. The protonated form may refer to a salt thereof throughout the disclosure.
EP可包含至少两个、至少三个或至少四个或更多个赖氨酸残基。EP可包含2、3或4个赖氨酸残基。每个赖氨酸残基的侧链上的氨基基团可被保护基团取代，所述保护基团包括例如三氟乙酰基(-COCF3)、烯丙氧基羰基(Alloc)、1-(4，4-二甲基-2，6-二氧代亚环己基)乙基(Dde)或(4，4-二甲基-2，6-二氧代环己-1-亚基-3)-甲基丁基(ivDde)基团。每个赖氨酸残基的侧链上的氨基基团可被三氟乙酰基(-COCF3)取代。可包含保护基团以实现酰胺缀合。可在EP与cCPP缀合后去除保护基团。EP may contain at least two, at least three or at least four or more lysine residues. EP may contain 2, 3 or 4 lysine residues. The amino group on the side chain of each lysine residue may be substituted by a protecting group, and the protecting group includes, for example, trifluoroacetyl (-COCF3 ), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl (Dde) or (4,4-dimethyl-2,6-dioxocyclohexyl-1-ylidene-3)-methylbutyl (ivDde) groups. The amino group on the side chain of each lysine residue may be substituted by a trifluoroacetyl (-COCF3 ). Protective groups may be included to achieve amide conjugation. The protecting groups may be removed after EP is conjugated to cCPP.
EP可包含至少2个具有疏水侧链的氨基酸残基。具有疏水侧链的氨基酸残基可选自缬氨酸、脯氨酸、丙氨酸、亮氨酸、异亮氨酸和甲硫氨酸。具有疏水侧链的氨基酸残基可以是缬氨酸或脯氨酸。EP may contain at least 2 amino acid residues with hydrophobic side chains. The amino acid residue with a hydrophobic side chain may be selected from valine, proline, alanine, leucine, isoleucine and methionine. The amino acid residue with a hydrophobic side chain may be valine or proline.
EP可包含至少一个带正电荷的氨基酸残基，例如至少一个赖氨酸残基和/或至少一个精氨酸残基。EP可包含至少两个、至少三个或至少四个或更多个赖氨酸残基和/或精氨酸残基。EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one arginine residue. EP may comprise at least two, at least three or at least four or more lysine residues and/or arginine residues.
EP可包含KK、KR、RR、HH、HK、HR、RH、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKH、KHK、HKK、HRR、HRH、HHR、HBH、HHH、HHHH、KHKK、KKHK、KKKH、KHKH、HKHK、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、HBHBH、HBKBH、RRRRR、KKKKK、KKKRK、RKKKK、KRKKK、KKRKK、KKKKR、KBKBK、RKKKKG、KRKKKG、KKRKKG、KKKKRG、RKKKKB、KRKKKB、KKRKKB、KKKKRB、KKKRKV、RRRRRR、HHHHHH、RHRHRH、HRHRHR、KRKRKR、RKRKRK、RBRBRB、KBKBKB、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV或PKKKRKG，其中B是β-丙氨酸。EP中的氨基酸可具有D或L立体化学。EP can contain KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH , KHKK, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, KKKKR, KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, wherein B is β-alanine. The amino acids in EP can have D or L stereochemistry.
EP可包含KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、KKKKK、KKKRK、KBKBK、KKKRKV、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV或PKKKRKG。EP可包含PKKKRKV、PR、RRR、RHR、RBR、RBRBR、RBHBR或HBRBH，其中B是β-丙氨酸。EP中的氨基酸可具有D或L立体化学。The EP may comprise KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. The EP may comprise PKKKRKV, PR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, wherein B is β-alanine. The amino acids in the EP may have D or L stereochemistry.
EP可由KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、KKKKK、KKKRK、KBKBK、KKKRKV、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV或PKKKRKG组成。EP可由PKKKRKV、RR、RRR、RHR、RBR、RBRBR、RBHBR或HBRBH组成，其中B是β-丙氨酸。EP中的氨基酸可具有D或L立体化学。EP may consist of KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP may consist of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, wherein B is β-alanine. The amino acids in EP may have D or L stereochemistry.
EP可包含本领域中鉴定为核定位序列(NLS)的氨基酸序列。EP可由本领域中鉴定为核定位序列(NLS)的氨基酸序列组成。EP可包含含有氨基酸序列PKKKRKV的NLS。EP可由包含氨基酸序列PKKKRKV的NLS组成。EP可包含含有选自NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK和RKCLQAGMNLEARKTKK的氨基酸序列的NLS。EP可由包含选自NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK和RKCLQAGMNLEARKTKK的氨基酸序列的NLS组成The EP may comprise an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. The EP may consist of an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. The EP may comprise an NLS comprising an amino acid sequence PKKKRKV. The EP may consist of an NLS comprising an amino acid sequence PKKKRKV. The EP may comprise an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK. The EP may consist of an NLS comprising an amino acid sequence selected from the group consisting of NLSKRPAA IKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK
所有环外序列还可含有N-末端乙酰基基团。因此，例如，EP可具有以下结构：Ac-PKKKRKV。All exocyclic sequences may also contain an N-terminal acetyl group. Thus, for example, EP may have the following structure: Ac-PKKKRKV.
细胞穿透肽(CPP)Cell Penetrating Peptides (CPP)
细胞穿透肽(CPP)可包含6至20个氨基酸残基。细胞穿透肽可以是环状细胞穿透肽(cCPP)。cCPP能够穿透细胞膜。环外肽(EP)可与cCPP缀合，并且所得构建体可称为内体逃逸载体(EEV)。cCPP可引导AC穿透细胞膜。cCPP可将AC递送至细胞的胞质。cCPP可将AC递送至靶(例如，前mRNA)所在的细胞位置。为了将cCPP与AC缀合，可替换cCPP上的至少一个键或孤对电子。The cell penetrating peptide (CPP) may comprise 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). The cCPP is capable of penetrating the cell membrane. An exocyclic peptide (EP) may be conjugated to the cCPP, and the resulting construct may be referred to as an endosomal escape vector (EEV). The cCPP may guide the AC to penetrate the cell membrane. The cCPP may deliver the AC to the cytoplasm of the cell. The cCPP may deliver the AC to the cellular location where the target (e.g., pre-mRNA) is located. In order to conjugate the cCPP to the AC, at least one bond or lone pair of electrons on the cCPP may be replaced.
cCPP中氨基酸残基的总数在6至20个氨基酸残基的范围内，例如6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个氨基酸残基，包括其间的所有范围和子范围。cCPP可包含6至13个氨基酸残基。本文公开的cCPP可包含6至10个氨基酸。以举例的方式，包含6-10个氨基酸残基的cCPP可具有根据式I-A至I-E中任一种的结构：The total number of amino acid residues in the cCPP is in the range of 6 to 20 amino acid residues, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues, including all ranges and subranges therebetween. The cCPP may comprise 6 to 13 amino acid residues. The cCPP disclosed herein may comprise 6 to 10 amino acids. By way of example, a cCPP comprising 6-10 amino acid residues may have a structure according to any one of Formulas I-A to I-E:
其中AA1、AA2、AA3、AA4、AA5、AA6、AA7、AA8、AA9和AA10是氨基酸残基。 Among them,AA1 ,AA2 ,AA3 ,AA4 ,AA5 ,AA6 ,AA7 ,AA8 ,AA9 andAA10 are amino acid residues.
cCPP可包含6至8个氨基酸。cCPP可包含8个氨基酸。cCPP may comprise 6 to 8 amino acids. cCPP may comprise 8 amino acids.
cCPP中的每个氨基酸可以是天然的或非天然的氨基酸。术语“非天然氨基酸”是指一种有机化合物，它是天然氨基酸的同源物，因为它具有与天然氨基酸类似的结构，从而模拟天然氨基酸的结构和反应性。非天然氨基酸可以是修饰的氨基酸和/或氨基酸类似物，其不是20种常见天然存在的氨基酸中的一种，也不是稀有天然氨基酸硒代半胱氨酸或吡咯赖氨酸。非天然氨基酸也可以是天然氨基酸的D-异构体。合适的氨基酸的实例包括但不限于丙氨酸、别亮氨酸、精氨酸、瓜氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、萘基丙氨酸、苯丙氨酸、脯氨酸、焦谷氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、它们的衍生物或它们的组合。这些和其他氨基酸连同它们在本文中使用的缩写列于表1中。Each amino acid in cCPP can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a homologue of a natural amino acid because it has a structure similar to that of a natural amino acid, thereby simulating the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and/or amino acid analogs that are not one of the 20 common naturally occurring amino acids, nor are they rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives, or combinations thereof. These and other amino acids are listed in Table 1 together with their abbreviations used herein.
表1.氨基酸缩写Table 1. Amino acid abbreviations
*单字母缩写：当在本文中以大写字母示出时，其表示L-氨基酸形式，当在本文中以小写字母示出时，其表示D-氨基酸形式。*Single letter abbreviations: When shown herein with a capital letter, it refers to the L-amino acid form, and when shown herein with a lower case letter, it refers to the D-amino acid form.
cCPP可包含4至20个氨基酸，其中：(i)至少一个氨基酸具有包含胍基或其质子化形式的侧链；(ii)至少一个氨基酸不具有侧链或具有包含或其质子化形式的侧链；并且(iii)至少两个氨基酸独立地具有包含芳族或杂芳族基团的侧链。The cCPP may comprise 4 to 20 amino acids, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid has no side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acids independently have a side chain comprising an aromatic or heteroaromatic group.
至少两个氨基酸可不具有侧链或具有包含或其质子化形式的侧链。如本文所用，当不存在侧链时，氨基酸在连接胺和羧酸的碳原子上具有两个氢原子(例如，-CH2-)。At least two amino acids may have no side chains or have side chains comprising or a protonated form of its side chain. As used herein, when no side chain is present, an amino acid has two hydrogen atoms on the carbon atom connecting the amine and the carboxylic acid (eg,-CH2- ).
不具有侧链的氨基酸可以是甘氨酸或β-丙氨酸。The amino acid having no side chain may be glycine or β-alanine.
cCPP可包含形成cCPP的6至20个氨基酸残基，其中：(i)至少一个氨基酸可以是甘氨酸、β-丙氨酸或4-氨基丁酸残基；(ii)至少一个氨基酸可具有包含芳基或杂芳基基团的侧链；并且(iii、)至少一个氨基酸具有包含胍基、或其质子化形式的侧链。The cCPP may comprise 6 to 20 amino acid residues forming the cCPP, wherein: (i) at least one amino acid may be a glycine, β-alanine or 4-aminobutyric acid residue; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.
cCPP可包含形成cCPP的6至20个氨基酸残基，其中：(i)至少两个氨基酸可独立地是甘氨酸、β-丙氨酸或4-氨基丁酸残基；(ii)至少一个氨基酸可具有包含芳基或杂芳基基团的侧链；并且(iii)至少一个氨基酸具有包含胍基、或其质子化形式的侧链。The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least two amino acids may independently be glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.
cCPP可包含形成cCPP的6至20个氨基酸残基，其中：(i)至少三个氨基酸可独立地是甘氨酸、β-丙氨酸或4-氨基丁酸残基；(ii)至少一个氨基酸可具有包含芳族或杂芳族基团的侧链；并且(iii)至少一个氨基酸可具有包含胍基、或其质子化形式的侧链。The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least three amino acids may independently be glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aromatic or heteroaromatic group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.
甘氨酸和相关的氨基酸残基Glycine and related amino acid residues
cCPP可包含(i)1、2、3、4、5或6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)2个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3、4或5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3或4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP may contain (i) 1, 2, 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 2 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.
cCPP可包含(i)1、2、3、4、5或6个甘氨酸残基。cCPP可包含(i)2个甘氨酸残基。cCPP可包含(i)3个甘氨酸残基。cCPP可包含(i)4个甘氨酸残基。cCPP可包含(i)5个甘氨酸残基。cCPP可包含(i)6个甘氨酸残基。cCPP可包含(i)3、4或5个甘氨酸残基。cCPP可包含(i)3或4个甘氨酸残基。cCPP可包含(i)2或3个甘氨酸残基。cCPP可包含(i)1或2个甘氨酸残基。cCPP may comprise (i) 1, 2, 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 2 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues. cCPP may comprise (i) 2 or 3 glycine residues. cCPP may comprise (i) 1 or 2 glycine residues.
cCPP可包含(i)3、4、5或6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3、4或5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3或4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP may contain (i) 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.
cCPP可包含至少三个甘氨酸残基。cCPP可包含(i)3、4、5或6个甘氨酸残基。cCPP可包含(i)3个甘氨酸残基。cCPP可包含(i)4个甘氨酸残基。cCPP可包含(i)5个甘氨酸残基。cCPP可包含(i)6个甘氨酸残基。cCPP可包含(i)3、4或5个甘氨酸残基。cCPP可包含(i)3或4个甘氨酸残基cCPP may comprise at least three glycine residues. cCPP may comprise (i) 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues
在实施方案中，cCPP中的甘氨酸、β-丙氨酸或4-氨基丁酸残基都不是邻接的。两个或三个甘氨酸、β-丙氨酸或4-氨基丁酸残基可以是邻接的。两个甘氨酸、β-丙氨酸或4-氨基丁酸残基可以是邻接的。In an embodiment, none of the glycine, β-alanine or 4-aminobutyric acid residues in the cCPP are contiguous. Two or three glycine, β-alanine or 4-aminobutyric acid residues may be contiguous. Two glycine, β-alanine or 4-aminobutyric acid residues may be contiguous.
在实施方案中，cCPP中没有一个甘氨酸残基是邻接的。cCPP中的每个甘氨酸残基可被不能是甘氨酸的氨基酸残基分开。两个或三个甘氨酸残基可以是邻接的。两个甘氨酸残基可以是邻接的。In an embodiment, no glycine residues in a cCPP are contiguous. Each glycine residue in a cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be contiguous. Two glycine residues may be contiguous.
具有芳族或杂芳族基团的氨基酸侧链Amino acid side chains with aromatic or heteroaromatic groups
cCPP可包含(ii)2、3、4、5或6个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)3个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)4个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)5个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)6个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2、3或4个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2或3个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2, 3 or 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group.
cCPP可包含(ii)2、3、4、5或6个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)3个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)4个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)5个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)6个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2、3或4个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2或3个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP may contain (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 2 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 3 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 4 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 5 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 6 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 2, 3 or 4 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 2 or 3 amino acid residues independently having a side chain containing an aromatic group.
芳族基团可以是6至14元芳基。芳基可以是苯基、萘基或蒽基，它们各自任选地被取代。芳基可以是苯基或萘基，它们各自任选地被取代。杂芳族基团可以是具有1、2或3个选自N、O和S的杂原子的6至14元杂芳基。杂芳基可以是吡啶基、喹啉基或异喹啉基。The aromatic group may be a 6 to 14 membered aryl group. The aryl group may be a phenyl, naphthyl or anthracenyl group, each of which may be optionally substituted. The aryl group may be a phenyl or naphthyl group, each of which may be optionally substituted. The heteroaromatic group may be a 6 to 14 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S. The heteroaryl group may be a pyridyl, quinolyl or isoquinolyl group.
具有包含芳族或杂芳族基团的侧链的氨基酸残基可各自独立地是双(高萘基丙氨酸)、高萘基丙氨酸、萘基丙氨酸、苯基甘氨酸、双(高苯丙氨酸)、高苯丙氨酸、苯丙氨酸、色氨酸、3-(3-苯并噻吩基)-丙氨酸、3-(2-喹啉基)-丙氨酸、O-苄基丝氨酸、3-(4-(苄氧基)苯基)-丙氨酸、S-(4-甲基苄基)半胱氨酸、N-(萘-2-基)谷氨酰胺、3-(1，1’-联苯-4-基)-丙氨酸、3-(3-苯并噻吩基)-丙氨酸或酪氨酸，它们各自任选地被一个或多个取代基取代。具有包含芳族或杂芳族基团的侧链的氨基酸可各自独立地选自：The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be bis(homonaphthylalanine), homonaphthylalanine, naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalene-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, 3-(3-benzothienyl)-alanine or tyrosine, each of which is optionally substituted with one or more substituents. The amino acids having side chains containing aromatic or heteroaromatic groups may each independently be selected from:
其中N-末端的H和/或C-末端的H被肽键替换。 wherein the N-terminal H and/or the C-terminal H are replaced by a peptide bond.
具有包含芳族或杂芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸、萘基丙氨酸、苯基甘氨酸、高苯丙氨酸、高萘基丙氨酸、双(高苯丙氨酸)、双-(高萘基丙氨酸)、色氨酸或酪氨酸的残基，它们各自任选地被一个或多个取代基取代。具有包含芳族基团的侧链的氨基酸残基可各自独立地是酪氨酸、苯丙氨酸、1-萘基丙氨酸、2-萘基丙氨酸、色氨酸、3-苯并噻吩基丙氨酸、4-苯基苯丙氨酸、3，4-二氟苯丙氨酸、4-三氟甲基苯丙氨酸、2，3，4，5，6-五氟苯丙氨酸、高苯丙氨酸、β-高苯丙氨酸、4-叔丁基-苯丙氨酸、4-吡啶基丙氨酸、3-吡啶基丙氨酸、4-甲基苯丙氨酸、4-氟苯丙氨酸、4-氯苯丙氨酸、3-(9-蒽基)-丙氨酸的残基。具有包含芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸、萘基丙氨酸、苯基甘氨酸、高苯丙氨酸或高萘基丙氨酸的残基，它们各自任选地被一个或多个取代基取代。具有包含芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸、萘基丙氨酸、高苯丙氨酸、高萘基丙氨酸、双(高萘基丙氨酸)或双(高萘基丙氨酸)的残基，它们各自任选地被一个或多个取代基取代。具有包含芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸或萘基丙氨酸的残基，它们各自任选地被一个或多个取代基取代。至少一个具有包含芳族基团的侧链的氨基酸残基可以是苯丙氨酸的残基。至少两个具有包含芳族基团的侧链的氨基酸残基可以是苯丙氨酸的残基。每个具有包含芳族基团的侧链的氨基酸残基可以是苯丙氨酸的残基。The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, homophenylalanine, homonaphthylalanine, bis(homonaphthylalanine) or bis(homonaphthylalanine), each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine or naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine. At least two amino acid residues having a side chain containing an aromatic group may be a residue of phenylalanine. Each amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine.
在实施方案中，具有包含芳族或杂芳族基团的侧链的氨基酸没有一个是邻接的。两个具有包含芳族或杂芳族基团的侧链的氨基酸可以是邻接的。两个邻接的氨基酸可具有相反的立体化学。两个邻接的氨基酸可具有相同的立体化学。三个具有包含芳族或杂芳族基团的侧链的氨基酸可以是邻接的。三个邻接的氨基酸可具有相同的立体化学。三个邻接的氨基酸可具有交替的立体化学。In an embodiment, none of the amino acids having side chains comprising aromatic or heteroaromatic groups are adjacent. Two amino acids having side chains comprising aromatic or heteroaromatic groups can be adjacent. Two adjacent amino acids can have opposite stereochemistry. Two adjacent amino acids can have the same stereochemistry. Three amino acids having side chains comprising aromatic or heteroaromatic groups can be adjacent. Three adjacent amino acids can have the same stereochemistry. Three adjacent amino acids can have alternating stereochemistry.
包含芳族或杂芳族基团的氨基酸残基可以是L-氨基酸。包含芳族或杂芳族基团的氨基酸残基可以是D-氨基酸。包含芳族或杂芳族基团的氨基酸残基可以是D-氨基酸和L-氨基酸的混合物。The amino acid residue containing an aromatic or heteroaromatic group can be an L-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a D-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a mixture of a D-amino acid and an L-amino acid.
任选的取代基可以是任何不显著降低(例如超过50％)cCPP的胞质递送效率的原子或基团，例如与不具有所述取代基的其他相同序列相比。任选的取代基可以是疏水取代基或亲水取代基。任选的取代基可以是疏水取代基。取代基可增加疏水氨基酸的溶剂可及表面积(如本文所定义)。取代基可以是卤素、烷基、烯基、炔基、环烷基、环烯基、环炔基、杂环基、芳基、杂芳基、烷氧基、芳氧基、酰基、烷基氨基甲酰基、烷基羧酰胺基(alkylcarboxamidyl)、烷氧羰基、烷硫基或芳硫基。取代基可以是卤素。The optional substituent may be any atom or group that does not significantly reduce (e.g., more than 50%) the cytoplasmic delivery efficiency of the cCPP, for example, compared to an otherwise identical sequence without the substituent. The optional substituent may be a hydrophobic substituent or a hydrophilic substituent. The optional substituent may be a hydrophobic substituent. The substituent may increase the solvent accessible surface area (as defined herein) of the hydrophobic amino acid. The substituent may be a halogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, a cycloalkynyl, a heterocyclyl, an aryl, a heteroaryl, an alkoxy, an aryloxy, an acyl, an alkylcarbamoyl, an alkylcarboxamidyl, an alkoxycarbonyl, an alkylthio, or an arylthio. The substituent may be a halogen.
尽管不希望受理论束缚，但据信相对于具有较低疏水性值的氨基酸，有具有较高疏水性值的芳族或杂芳族基团的氨基酸(即具有包含芳族或杂芳族基团的侧链的氨基酸)可改善cCPP的胞质递送效率。每个疏水氨基酸可独立地具有大于甘氨酸的疏水性值。每个疏水氨基酸可独立地是疏水性值大于丙氨酸的疏水氨基酸。每个疏水氨基酸可独立地具有大于或等于苯丙氨酸的疏水性值。疏水性可使用本领域已知的疏水性标度来测量。表2列出了由Eisenberg和Weiss(Proc.Natl.Acad.Sci.U.S.A.1984；81(1)：140-144)、Engleman等人(Ann.Rev.ofBiophys.Biophys.Chem.1986；1986(15)：321-53)、Kyte和Doolittle(J.Mol.Biol.1982；157(1)：105-132)、Hoop和Woods(Proc.Natl.Acad.Sci.U.S.A.1981；78(6)：3824-3828)以及Janin(Nature.1979；277(5696)：491-492)报道的各种氨基酸的疏水性值，其各自的全部内容以引用方式并入本文。疏水性可使用Engleman等人报道的疏水性标度来测量。Although not wishing to be bound by theory, it is believed that amino acids with aromatic or heteroaromatic groups (i.e., amino acids with side chains comprising aromatic or heteroaromatic groups) having higher hydrophobicity values relative to amino acids with lower hydrophobicity values can improve the cytoplasmic delivery efficiency of cCPPs. Each hydrophobic amino acid can independently have a hydrophobicity value greater than glycine. Each hydrophobic amino acid can independently be a hydrophobic amino acid with a hydrophobicity value greater than alanine. Each hydrophobic amino acid can independently have a hydrophobicity value greater than or equal to phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 2 lists the hydrophobicity values of various amino acids reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S.A. 1984; 81(1): 140-144), Engleman et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986; 1986(15): 321-53), Kyte and Doolittle (J. Mol. Biol. 1982; 157(1): 105-132), Hoop and Woods (Proc. Natl. Acad. Sci. U.S.A. 1981; 78(6): 3824-3828), and Janin (Nature. 1979; 277(5696): 491-492), the entire contents of each of which are incorporated herein by reference. Hydrophobicity can be measured using the hydrophobicity scale reported by Engleman et al.
表2.氨基酸疏水性Table 2. Amino Acid Hydrophobicity
可选择芳族或杂芳族基团的大小以改善cCPP的胞质递送效率。尽管不希望受理论束缚，但据信与具有较小疏水氨基酸的其他相同序列相比，氨基酸侧链上的较大芳族或杂芳族基团可改善胞质递送效率。疏水氨基酸的大小可根据疏水氨基酸的分子量、疏水氨基酸的位阻效应、侧链的溶剂可及表面积(SASA)或它们的组合来测量。疏水氨基酸的大小可根据疏水氨基酸的分子量来测量，并且较大的疏水氨基酸具有分子量为至少约90g/mol、或至少约130g/mol、或至少约141g/mol的侧链。氨基酸的大小可根据疏水侧链的SASA来测量。疏水氨基酸可具有SASA大于或等于丙氨酸、或大于或等于甘氨酸的侧链。较大的疏水氨基酸可具有SASA大于丙氨酸或大于甘氨酸的侧链。疏水氨基酸可具有SASA大于或等于约哌啶-2-羧酸、大于或等于约色氨酸、大于或等于约苯丙氨酸、或大于或等于约萘基丙氨酸的芳族或杂芳族基团。第一疏水氨基酸(AAH1)可具有SASA为至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约或至少约的侧链。第二疏水氨基酸(AAH2)可具有SASA为至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约或至少约的侧链。AAH1和AAH2的侧链可具有至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约大于约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约大于约至少约至少约至少约至少约或至少约的组合SASA。AAH2可以是疏水氨基酸残基，其侧链的SASA小于或等于AAH1的疏水侧链的SASA。以举例的方式而非限制，与具有Phe-Arg基序的其他方面相同的cCPP相比，具有Nal-Arg基序的cCPP可表现出改善的胞质递送效率；与具有Nal-Phe-Arg基序的其他方面相同的cCPP相比，具有Phe-Nal-Arg基序的cCPP可表现出改善的胞质递送效率；并且与具有nal-Phe-Arg基序的其他方面相同的cCPP相比，phe-Nal-Arg基序可表现出改善的胞质递送效率。The size of the aromatic or heteroaromatic group can be selected to improve the cytoplasmic delivery efficiency of the cCPP. Although it is not desired to be bound by theory, it is believed that the larger aromatic or heteroaromatic group on the amino acid side chain can improve the cytoplasmic delivery efficiency compared to other identical sequences with smaller hydrophobic amino acids. The size of the hydrophobic amino acid can be measured according to the molecular weight of the hydrophobic amino acid, the steric effect of the hydrophobic amino acid, the solvent accessible surface area (SASA) of the side chain, or a combination thereof. The size of the hydrophobic amino acid can be measured according to the molecular weight of the hydrophobic amino acid, and the larger hydrophobic amino acid has a side chain with a molecular weight of at least about 90g/mol, or at least about 130g/mol, or at least about 141g/mol. The size of the amino acid can be measured according to the SASA of the hydrophobic side chain. The hydrophobic amino acid may have a side chain with a SASA greater than or equal to alanine, or greater than or equal to glycine. The larger hydrophobic amino acid may have a side chain with a SASA greater than alanine or greater than glycine. The hydrophobic amino acid may have an aromatic or heteroaromatic group with a SASA greater than or equal to about piperidine-2-carboxylic acid, greater than or equal to about tryptophan, greater than or equal to about phenylalanine, or greater than or equal to about naphthylalanine. The first hydrophobic amino acid (AAH1 ) may have a SASA of at least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about or at least about The second hydrophobic amino acid (AAH2 ) may have a SASA of at least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about or at least about The side chains of AAH1 and AAH2 may have at least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about Greater than about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about Greater than about At least about At least about At least about At least about or at least about AAH2 may be a hydrophobic amino acid residue having a side chain SASA less than or equal to the SASA of the hydrophobic side chain of AAH1 . By way of example and not limitation, a cCPP having a Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a Phe-Arg motif; a cCPP having a Phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a Nal-Phe-Arg motif; and a phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a nal-Phe-Arg motif.
如本文所用，“疏水表面积”或“SASA”是指氨基酸侧链的溶剂可及的表面积(报告为平方埃；)。SASA可使用由Shrake & Rupley(J Mol Biol.79(2)：351-71)开发的’滚球’算法来计算，其全文以引用方式并入本文用于所有目的。这种算法使用特定半径的溶剂“球体”来探测分子表面。球体的典型值是其近似于水分子的半径。As used herein, "hydrophobic surface area" or "SASA" refers to the solvent accessible surface area of amino acid side chains (reported as square angstroms; ). SASA can be calculated using the 'rolling ball' algorithm developed by Shrake & Rupley (J Mol Biol. 79(2): 351-71), which is incorporated herein by reference in its entirety for all purposes. This algorithm uses a solvent "sphere" of a specific radius to probe the molecular surface. Typical values for the sphere are This is approximately the radius of a water molecule.
某些侧链的SASA值示于下表3中。本文所述的SASA值基于下表3中列出的理论值，如Tien等人(PLOS ONE 8(11)：e80635.https：//doi.org/10.1371/journal.pone.0080635)所报道的，其全文以引用方式并入本文用于所有目的。The SASA values of certain side chains are shown in Table 3 below. The SASA values described herein are based on the theoretical values listed in Table 3 below, as reported by Tien et al. (PLOS ONE 8(11): e80635. https://doi.org/10.1371/journal.pone.0080635), which is incorporated herein by reference in its entirety for all purposes.
表3.氨基酸SASA值Table 3. SASA values of amino acids
具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基Amino acid residues having a side chain containing a guanidine group, a guanidine replacement group or a protonated form thereof
如本文所用，胍是指以下结构：As used herein, guanidine refers to the following structure:
如本文所用，质子化形式的胍是指以下结构：As used herein, the protonated form of guanidine refers to the following structure:
胍置换基团是指氨基酸侧链上的官能团，其在生理pH或高于生理pH时将带正电荷，或者可再现胍鎓(guanidinium)基团的氢键给予和接受活性。The guanidine replacement group refers to a functional group on the amino acid side chain that will be positively charged at or above physiological pH, or can reproduce the hydrogen bond donation and acceptance activity of the guanidinium group.
胍置换基团促进细胞渗透和治疗剂的递送，同时降低与胍基或其质子化形式相关的毒性。cCPP可包含至少一个具有包含胍或胍鎓置换基团的侧链的氨基酸。cCPP可包含至少两个具有包含胍或胍鎓置换基团的侧链的氨基酸。cCPP可包含至少三个具有包含胍或胍鎓置换基团的侧链的氨基酸The guanidine replacement group promotes cell penetration and delivery of therapeutic agents while reducing the toxicity associated with the guanidine group or its protonated form. The cCPP may include at least one amino acid having a side chain comprising a guanidine or guanidinium replacement group. The cCPP may include at least two amino acids having side chains comprising a guanidine or guanidinium replacement group. The cCPP may include at least three amino acids having side chains comprising a guanidine or guanidinium replacement group.
胍或胍鎓基团可以是胍或胍鎓的电子等排体。胍或胍鎓置换基团的碱性可低于胍。The guanidine or guanidinium group may be an isostere of guanidine or guanidinium. The guanidine or guanidinium displacement group may be less basic than guanidine.
如本文所用，胍置换基团是指或其质子化形式。As used herein, a guanidine replacement group refers to or its protonated form.
本公开涉及包含4至20个氨基酸残基的cCPP，其中：(i)至少一个氨基酸具有包含胍基或其质子化形式的侧链；(ii)至少一个氨基酸残基不具有侧链或具有包含或其质子化形式的侧链；并且(iii)至少两个氨基酸残基独立地具有包含芳族或杂芳族基团的侧链。The present disclosure relates to cCPPs comprising 4 to 20 amino acid residues, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid residue does not have a side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acid residues independently have a side chain comprising an aromatic or heteroaromatic group.
至少两个氨基酸残基可不具有侧链或具有包含或其质子化形式的侧链。如本文所用，当不存在侧链时，氨基酸残基在连接胺和羧酸的碳原子上具有两个氢原子(例如，-CH2-)。At least two amino acid residues may have no side chains or have or a protonated form of its side chain. As used herein, when no side chain is present, the amino acid residue has two hydrogen atoms on the carbon atom connecting the amine and the carboxylic acid (eg,-CH2- ).
cCPP可包含至少一个具有包含以下部分之一的侧链的氨基酸：或其质子化形式。The cCPP may comprise at least one amino acid having a side chain comprising one of the following moieties: or its protonated form.
cCPP可包含至少两个氨基酸，每个氨基酸独立地具有以下部分之一：或其质子化形式。至少两个氨基酸可具有包含选自以下的相同部分的侧链：或其质子化形式。至少一个氨基酸可具有包含或其质子化形式的侧链。至少两个氨基酸可具有包含或其质子化形式的侧链。一个、两个、三个或四个氨基酸可具有包含或其质子化形式的侧链。一个氨基酸可具有包含或其质子化形式的侧链。两个氨基酸可具有包含或其质子化形式的侧链。或其质子化形式可附接到氨基酸侧链的末端。可附接到氨基酸侧链的末端。The cCPP may comprise at least two amino acids, each amino acid independently having one of the following moieties: or a protonated form thereof. At least two amino acids may have a side chain comprising the same moiety selected from: or its protonated form. At least one amino acid may have or their protonated side chains. At least two amino acids may have or their protonated forms. One, two, three or four amino acids may have a or its protonated form. An amino acid may have a side chain comprising or their protonated side chains. The two amino acids may have or its protonated form. Or its protonated form may be attached to the terminus of the amino acid side chain. Can be attached to the terminal end of an amino acid side chain.
cCPP可包含(iii)2、3、4、5或6个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)3个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)4个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)5个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)6个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2、3、4或5个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2、3或4个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2或3个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)至少一个具有包含胍基或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)两个具有包含胍基或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)三个具有包含胍基或其质子化形式的侧链的氨基酸残基。The cCPP may comprise (iii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3, 4 or 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3 or 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) two amino acid residues having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) three amino acid residues having a side chain comprising a guanidine group or a protonated form thereof.
氨基酸残基可独立地具有包含不邻接的胍基、胍置换基团或其质子化形式的侧链。两个氨基酸残基可独立地具有包含可以是邻接的胍基、胍置换基团或其质子化形式的侧链。三个氨基酸残基可独立地具有包含可以是邻接的胍基、胍置换基团或其质子化形式的侧链。四个氨基酸残基可独立地具有包含可以是邻接的胍基、胍置换基团或其质子化形式的侧链。邻接的氨基酸残基可具有相同的立体化学。邻接的氨基酸可具有交替的立体化学。The amino acid residues may independently have side chains comprising non-adjacent guanidine groups, guanidine replacement groups or their protonated forms. Two amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Three amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Four amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Adjacent amino acid residues may have the same stereochemistry. Adjacent amino acids may have alternating stereochemistry.
独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基可以是L-氨基酸。独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基可以是D-氨基酸。独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基可以是L-氨基酸或D-氨基酸的混合物。The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group, or a protonated form thereof can be an L-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a D-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a mixture of L-amino acids or D-amino acids.
每个具有包含胍基或其质子化形式的侧链的氨基酸残基可独立地是精氨酸、高精氨酸、2-氨基-3-丙酸、2-氨基-4-胍基丁酸或其质子化形式的残基。每个具有包含胍基或其质子化形式的侧链的氨基酸残基可独立地是精氨酸残基或其质子化形式的残基。Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof. Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be a residue of arginine residue or a protonated form thereof.
每个具有包含胍置换基团或其质子化形式的侧链的氨基酸可独立地是或其质子化形式。Each amino acid having a side chain containing a guanidine replacement group or a protonated form thereof may independently be or its protonated form.
不受理论的束缚，假设胍置换基团相对于精氨酸具有降低的碱度，并且在一些情况下在生理pH下不带电荷(例如，-N(H)C(O))，并且能够维持与质膜上的磷脂的双齿氢键合相互作用，这被认为促进有效的膜结合和随后的内化。正电荷的去除也被认为降低了cCPP的毒性。Without being bound by theory, it is hypothesized that the guanidine replacement group has reduced basicity relative to arginine and, in some cases, is uncharged (e.g., -N(H)C(O)) at physiological pH and is able to maintain bidentate hydrogen bonding interactions with phospholipids on the plasma membrane, which is thought to promote efficient membrane binding and subsequent internalization. The removal of the positive charge is also thought to reduce the toxicity of cCPP.
本领域技术人员将理解，上述非天然芳族疏水氨基酸的N-末端/或C-末端在掺入本文公开的肽中后形成酰胺键。It will be understood by those skilled in the art that the N-terminus and/or C-terminus of the above-mentioned non-natural aromatic hydrophobic amino acids form an amide bond after incorporation into the peptides disclosed herein.
cCPP可包含具有包含芳族或杂芳族基团的侧链的第一氨基酸和具有包含芳族或杂芳族基团的侧链的第二氨基酸，其中第一甘氨酸的N-末端与具有包含芳族或杂芳族基团的侧链的第一氨基酸形成肽键，并且第一甘氨酸的C-末端与具有包含芳族或杂芳族基团的侧链的第二氨基酸形成肽键。尽管按照惯例，术语“第一氨基酸”通常是指肽序列的N-末端氨基酸，但如本文所用，“第一氨基酸”用于将所指氨基酸与cCPP中的另一氨基酸(例如，“第二氨基酸”)区分开，使得术语“第一氨基酸”可以是或可指位于肽序列的N-末端的氨基酸。The cCPP may comprise a first amino acid having a side chain comprising an aromatic or heteroaromatic group and a second amino acid having a side chain comprising an aromatic or heteroaromatic group, wherein the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain comprising an aromatic or heteroaromatic group. Although by convention, the term "first amino acid" generally refers to the N-terminal amino acid of a peptide sequence, as used herein, "first amino acid" is used to distinguish the referred amino acid from another amino acid (e.g., "second amino acid") in the cCPP, such that the term "first amino acid" may be or may refer to an amino acid located at the N-terminus of the peptide sequence.
cCPP可包含：第二甘氨酸的N-末端与具有包含芳族或杂芳族基团的侧链的氨基酸形成肽键，并且第二甘氨酸的C-末端与具有包含胍基或其质子化形式的侧链的氨基酸形成肽键。The cCPP may comprise: the N-terminus of the second glycine forming a peptide bond with an amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the second glycine forming a peptide bond with an amino acid having a side chain comprising a guanidino group or a protonated form thereof.
cCPP可包含具有包含胍基或其质子化形式的侧链的第一氨基酸，和具有包含胍基或其质子化形式的侧链的第二氨基酸，其中第三甘氨酸的N-末端与具有包含胍基或其质子化形式的侧链的第一氨基酸形成肽键，并且第三甘氨酸的C-末端与具有包含胍基或其质子化形式的侧链的第二氨基酸形成肽键。The cCPP may comprise a first amino acid having a side chain comprising a guanidine group or a protonated form thereof, and a second amino acid having a side chain comprising a guanidine group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidine group or a protonated form thereof, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain comprising a guanidine group or a protonated form thereof.
cCPP可包含天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸或高谷氨酰胺的残基。cCPP可包含天冬酰胺的残基。cCPP可包含谷氨酰胺的残基。The cCPP may comprise residues of asparagine, aspartic acid, glutamine, glutamic acid or homoglutamine. The cCPP may comprise residues of asparagine. The cCPP may comprise residues of glutamine.
cCPP可包含酪氨酸、苯丙氨酸、1-萘基丙氨酸、2-萘基丙氨酸、色氨酸、3-苯并噻吩基丙氨酸、4-苯基苯丙氨酸、3，4-二氟苯丙氨酸、4-三氟甲基苯丙氨酸、2，3，4，5，6-五氟苯丙氨酸、高苯丙氨酸、β-高苯丙氨酸、4-叔丁基-苯丙氨酸、4-吡啶基丙氨酸、3-吡啶基丙氨酸、4-甲基苯丙氨酸、4-氟苯丙氨酸、4-氯苯丙氨酸、3-(9-蒽基)-丙氨酸的残基。cCPP may comprise residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.
尽管不希望受理论束缚，但据信cCPP中氨基酸的手性可影响胞质摄取效率。cCPP可包含至少一个D氨基酸。cCPP可包含一至十五个D氨基酸。cCPP可包含一至十个D氨基酸。cCPP可包含1、2、3或4个D氨基酸。cCPP可包含具有交替的D和L手性的2、3、4、5、6、7或8个邻接的氨基酸。cCPP可包含具有相同手性的三个邻接的氨基酸。cCPP可包含具有相同手性的两个邻接的氨基酸。至少两个氨基酸可具有相反的手性。具有相反手性的至少两个氨基酸可彼此相邻。至少三个氨基酸可相对于彼此具有交替的立体化学。相对于彼此具有交替手性的至少三个氨基酸可彼此相邻。至少四个氨基酸相对于彼此具有交替的立体化学。相对于彼此具有交替手性的至少四个氨基酸可彼此相邻。至少两个氨基酸可具有相同的手性。具有相同手性的至少两个氨基酸可彼此相邻。至少两个氨基酸具有相同的手性并且至少两个氨基酸具有相反的手性。具有相反手性的至少两个氨基酸可与具有相同手性的至少两个氨基酸相邻。因此，cCPP中的相邻氨基酸可具有以下序列中的任一种：D-L；L-D；D-L-L-D；L-D-D-L；L-D-L-L-D；D-L-D-D-L；D-L-L-D-L；或L-D-D-L-D。形成cCPP的氨基酸残基都可以是L-氨基酸。形成cCPP的氨基酸残基都可以是D-氨基酸。Although not wishing to be bound by theory, it is believed that the chirality of the amino acids in the cCPP can affect the efficiency of cytoplasmic uptake. The cCPP may comprise at least one D amino acid. The cCPP may comprise one to fifteen D amino acids. The cCPP may comprise one to ten D amino acids. The cCPP may comprise 1, 2, 3 or 4 D amino acids. The cCPP may comprise 2, 3, 4, 5, 6, 7 or 8 adjacent amino acids with alternating D and L chirality. The cCPP may comprise three adjacent amino acids with the same chirality. The cCPP may comprise two adjacent amino acids with the same chirality. At least two amino acids may have opposite chirality. At least two amino acids with opposite chirality may be adjacent to each other. At least three amino acids may have alternating stereochemistry relative to each other. At least three amino acids with alternating chirality relative to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry relative to each other. At least four amino acids with alternating chirality relative to each other may be adjacent to each other. At least two amino acids may have the same chirality. At least two amino acids with the same chirality may be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Thus, the adjacent amino acids in a cCPP may have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D. The amino acid residues that form the cCPP may all be L-amino acids. The amino acid residues that form the cCPP may all be D-amino acids.
至少两个氨基酸可具有不同的手性。具有不同手性的至少两个氨基酸可彼此相邻。至少三个氨基酸可相对于相邻氨基酸具有不同的手性。至少四个氨基酸可相对于相邻氨基酸具有不同的手性。至少两个氨基酸具有相同的手性并且至少两个氨基酸具有不同的手性。形成cCPP的一个或多个氨基酸残基可以是非手性的。cCPP可包含3、4或5个氨基酸的基序，其中具有相同手性的两个氨基酸可被非手性氨基酸分开。cCPP可包含以下序列：D-X-D；D-X-D-X；D-X-D-X-D；L-X-L；L-X-L-X；或L-X-L-X-L，其中X是非手性氨基酸。非手性氨基酸可以是甘氨酸。At least two amino acids may have different chirality. At least two amino acids with different chirality may be adjacent to each other. At least three amino acids may have different chirality relative to adjacent amino acids. At least four amino acids may have different chirality relative to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chirality. One or more amino acid residues forming a cCPP may be achiral. The cCPP may comprise a motif of 3, 4 or 5 amino acids, wherein two amino acids with the same chirality may be separated by an achiral amino acid. The cCPP may comprise the following sequence: D-X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L, wherein X is an achiral amino acid. The achiral amino acid may be glycine.
具有包含以下的侧链的氨基酸：Amino acids having side chains comprising:
或其质子化形式，可与具有包含芳族或杂芳族基团的侧链的氨基酸相邻。具有包含以下的侧链的氨基酸：或其质子化形式，可与至少一个具有包含胍或其质子化形式的侧链的氨基酸相邻。具有包含胍或其质子化形式的侧链的氨基酸可与具有包含芳族或杂芳族基团的侧链的氨基酸相邻。两个具有包含以下的侧链的氨基酸：或其质子化形式，可彼此相邻。两个具有包含胍或其质子化形式的侧链的氨基酸彼此相邻。cCPP可包含至少两个具有可包含芳族或杂芳族基团的侧链的邻接氨基酸，和至少两个具有包含以下的侧链的不相邻氨基酸：或其质子化形式。cCPP可包含至少两个具有包含芳族或杂芳族基团的侧链的邻接氨基酸和至少两个具有包含或其质子化形式的侧链的不相邻氨基酸。相邻氨基酸可具有相同的手性。相邻氨基酸可具有相反的手性。氨基酸的其他组合可具有D和L氨基酸的任何排列，例如，在前述段落中描述的任何序列。or its protonated form, adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. An amino acid having a side chain comprising: or a protonated form thereof, may be adjacent to at least one amino acid having a side chain comprising guanidine or a protonated form thereof. An amino acid having a side chain comprising guanidine or a protonated form thereof may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Two amino acids having side chains comprising: or a protonated form thereof, may be adjacent to each other. Two amino acids having side chains comprising guanidine or a protonated form thereof are adjacent to each other. A cCPP may comprise at least two adjacent amino acids having side chains that may comprise an aromatic or heteroaromatic group, and at least two non-adjacent amino acids having side chains comprising: or a protonated form thereof. The cCPP may comprise at least two contiguous amino acids having side chains comprising an aromatic or heteroaromatic group and at least two amino acids having side chains comprising or non-adjacent amino acids with side chains in their protonated form. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, for example, any of the sequences described in the preceding paragraphs.
至少两个具有包含以下的侧链的氨基酸：At least two amino acids having side chains comprising:
或其质子化形式，与至少两个具有包含胍基或其质子化形式的侧链的氨基酸交替。or a protonated form thereof, alternating with at least two amino acids having a side chain comprising a guanidine group or a protonated form thereof.
cCPP可包含式(A)的结构：The cCPP may comprise a structure of formula (A):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3各自独立地是H或氨基酸的芳族或杂芳族侧链；R1 , R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4、R5、R6、R7独立地是H或氨基酸侧链；R4 , R5 , R6 , and R7 are independently H or an amino acid side chain;
R4、R5、R6、R7中的至少一者是3-胍基-2-氨基丙酸、4-胍基-2-氨基丁酸、精氨酸、高精氨酸、N-甲基精氨酸、N，N-二甲基精氨酸、2，3-二氨基丙酸、2，4-二氨基丁酸、赖氨酸、N-甲基赖氨酸、N，N-二甲基赖氨酸、N-乙基赖氨酸、N，N，N-三甲基赖氨酸、4-胍基苯丙氨酸、瓜氨酸、N，N-二甲基赖氨酸、β-高精氨酸、3-(1-哌啶基)丙氨酸的侧链；At least one of R4 , R5 , R6 , and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidinyl)alanine;
AASC是氨基酸侧链；并且AASC is an amino acid side chain; and
q是1、2、3或4。q is 1, 2, 3, or 4.
在实施方案中，R4、R5、R6、R7中的至少一者独立地是氨基酸的不带电荷的非芳族侧链。在实施方案中，R4、R5、R6、R7中的至少一者独立地是H或瓜氨酸的侧链。In an embodiment, at least one of R4 , R5 , R6 , R7 is independently an uncharged non-aromatic side chain of an amino acid. In an embodiment, at least one of R4 , R5 , R6 , R7 is independently H or a side chain of citrulline.
在实施方案中，提供了包含具有6至12个氨基酸的环肽的化合物，其中环肽的至少两个氨基酸是带电荷的氨基酸，环肽的至少两个氨基酸是芳族疏水氨基酸，并且环肽的至少两个氨基酸是不带电荷的非芳族氨基酸。在实施方案中，环肽的至少两个带电荷的氨基酸是精氨酸。在实施方案中，环肽的至少两个芳族疏水氨基酸是苯丙氨酸、萘丙氨酸(3-萘-2-基-丙氨酸)或它们的组合。在实施方案中，环肽的至少两个不带电荷的非芳族氨基酸是瓜氨酸、甘氨酸或它们的组合。在实施方案中，化合物是具有6至12个氨基酸的环肽，其中环肽的两个氨基酸是精氨酸，至少两个氨基酸是选自苯丙氨酸、萘丙氨酸和它们的组合的芳族疏水氨基酸，并且至少两个氨基酸是选自瓜氨酸、甘氨酸及它们的组合的不带电荷的非芳族氨基酸。In an embodiment, a compound comprising a cyclic peptide with 6 to 12 amino acids is provided, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids. In an embodiment, at least two charged amino acids of the cyclic peptide are arginine. In an embodiment, at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine (3-naphthyl-2-yl-alanine) or a combination thereof. In an embodiment, at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, glycine or a combination thereof. In an embodiment, the compound is a cyclic peptide with 6 to 12 amino acids, wherein two amino acids of the cyclic peptide are arginine, at least two amino acids are aromatic hydrophobic amino acids selected from phenylalanine, naphthylalanine and a combination thereof, and at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine and a combination thereof.
在实施方案中，式(A)的环肽不是具有以下序列的环肽：In an embodiment, the cyclic peptide of formula (A) is not a cyclic peptide having the sequence:
其中F是L-苯丙氨酸，f是D-苯丙氨酸，Φ是L-3-(2-萘基)-丙氨酸，Φ是D-3-(2-萘基)-丙氨酸，R是L-精氨酸，r是D-精氨酸，Q是L-谷氨酰胺，q是D-谷氨酰胺，C是L-半胱氨酸，U是L-硒代半胱氨酸，W是L-色氨酸，K是L-赖氨酸，D是L-天冬氨酸，并且Ω是L-正亮氨酸。wherein F is L-phenylalanine, f is D-phenylalanine, Φ is L-3-(2-naphthyl)-alanine, Φ is D-3-(2-naphthyl)-alanine, R is L-arginine, r is D-arginine, Q is L-glutamine, q is D-glutamine, C is L-cysteine, U is L-selenocysteine, W is L-tryptophan, K is L-lysine, D is L-aspartic acid, and Ω is L-norleucine.
cCPP可包含式(I)的结构：The cCPP may comprise the structure of formula (I):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；并且q is 1, 2, 3, or 4; and
每个m独立地是整数0、1、2或3。Each m is independently an integer of 0, 1, 2 or 3.
R1、R2和R3可各自独立地是H、-亚烷基-芳基或-亚烷基-杂芳基。R1、R2和R3可各自独立地是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R1、R2和R3可各自独立地是H或-亚烷基-芳基。R1、R2和R3可各自独立地是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R1、R2和R3可各自独立地是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R1、R2和R3可各自独立地是H、-CH2Ph或-CH2萘基。R1、R2和R3可各自独立地是H或-CH2Ph。R1 , R2 and R3 may each independently be H, -alkylene-aryl or -alkylene-heteroaryl. R1 , R2 and R3 may each independently be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R1 , R2 and R3 may each independently be H or -alkylene-aryl.R 1 , R2 and R3 may each independently be H or -C1-3 alkylene-aryl. C1-3 alkylene may be a methylene group. The aryl group may be a 6 to 14-membered aryl group. The heteroaryl group may be a 6 to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R1 , R2 and R3 may each independently be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R1 , R2 and R3 may each independently be H, -CH2 Ph or -CH2 naphthyl. R1 , R2 and R3 may each independently be H or -CH2 Ph.
R1、R2和R3可各自独立地是酪氨酸、苯丙氨酸、1-萘基丙氨酸、2-萘基丙氨酸、色氨酸、3-苯并噻吩基丙氨酸、4-苯基苯丙氨酸、3，4-二氟苯丙氨酸、4-三氟甲基苯丙氨酸、2，3，4，5，6-五氟苯丙氨酸、高苯丙氨酸、β-高苯丙氨酸、4-叔丁基-苯丙氨酸、4-吡啶基丙氨酸、3-吡啶基丙氨酸、4-甲基苯丙氨酸、4-氟苯丙氨酸、4-氯苯丙氨酸、3-(9-蒽基)-丙氨酸的侧链。R1 , R2 and R3 may each independently be a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 3-(9-anthryl)-alanine.
R1可以是酪氨酸的侧链。R1可以是苯丙氨酸的侧链。R1可以是1-萘基丙氨酸的侧链。R1可以是2-萘基丙氨酸的侧链。R1可以是色氨酸的侧链。R1可以是3-苯并噻吩基丙氨酸的侧链。R1可以是4-苯基苯丙氨酸的侧链。R1可以是3，4-二氟苯丙氨酸的侧链。R1可以是4-三氟甲基苯丙氨酸的侧链。R1可以是2，3，4，5，6-五氟苯丙氨酸的侧链。R1可以是高苯丙氨酸的侧链。R1可以是β-高苯丙氨酸的侧链。R1可以是4-叔丁基-苯丙氨酸的侧链。R1可以是4-吡啶基丙氨酸的侧链。R1可以是3-吡啶基丙氨酸的侧链。R1可以是4-甲基苯丙氨酸的侧链。R1可以是4-氟苯丙氨酸的侧链。R1可以是4-氯苯丙氨酸的侧链。R1可以是3-(9-蒽基)-丙氨酸的侧链。R1 may be a side chain of tyrosine.R1 may be a side chain of phenylalanine. R 1 may be a side chain of 1-naphthylalanine.R 1 may be a side chain of 2-naphthylalanine. R1 may be a side chain of tryptophan.R 1 may be a side chain of 3-benzothienylalanine. R1 may be a side chain of 4-phenylphenylalanine. R 1 may be a side chain of 3,4-difluorophenylalanine. R 1maybe a side chain of 4-trifluoromethylphenylalanine.R 1 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R1 may be a side chain of homophenylalanine. R1 may be a side chain of β-homophenylalanine.R 1 may be a side chain of 4-tert-butyl-phenylalanine.R 1 may be a side chain of 4-pyridylalanine.R 1 may be a side chain of 3-pyridylalanine.R 1 may be a side chain of 4-methylphenylalanine.R1 may be the side chain of 4-fluorophenylalanine.R1 may be the side chain of 4-chlorophenylalanine.R1 may be the side chain of 3-(9-anthryl)-alanine.
R2可以是酪氨酸的侧链。R2可以是苯丙氨酸的侧链。R2可以是1-萘基丙氨酸的侧链。R1可以是2-萘基丙氨酸的侧链。R2可以是色氨酸的侧链。R2可以是3-苯并噻吩基丙氨酸的侧链。R2可以是4-苯基苯丙氨酸的侧链。R2可以是3，4-二氟苯丙氨酸的侧链。R2可以是4-三氟甲基苯丙氨酸的侧链。R2可以是2，3，4，5，6-五氟苯丙氨酸的侧链。R2可以是高苯丙氨酸的侧链。R2可以是β-高苯丙氨酸的侧链。R2可以是4-叔丁基-苯丙氨酸的侧链。R2可以是4-吡啶基丙氨酸的侧链。R2可以是3-吡啶基丙氨酸的侧链。R2可以是4-甲基苯丙氨酸的侧链。R2可以是4-氟苯丙氨酸的侧链。R2可以是4-氯苯丙氨酸的侧链。R2可以是3-(9-蒽基)-丙氨酸的侧链。R2 may be a side chain of tyrosine.R2 may be a side chain of phenylalanine.R2 may be a side chain of 1-naphthylalanine.R1 may be a side chain of 2-naphthylalanine.R2 may be a side chain of tryptophan.R2 may be a side chain of 3-benzothienylalanine.R2 may be a side chain of 4-phenylphenylalanine.R2 may be a side chain of 3,4-difluorophenylalanine.R2 may be a side chain of 4-trifluoromethylphenylalanine.R2 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine.R2 may be a side chain of homophenylalanine.R2 may be a side chain of β-homophenylalanine.R2 may be a side chain of 4-tert-butyl-phenylalanine.R2 may be a side chain of 4-pyridylalanine.R2 may be a side chain of 3-pyridylalanine.R2 may be a side chain of 4-methylphenylalanine.R2 may be the side chain of 4-fluorophenylalanine.R2 may be the side chain of 4-chlorophenylalanine.R2 may be the side chain of 3-(9-anthryl)-alanine.
R3可以是酪氨酸的侧链。R3可以是苯丙氨酸的侧链。R3可以是1-萘基丙氨酸的侧链。R3可以是2-萘基丙氨酸的侧链。R3可以是色氨酸的侧链。R3可以是3-苯并噻吩基丙氨酸的侧链。R3可以是4-苯基苯丙氨酸的侧链。R3可以是3，4-二氟苯丙氨酸的侧链。R3可以是4-三氟甲基苯丙氨酸的侧链。R3可以是2，3，4，5，6-五氟苯丙氨酸的侧链。R3可以是高苯丙氨酸的侧链。R3可以是β-高苯丙氨酸的侧链。R3可以是4-叔丁基-苯丙氨酸的侧链。R3可以是4-吡啶基丙氨酸的侧链。R3可以是3-吡啶基丙氨酸的侧链。R3可以是4-甲基苯丙氨酸的侧链。R3可以是4-氟苯丙氨酸的侧链。R3可以是4-氯苯丙氨酸的侧链。R3可以是3-(9-蒽基)-丙氨酸的侧链。R3 may be a side chain of tyrosine.R 3 may be a side chain of phenylalanine.R 3 may be a side chain of 1-naphthylalanine. R3 may be a side chain of 2-naphthylalanine. R3 may be a side chain of tryptophan.R 3 may be a side chain of 3-benzothienylalanine. R3 may be a side chain of 4-phenylphenylalanine. R 3 may be a side chain of 3,4-difluorophenylalanine. R 3maybe a side chain of 4-trifluoromethylphenylalanine.R 3 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 may be a side chain of homophenylalanine. R3 may be a side chain of β-homophenylalanine.R 3 may be a side chain of 4-tert-butyl-phenylalanine.R 3 may be a side chain of 4-pyridylalanine.R 3 may be a side chain of 3-pyridylalanine.R 3 may be a side chain of 4-methylphenylalanine. R3 may be the side chain of 4-fluorophenylalanine. R3 may be the side chain of 4-chlorophenylalanine. R3 may be the side chain of 3-(9-anthryl)-alanine.
R4可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R4可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R4可以是H或-亚烷基-芳基。R4可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R4可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R4可以是H或表1或表3中氨基酸的侧链。R4可以是H或具有包含芳族基团的侧链的氨基酸残基。R4可以是H、-CH2Ph或-CH2萘基。R4可以是H或-CH2Ph。R4 may be H, -alkylene-aryl, -alkylene-heteroaryl. R4 may be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R4 may be H or -alkylene-aryl.R 4 may be H or -C1-3 alkylene-aryl. C1-3 alkylene may be methylene. Aryl may be a 6- to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S. Aryl may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl may be pyridyl, quinolyl and isoquinolyl.R 4 may be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R4 may be H or a side chain of an amino acid in Table 1 or Table 3. R4 may be H or an amino acid residue having a side chain containing an aromatic group.R4 may be H,-CH2Ph or-CH2naphthyl .R4 may be H or-CH2Ph .
R5可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R5可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R5可以是H或-亚烷基-芳基。R5可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R5可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R5可以是H或表1或表3中氨基酸的侧链。R4可以是H或具有包含芳族基团的侧链的氨基酸残基。R5可以是H、-CH2Ph或-CH2萘基。R4可以是H或-CH2Ph。R5 may be H, -alkylene-aryl, -alkylene-heteroaryl. R5 may be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R5 may be H or -alkylene-aryl. R5 may be H or -C1-3 alkylene-aryl. C1-3 alkylene may be a methylene group. The aryl group may be a 6- to 14-membered aryl group. The heteroaryl group may be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R5 may be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R5 may be H or a side chain of an amino acid in Table 1 or Table 3. R4 may be H or an amino acid residue having a side chain containing an aromatic group. R5 may be H, -CH2 Ph or -CH2 naphthyl. R4 may be H or -CH2 Ph.
R6可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R6可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R6可以是H或-亚烷基-芳基。R6可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R6可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R6可以是H或表1或表3中氨基酸的侧链。R6可以是H或具有包含芳族基团的侧链的氨基酸残基。R6可以是H、-CH2Ph或-CH2萘基。R6可以是H或-CH2Ph。R6 can be H, -alkylene-aryl, -alkylene-heteroaryl. R6 can be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R6 can be H or -alkylene-aryl.R 6 can be H or -C1-3 alkylene-aryl. C1-3 alkylene can be methylene. Aryl can be 6 to 14-membered aryl. Heteroaryl can be 6 to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S. Aryl can be selected from phenyl, naphthyl or anthracenyl. Aryl can be phenyl or naphthyl. Aryl can be phenyl. Heteroaryl can be pyridyl, quinolyl and isoquinolyl. R6 can be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R6 can be H or a side chain of an amino acid in Table 1 or Table 3. R6 can be H or an amino acid residue having a side chain containing an aromatic group.R6 may be H,-CH2Ph or-CH2naphthyl .R6 may be H or-CH2Ph .
R7可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R7可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R7可以是H或-亚烷基-芳基。R7可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R7可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R7可以是H或表1或表3中氨基酸的侧链。R7可以是H或具有包含芳族基团的侧链的氨基酸残基。R7可以是H、-CH2Ph或-CH2萘基。R7可以是H或-CH2Ph。R7 may be H, -alkylene-aryl, -alkylene-heteroaryl. R7 may be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R7 may be H or -alkylene-aryl. R7 may be H or -C1-3 alkylene-aryl. C1-3 alkylene may be methylene. Aryl may be a 6- to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S. Aryl may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl may be pyridyl, quinolyl and isoquinolyl.R 7 may be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R7 may be H or a side chain of an amino acid in Table 1 or Table 3. R7 may be H or an amino acid residue having a side chain containing an aromatic group.R7 may be H,-CH2Ph or-CH2naphthyl .R7 may be H or-CH2Ph .
R1、R2、R3、R4、R5、R6和R7中的一者、两者或三者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的一者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的两者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的三者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的至少一者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中不超过四者可以是-CH2Ph。One, two or three of R1 , R2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. One of R1 , R 2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. Two ofR 1 , R2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. Three of R1 , R2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. At least one of R1 , R2 , R3 , R4, R5 , R6 and R7 may be -CH2 Ph. No more than four ofR1 ,R2 ,R3 ,R4 ,R5 ,R6 andR7 may be-CH2Ph .
R1、R2、R3和R4中的一者、两者或三者是-CH2Ph。R1、R2、R3和R4中的一者是-CH2Ph。R1、R2、R3和R4中的两者是-CH2Ph。R1、R2、R3和R4中的三者是-CH2Ph。R1、R2、R3和R4中的至少一者是-CH2Ph。One, two or three of R1 , R2 , R3 and R4 are -CH2 Ph. One of R1 , R2 , R3 and R4 is -CH 2 Ph. Two of R 1 , R 2 , R 3 and R 4are -CH 2Ph.ThreeofR1 , R2 , R3 and R4 are -CH2 Ph. At least one ofR 1 , R2 , R3 and R4 is -CH2 Ph.
R1、R2、R3、R4、R5、R6和R7中的一者、两者或三者可以是H。R1、R2、R3、R4、R5、R6和R7中的一者可以是H。R1、R2、R3、R4、R5、R6和R7中的两者可以是H。R1、R2、R3、R4、R5、R6和R7中的三者可以是H。R1、R2、R3、R4、R5、R6和R7中的至少一者可以是H。R1、R2、R3、R4、R5、R6和R7中不超过三者可以是-CH2Ph。R1 , R2 , R3 , R4 , R5 , R6 , and R7 may be H. One, two, or three of R1 , R2 , R3 , R4 , R5 , R6 , and R7 may be H. Two ofR 1 , R2 , R 3, R4 , R5 , R6 , and R7 may be H. Three ofR 1 , R2 , R3 , R4 , R5 , R6 , and R7 may be H. At least one of R1 , R2 , R3 , R4 , R5 , R6 , and R7 may be H. No more than three of R1 , R2 , R3 , R4 , R5 , R6,and R7 may be -CH2 Ph.
R1、R2、R3和R4中的一者、两者或三者是H。R1、R2、R3和R4中的一者是H。R1、R2、R3和R4中的两者是H。R1、R2、R3和R4中的三者是H。R1、R2、R3和R4中的至少一者是H。One, two or three of R1 , R2 , R3 and R4 are H. One of R1 , R2 , R3 and R4 is H. Two of R1 , R2 , R3 and R4 are H. Three of R1 , R2 , R3 and R4 are H. At least one of R1 , R2 , R3 and R4 is H.
R4、R5、R6和R7中的至少一者可以是3-胍基-2-氨基丙酸的侧链。R4、R5、R6和R7中的至少一者可以是4-胍基-2-氨基丙酸的侧链。R4、R5、R6和R7中的至少一者可以是精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是高精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N-甲基精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N，N-二甲基精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是2，3-二氨基丙酸的侧链。R4、R5、R6和R7中的至少一者可以是2，4-二氨基丁酸、赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N-甲基赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N，N-二甲基赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N-乙基赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N，N，N-三甲基赖氨酸、4-胍基苯丙氨酸的侧链。R4、R5、R6和R7中的至少一者可以是瓜氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N，N-二甲基赖氨酸、β-高精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是3-(1-哌啶基)丙氨酸的侧链。At least one of R4 , R5 , R6 and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R4 , R5 , R6 and R7 may be a side chain of 4-guanidino-2-aminopropionic acid. At least one of R4 , R5 , R6 and R7 may be a side chain of arginine. At least one of R4 , R5 , R6 and R7 may be a side chain of homoarginine. At least one ofR 4 , R5 , R6 and R7 may be a side chain of N-methylarginine. At least one ofR 4 , R5 , R6 and R7 may be a side chain of N,N-dimethylarginine. At least one of R4 , R5 , R6 and R7 may be a side chain of 2,3-diaminopropionic acid. At least one of R4 , R5 , R6 and R7 may be a side chain of 2,4-diaminobutyric acid or lysine. At least one of R4 , R 5 , R6 and R7 may be a side chain of N-methyllysine. At least one ofR 4 , R5 , R6 and R7 may be a side chain of N,N-dimethyllysine. At least one of R4 , R5 , R6 and R7 may be a side chain of N-ethyllysine. At least one of R4 , R5 , R6 and R7 may be a side chain of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least one of R4 , R5, R6 and R7 may be a side chain of citrulline. At least one of R4 , R5 , R6 and R7 may be a side chain of N,N-dimethyllysine or β-homoarginine. At least one of R4 , R5 , R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.
R4、R5、R6和R7中的至少两者可以是3-胍基-2-氨基丙酸的侧链。R4、R5、R6和R7中的至少两者可以是4-胍基-2-氨基丁酸的侧链。R4、R5、R6和R7中的至少两者可以是精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是高精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N-甲基精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N，N-二甲基精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是2，3-二氨基丙酸的侧链。R4、R5、R6和R7中的至少两者可以是2，4-二氨基丁酸、赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N-甲基赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N，N-二甲基赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N-乙基赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N，N，N-三甲基赖氨酸、4-胍基苯丙氨酸的侧链。R4、R5、R6和R7中的至少两者可以是瓜氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N，N-二甲基赖氨酸、β-高精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是3-(1-哌啶基)丙氨酸的侧链。At least two of R4 , R5 , R6 and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least two of R4 , R5 , R 6 and R7 may be a side chain of 4-guanidino-2-aminobutyric acid. At least two ofR 4 , R5 , R6 and R 7 may be a side chain of arginine. At least two ofR 4 , R5 , R6 and R7 may be a side chain of homoarginine. At least two ofR 4 , R 5 , R6 and R7 may be a side chain of N-methylarginine. At least two ofR4 , R 5 , R 6 and R 7 may be a side chain of N,N-dimethylarginine. At least two of R 4,R5,R6 andR 7may be a side chain of 2,3- diaminopropionic acid. At least two of R4 , R5 , R6 and R7 may be side chains of 2,4-diaminobutyric acid or lysine. At least two of R4 , R 5 , R6 and R7 may be side chains of N-methyllysine. At least two of R4 , R 5 , R 6 and R 7 may be side chains of N,N-dimethyllysine. At least two of R 4,R5,R6 and R 7 may be side chains of N-ethyllysine. At least two of R 4 , R 5 , R 6and R 7maybesidechains of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least two ofR 4 , R 5 , R 6 and R 7 may be side chains of citrulline. At least two of R 4,R 5,R 6andR7may be side chains of N,N-dimethyllysine or β-homoarginine. At least two of R4 , R5 , R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.
R4、R5、R6和R7中的至少三者可以是3-胍基-2-氨基丙酸的侧链。R4、R5、R6和R7中的至少三者可以是4-胍基-2-氨基丁酸的侧链。R4、R5、R6和R7中的至少三者可以是精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是高精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N-甲基精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N，N-二甲基精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是2，3-二氨基丙酸的侧链。R4、R5、R6和R7中的至少三者可以是2，4-二氨基丁酸、赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N-甲基赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N，N-二甲基赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N-乙基赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N，N，N-三甲基赖氨酸、4-胍基苯丙氨酸的侧链。R4、R5、R6和R7中的至少三者可以是瓜氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N，N-二甲基赖氨酸、β-高精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是3-(1-哌啶基)丙氨酸的侧链。At least three of R4 , R5 , R6 and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least three of R4 , R5 , R6 and R7 may be a side chain of 4-guanidino-2-aminobutyric acid. At least three ofR 4 , R5 , R6 and R7 may be a side chain of arginine. At least three of R 4 , R5 , R6 and R7 may be a side chain of homoarginine. At least three ofR 4 , R5 , R6 and R 7 may be a side chain of N-methylarginine. At least three ofR 4 , R 5 , R 6 and R 7 may be a side chain of N,N-dimethylarginine. At least three of R 4,R 5, R 6andR7maybe a side chain of 2,3- diaminopropionic acid. At least three of R4 , R5 , R6 and R7 may be a side chain of 2,4-diaminobutyric acid or lysine. At least three of R4 , R5 , R6 and R 7 may be a side chain of N-methyllysine. At least three of R4 , R5 , R6 and R7 may be a side chain of N,N-dimethyllysine. At least three of R4 , R5 , R6 and R7 may be a side chain of N-ethyllysine. At least three of R 4 , R5 , R6 and R7 may be a side chain of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least three of R 4 , R 5 , R 6 and R 7 may be a side chain of citrulline. At least three of R 4, R 5 , R 6 and R 7maybeasidechainofN, N-dimethyllysine or β-homoarginine. At least three of R4 , R5 , R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.
AASC可以是天冬酰胺、谷氨酰胺或高谷氨酰胺残基的侧链。AASC可以是谷氨酰胺残基的侧链。cCPP还可包含与AASC(例如，天冬酰胺、谷氨酰胺或高谷氨酰胺的残基)缀合的接头。因此，cCPP还可包含与天冬酰胺、谷氨酰胺或高谷氨酰胺残基缀合的接头。cCPP还可包含与谷氨酰胺残基缀合的接头。AASC can be a side chain of an asparagine, glutamine or homoglutamine residue. AASC can be a side chain of a glutamine residue. cCPP can also comprise a linker conjugated to AASC (e.g., a residue of asparagine, glutamine or homoglutamine). Thus, cCPP can also comprise a linker conjugated to an asparagine, glutamine or homoglutamine residue. cCPP can also comprise a linker conjugated to a glutamine residue.
q可以是1、2或3。q可以是1或2。q可以是1。q可以是2。q可以是3。q可以是4。q can be 1, 2, or 3. q can be 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.
m可以是1-3。m可以是1或2。m可以是0，m可以是1。m可以是2。m可以是3。m can be 1-3. m can be 1 or 2. m can be 0, m can be 1. m can be 2. m can be 3.
式(A)的cCPP可包含式(I)的结构：或其质子化形式，其中AASC、R1、R2、R3、R4、R7、m和q如本文所定义The cCPP of formula (A) may comprise the structure of formula (I): or a protonated form thereof, wherein AASC , R1 , R2 , R3 , R4 , R7 , m and q are as defined herein
式(A)的cCPP可包含式(I-a)或式(I-b)的结构：The cCPP of formula (A) may comprise the structure of formula (I-a) or formula (I-b):
或其质子化形式，其中AASC、R1、R2、R3、R4和m如本文所定义。or a protonated form thereof, wherein AASC , R1 , R2 , R3 , R4 and m are as defined herein.
式(A)的cCPP可包含式(I-1)、(I-2)、(I-3)或(I-4)的结构：The cCPP of formula (A) may comprise the structure of formula (I-1), (I-2), (I-3) or (I-4):
或其质子化形式，其中AASC和m如本文所定义。or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可包含式(I-5)或(I-6)的结构：The cCPP of formula (A) may comprise a structure of formula (I-5) or (I-6):
或其质子化形式，其中AASC如本文所定义。 or a protonated form thereof, wherein AASC is as defined herein.
式(A)的cCPP可包含式(I-1)的结构：The cCPP of formula (A) may comprise the structure of formula (I-1):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可包含式(I-2)的结构：The cCPP of formula (A) may comprise the structure of formula (I-2):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可包含式(I-3)的结构：The cCPP of formula (A) may comprise the structure of formula (I-3):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可包含式(I-4)的结构：The cCPP of formula (A) may comprise the structure of formula (I-4):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可包含式(I-5)的结构：The cCPP of formula (A) may comprise a structure of formula (I-5):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可包含式(I-6)的结构：The cCPP of formula (A) may comprise the structure of formula (I-6):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
cCPP可包含以下序列之一：FGFGRGR；GfFGrGr；FfΦGRGR；FfFGRGR；或FfΦGrGr。cCPP可具有以下序列之一：FGFGRGRQ；GfFGrGrQ；FfΦGRGRQ；FfFGRGRQ；或FfΦGrGrQ。cCPP may comprise one of the following sequences: FGFGRGR; GfFGrGr; FfΦGRGR; FfFGRGR; or FfΦGrGr. cCPP may have one of the following sequences: FGFGRGRQ; GfFGrGrQ; FfΦGRGRQ; FfFGRGRQ; or FfΦGrGrQ.
本公开还涉及具有式(II)的结构的cCPP：The present disclosure also relates to a cCPP having the structure of formula (II):
其中：in:
AASC是氨基酸侧链；AASC is the amino acid side chain;
R1a、R1b和R1c各自独立地是6至14元芳基或6至14元杂芳基；R1a , R1b and R1c are each independently 6- to 14-membered aryl or 6- to 14-membered heteroaryl;
R2a、R2b、R2c和R2d独立地是氨基酸侧链；R2a , R2b , R2c and R2d are independently amino acid side chains;
R2a、R2b、R2c和R2d中的至少一者是或其质子化形式；At least one of R2a , R2b , R2c and R2d is or its protonated form;
R2a、R2b、R2c和R2d中的至少一者是胍或其质子化形式；At least one of R2a , R2b , R2c and R2d is guanidine or a protonated form thereof;
每个n”独立地是整数0、1、2、3、4或5；Each n" is independently an integer 0, 1, 2, 3, 4 or 5;
每个n’独立地是0、1、2或3的整数；并且Each n' is independently an integer of 0, 1, 2 or 3; and
如果n’是0，则R2a、R2b、R2b或R2d不存在。If n' is 0, then R2a , R2b , R2d or R2d is not present.
R2a、R2b、R2c和R2d中的至少两者可以是或其质子化形式。R2a、R2b、R2c和R2d中的两者或三者可以是或其质子化形式。R2a、R2b、R2c和R2d中的一者可以是或其质子化形式。R2a、R2b、R2c和R2d中的至少一者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余各者可以是胍或其质子化形式。R2a、R2b、R2c和R2d中的至少两者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余各者可以是胍或其质子化形式。At least two of R2a , R2b , R2c and R2d may be or a protonated form thereof. Two or three of R2a , R2b , R2c and R2d may be or a protonated form thereof. One of R2a , R2b , R2c and R2d may be or a protonated form thereof. At least one ofR 2a , R2b , R2c and R2d may be or a protonated form thereof, and the remaining R2a , R2b , R2c and R2d may each be guanidine or a protonated form thereof. At least two of R2a , R2b , R2c and R2d may be or a protonated form thereof, and the remaining each of R2a , R2b , R2c and R2d may be guanidine or a protonated form thereof.
所有R2a、R2b、R2c和R2d可以是或其质子化形式。R2a、R2b、R2c和R2d中的至少一者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余各者可以是胍或其质子化形式。R2a、R2b、R2c和R2d中的至少两者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余各者可以是胍或其质子化形式。All of R2a , R2b , R2c and R2d may be or a protonated form thereof. At least one ofR 2a , R2b , R2c and R2d may be or a protonated form thereof, and the remaining R2a , R2b , R2c and R2d may each be guanidine or a protonated form thereof. At least two of R2a , R2b , R2c and R2d may be or a protonated form thereof, and the remaining each of R2a , R2b , R2c and R2d may be guanidine or a protonated form thereof.
R2a、R2b、R2c和R2d中的每一者可独立地是2，3-二氨基丙酸、2，4-二氨基丁酸、以下酸的侧链：鸟氨酸、赖氨酸、甲基赖氨酸、二甲基赖氨酸、三甲基赖氨酸、高赖氨酸、丝氨酸、高丝氨酸、苏氨酸、别苏氨酸、组氨酸、1-甲基组氨酸、2-氨基丁酸、天冬氨酸、谷氨酸或高谷氨酸。Each of R2a , R2b , R2c and R2d can independently be 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, the side chain of ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homolysine, serine, homoserine, threonine, allothreonine, histidine, 1-methylhistidine, 2-aminobutyric acid, aspartic acid, glutamic acid or homoglutamic acid.
AASC可以是其中t可以是0至5的整数。AASC可以是其中t可以是0至5的整数。t可以是1至5。t是2或3。t可以是2。t可以是3。AASC can be Where t can be an integer from 0 to 5. AASC can be Wherein t may be an integer from 0 to 5. t may be from 1 to 5. t is 2 or 3. t may be 2. t may be 3.
本文所述的AC可偶联到AASC。在实施方案中，接头(L)将AC偶联到AASC。在实施方案中，接头(L)共价结合到AC的主链。AC described herein may be coupled to AASC . In an embodiment, a linker (L) couples AC to AASC . In an embodiment, a linker (L) is covalently bound to the backbone of AC.
AASC可以是天冬酰胺、谷氨酰胺或高谷氨酰胺残基的侧链。AASC可以是谷氨酰胺残基的侧链。环肽可包含与AASC(例如，天冬酰胺、谷氨酰胺或高谷氨酰胺的残基)缀合的接头。AASC can be a side chain of an asparagine, glutamine or homoglutamine residue. AASC can be a side chain of a glutamine residue. The cyclic peptide can comprise a linker conjugated to AASC (e.g., a residue of asparagine, glutamine or homoglutamine).
R1a、R1b和R1c可各自独立地是6至14元芳基。R1a、R1b和R1c可各自独立地是具有一个或多个选自N、O或S的杂原子的6至14元杂芳基。R1a、R1b和R1c可各自独立地选自苯基、萘基、蒽基、吡啶基、喹啉基或异喹啉基。R1a、R1b和R1c可各自独立地选自苯基、萘基或蒽基。R1a、R1b和R1c可各自独立地是苯基或萘基。R1a、R1b和R1c可以是各自独立选择的吡啶基、喹啉基或异喹啉基。R1a , R1b and R1c may each independently be a 6- to 14-membered aryl group. R1a , R1b and R1c may each independently be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O or S. R1a , R 1b and R 1c may each independently be selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl or isoquinolyl. R 1a, R 1bandR1c may each independently be selected from phenyl, naphthyl or anthracenyl. R 1a , R 1b and R 1cmay each independently be phenyl or naphthyl. R 1a , R 1bandR1cmayeachindependently be a pyridyl, quinolyl or isoquinolyl group selected from each independently.
每个n’可独立地是1或2。每个n’可以是1。每个n’可以是2。至少一个n’可以是0。至少一个n’可以是1。至少一个n’可以是2。至少一个n’可以是3。至少一个n’可以是4。至少一个n’可以是5。Each n' may independently be 1 or 2. Each n' may be 1. Each n' may be 2. At least one n' may be 0. At least one n' may be 1. At least one n' may be 2. At least one n' may be 3. At least one n' may be 4. At least one n' may be 5.
每个n”可独立地是1至3的整数。每个n”可独立地是2或3。每个n”可以是2。每个n”可以是3。至少一个n”可以是0。至少一个n”可以是1。至少一个n”可以是2。至少一个n”可以是3。Each n" may independently be an integer from 1 to 3. Each n" may independently be 2 or 3. Each n" may be 2. Each n" may be 3. At least one n" may be 0. At least one n" may be 1. At least one n" may be 2. At least one n" may be 3.
每个n”可独立地是1或2，并且每个n’可独立地是2或3。每个n”可以是1并且每个n’可独立地是2或3。每个n”可以是1并且每个n’可以是2。每个n”是1并且每个n’是3。Each n" may independently be 1 or 2, and each n' may independently be 2 or 3. Each n" may be 1 and each n' may independently be 2 or 3. Each n" may be 1 and each n' may be 2. Each n" is 1 and each n' is 3.
式(II)的cCPP可具有式(II-1)的结构：The cCPP of formula (II) may have the structure of formula (II-1):
其中R1a、R1b、R1c、R2a、R2b、R2c、R2d、AASC、n’和n”如本文所定义。wherein R1a ,R1b , R1c , R 2a , R2b , R2c , R2d , AASC , n′ and n″ are as defined herein.
式(II)的cCPP可具有式(IIa)的结构：The cCPP of formula (II) may have the structure of formula (IIa):
其中R1a、R1b、R1c、R2a、R2b、R2c、R2d、AASC和n’如本文所定义。wherein R1a ,R1b , R1c , R 2a , R2b , R2c , R2d , AASC and n′ are as defined herein.
式(II)的cCPP可具有式(IIb)的结构：The cCPP of formula (II) may have the structure of formula (IIb):
其中R2a、R2b、AASC和n’如本文所定义。wherein R2a , R2b , AASC and n′ are as defined herein.
cCPP可具有式(IIb)的结构：The cCPP may have the structure of formula (IIb):
或其质子化形式， or its protonated form,
其中：in:
AASC和n’如本文所定义。AA,SC and n' are as defined herein.
式(IIa)的cCPP具有以下结构之一：The cCPP of formula (IIa) has one of the following structures:
其中AASC和n如本文所定义。 wherein AA,SC and n are as defined herein.
式(IIa)的cCPP具有以下结构之一：The cCPP of formula (IIa) has one of the following structures:
其中AASC和n如本文所定义 Wherein AASC and n are as defined herein
式(IIa)的cCPP具有以下结构之一：The cCPP of formula (IIa) has one of the following structures:
其中AASC和n如本文所定义。 wherein AA,SC and n are as defined herein.
式(II)的cCPP可具有以下结构：The cCPP of formula (II) may have the following structure:
式(II)的cCPP可具有以下结构：The cCPP of formula (II) may have the following structure:
cCPP可具有式(III)的结构：The cCPP may have the structure of formula (III):
其中：in:
AASC是氨基酸侧链；AASC is the amino acid side chain;
R1a、R1b和R1c各自独立地是6至14元芳基或6至14元杂芳基；R1a , R1b and R1c are each independently 6- to 14-membered aryl or 6- to 14-membered heteroaryl;
R2a和R2c各自独立地是H、或其质子化形式；R2a and R2c are each independently H, or its protonated form;
R2b和R2d各自独立地是胍或其质子化形式；R2b and R2d are each independently guanidine or a protonated form thereof;
每个n”独立地是1至3的整数；Each n" is independently an integer from 1 to 3;
每个n’独立地是1至5的整数；并且Each n' is independently an integer from 1 to 5; and
每个p’独立地是0至5的整数。Each p' is independently an integer from 0 to 5.
本文所述的AC可偶联到AASC。接头可将AC偶联到AASC。接头可共价结合到AC的主链、AC的5’端或AC的3’端。AC described herein may be coupled to AASC . A linker may couple AC to AASC . A linker may be covalently bound to the backbone of AC, the 5' end of AC, or the 3' end of AC.
式(III)的cCPP可具有式(III-1)的结构：The cCPP of formula (III) may have the structure of formula (III-1):
其中：in:
AASC、R1a、R1b、R1c、R2a、R2c、R2b、R2d、n’、n”和p’如本文所定义。AASC , R1a , R1b , R1c , R2a , R2c , R2b , R2d , n′, n″ and p′ are as defined herein.
式(III)的cCPP可具有式(IIIa)的结构：The cCPP of formula (III) may have the structure of formula (IIIa):
其中：in:
AASC、R2a、R2c、R2b、R2d、n’、n”和p’如本文所定义。AASC , R2a , R2c , R2b , R2d , n′, n″ and p′ are as defined herein.
在式(III)、(III-1)和(IIIa)中，Ra和Rc可以是H。Ra和Rc可以是H并且Rb和Rd可各自独立地是胍或其质子化形式。Ra可以是H。Rb可以是H。p’可以是0。Ra和Rc可以是H并且每个p’可以是0。In formula (III), (III-1) and (IIIa),Ra andRc may be H.Ra andRc may be H andRb andRd may each independently be guanidine or a protonated form thereof.Ra may be H.Rb may be H. p' may be 0.Ra andRc may be H and each p' may be 0.
在式(III)、(III-1)和(IIIa)中，Ra和Rc可以是H，Rb和Rd可各自独立地是胍或其质子化形式，n”可以是2或3，并且每个p’可以是0。In formula (III), (III-1) and (IIIa),Ra andRc may be H,Rb andRd may each independently be guanidine or a protonated form thereof, n" may be 2 or 3, and each p' may be 0.
p’可以是0。p’可以是1。p’可以是2。p’可以是3。p’可以是4。p’可以是5p’ can be 0. p’ can be 1. p’ can be 2. p’ can be 3. p’ can be 4. p’ can be 5
cCPP可具有以下结构：cCPP can have the following structure:
式(A)的cCPP可选自：The cCPP of formula (A) may be selected from:
式(A)的cCPP可选自：The cCPP of formula (A) may be selected from:
在实施方案中，cCPP选自：In an embodiment, the cCPP is selected from:
Ф＝L-萘基丙氨酸；Ф＝D-萘基丙氨酸；Ω＝L-正亮氨酸Ф＝L-naphthylalanine; Ф＝D-naphthylalanine; Ω＝L-norleucine
在实施方案中，cCPP并不选自：In an embodiment, the cCPP is not selected from:
Ф＝L-萘基丙氨酸；φ＝D-萘基丙氨酸；Ω＝L-正亮氨酸Φ＝L-naphthylalanine; φ＝D-naphthylalanine; Ω＝L-norleucine
cCPP可包含式(D)的结构：The cCPP may comprise a structure of formula (D):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
Y是Y is
q是1、2、3或4；q is 1, 2, 3, or 4;
每个m独立地是整数0、1、2或3，并且Each m is independently an integer 0, 1, 2 or 3, and
每个n独立地是整数0、1、2或3。Each n is independently an integer of 0, 1, 2 or 3.
式(D)的cCPP可具有式(D-I)的结构：The cCPP of formula (D) may have the structure of formula (D-I):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；q is 1, 2, 3, or 4;
每个m独立地是整数0、1、2或3，并且Each m is independently an integer 0, 1, 2 or 3, and
Y是Y is
式(D)的cCPP可具有式(D-II)的结构：The cCPP of formula (D) may have the structure of formula (D-II):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；q is 1, 2, 3, or 4;
每个m独立地是整数0、1、2或3，Each m is independently an integer 0, 1, 2 or 3,
每个n独立地是整数0、1、2或3，并且Each n is independently an integer 0, 1, 2 or 3, and
Y是Y is
式(D)的cCPP可具有式(D-III)的结构：The cCPP of formula (D) may have the structure of formula (D-III):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；q is 1, 2, 3, or 4;
每个m独立地是整数0、1、2或3，Each m is independently an integer 0, 1, 2 or 3,
每个n独立地是整数0、1、2或3，并且Each n is independently an integer 0, 1, 2 or 3, and
Y是Y is
式(D)的cCPP可具有式(D-IV)的结构：The cCPP of formula (D) may have the structure of formula (D-IV):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；q is 1, 2, 3, or 4;
每个m独立地是整数0、1、2或3，并且Each m is independently an integer 0, 1, 2 or 3, and
Y是Y is
式(D)的cCPP可具有式(D-V)的结构：The cCPP of formula (D) may have the structure of formula (D-V):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；q is 1, 2, 3, or 4;
每个m独立地是整数0、1、2或3，并且Each m is independently an integer 0, 1, 2 or 3, and
Y是Y is
AASC可与接头缀合。AASC can be conjugated to a linker.
接头Connectors
本公开的cCPP可与接头缀合。接头可将AC连接到cCPP。接头可附接到cCPP的氨基酸的侧链，并且AC可附接在接头上的合适位置处。The cCPP of the present disclosure can be conjugated with a linker. The linker can connect the AC to the cCPP. The linker can be attached to the side chain of the amino acid of the cCPP, and the AC can be attached at a suitable position on the linker.
接头可以是任何合适的部分，其可将cCPP与一个或多个附加部分(例如环外肽(EP)和/或AC)缀合。在与cCPP和一个或多个附加部分缀合之前，接头具有两个或更多个官能团，它们各自能够独立地与cCPP和一个或多个附加部分形成共价键。接头可共价结合到AC的5’端或AC的3’端。接头可共价结合到AC的5’端。接头可共价结合到AC的3’端。接头可以是将本文所述的cCPP与AC缀合的任何合适的部分。The linker can be any suitable moiety that can conjugate the cCPP to one or more additional moieties (e.g., an exocyclic peptide (EP) and/or AC). Prior to conjugation to the cCPP and the one or more additional moieties, the linker has two or more functional groups, each of which is independently capable of forming a covalent bond with the cCPP and the one or more additional moieties. The linker can be covalently bound to the 5' end of the AC or the 3' end of the AC. The linker can be covalently bound to the 5' end of the AC. The linker can be covalently bound to the 3' end of the AC. The linker can be any suitable moiety that conjugates the cCPP described herein to the AC.
接头可包括烃接头。The joint may comprise a hydrocarbon joint.
接头可包含切割位点。切割位点可以是二硫化物或胱天蛋白酶切割位点(例如，Val-Cit-PABC)。The linker may comprise a cleavage site. The cleavage site may be a disulfide or a caspase cleavage site (e.g., Val-Cit-PABC).
接头可包含：(i)一个或多个D或L氨基酸，它们各自任选地被取代；(ii)任选取代的亚烷基；(iii)任选取代的亚烯基；(iv)任选取代的亚炔基；(v)任选取代的碳环基；(vi)任选取代的杂环基；(vii)一个或多个-(R1-J-R2)z”-亚基，其中R1和R2在每种情况下各自独立地选自亚烷基、亚烯基、亚炔基、碳环基和杂环基，每个J独立地是C、NR3、-NR3C(O)-、S和O，其中R3独立地选自H、烷基、烯基、炔基、碳环基和杂环基，它们各自任选地被取代，并且z”是1至50的整数；(viii)-(R1-J)z”-或-(J-R1)z”-，其中R1在每种情况下各自独立地是亚烷基、亚烯基、亚炔基、碳环基或杂环基，每个J独立地是C、NR3、-NR3C(O)-、S或O，其中R3是H、烷基、烯基、炔基、碳环基或杂环基，它们各自任选地被取代，并且z”是1至50的整数；或(ix)接头可包含(i)至(x)中的一者或多者。The linker may comprise: (i) one or more D or L amino acids, each of which is optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) one or more -(R1- JR2 )z"-subunits, wherein R1 and R2 are each independently selected from alkylene, alkenylene, alkynylene, carbocyclyl and heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S and O, wherein R3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; (viii) -(R1- J)z"- or -(JR1 )z"-, wherein R1 is independently alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl in each case, each J is independently C, NR3 , -NR3 C(O)-, S or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; or (ix) the linker may comprise one or more of (i) to (x).
接头可包含一个或多个D或L氨基酸和/或-(R1-J-R2)z”-，其中R1和R2在每种情况下各自独立地是亚烷基，每个J独立地是C、NR3、-NR3C(O)-、S和O，其中R4独立地选自H和烷基，并且z”是1至50的整数；或它们的组合。The linker may comprise one or more D or L amino acids and/or -(R1- JR2 )z"-, wherein R1 and R2 are each independently alkylene, each J is independently C, NR3 , -NR3 C(O)-, S and O, wherein R4 is independently selected from H and alkyl, and z" is an integer from 1 to 50; or a combination thereof.
接头可包含-(OCH2CH2)z·-(例如，作为间隔区)，其中z’是1至23的整数，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23。“-(OCH2CH2)z”’也可称为聚乙二醇(PEG)。The linker may comprise -(OCH2CH2 )z- (e.g., as a spacer), wherein z' is an integerfrom 1 to 23, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. "-(OCH2CH2 )z"' may also be referred to as polyethylene glycol (PEG).
接头可包含一个或多个氨基酸。接头可包含肽。接头可包含-(OCH2CH2)z·-和肽，其中z’是1至23的整数。肽可包含2至10个氨基酸。接头还可包含能够通过点击化学反应的官能团(FG)。FG可以是叠氮化物或炔，并且当AC与接头缀合时形成三唑。The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise -(OCH2 CH2 )z ·- and a peptide, wherein z' is an integer from 1 to 23. The peptide may comprise 2 to 10 amino acids. The linker may also comprise a functional group (FG) capable of reacting by click chemistry. FG may be an azide or an alkyne, and a triazole is formed when AC is conjugated to the linker.
接头可包含(i)β丙氨酸残基和赖氨酸残基；(ii)-(J-R1)z”；或(iii)它们的组合。每个R1可独立地是亚烷基、亚烯基、亚炔基、碳环基或杂环基，每个J独立地是C、NR3、-NR3C(O)-、S或O，其中R3是H、烷基、烯基、炔基、碳环基或杂环基，它们各自任选地被取代，并且z”可以是1至50的整数。每个R1可以是亚烷基并且每个J可以是O。The linker may comprise (i) a β-alanine residue and a lysine residue; (ii) -(JR1 )z"; or (iii) a combination thereof. Each R1 may independently be an alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R1 may be an alkylene and each J may be O.
接头可包含(i)β-丙氨酸、甘氨酸、赖氨酸、4-氨基丁酸、5-氨基戊酸、6-氨基己酸或它们的组合的残基；和(ii)-(R1-J)z”-或-(J-R1)z”。每个R1可独立地是亚烷基、亚烯基、亚炔基、碳环基或杂环基，每个J独立地是C、NR3、-NR3C(O)-、S或O，其中R3是H、烷基、烯基、炔基、碳环基或杂环基，它们各自任选地被取代，并且z”可以是1至50的整数。每个R1可以是亚烷基并且每个J可以是O。接头可包含甘氨酸、β-丙氨酸、4-氨基丁酸、5-氨基戊酸、6-氨基己酸或它们的组合。The linker may comprise (i) the residue of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or a combination thereof; and (ii) -(R1- J)z"- or -(JR1 )z". Each R1 may independently be an alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S, or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R1 may be an alkylene and each J may be O. The linker may comprise glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or a combination thereof.
接头可以是三价接头。接头可具有以下结构：其中A1、B1和C1可独立地是烃接头(例如，NRH-(CH2)n-COOH)、PEG接头(例如，NRH-(CH2O)n-COOH，其中R是H、甲基或乙基)或一个或多个氨基酸残基，并且Z独立地是保护基团。接头还可掺入切割位点，包括二硫化物[NH2-(CH2O)n-S-S-(CH2O)n-COOH]或胱天蛋白酶切割位点(Val-Cit-PABC)。The linker can be a trivalent linker. The linker can have the following structure: WhereinA1 ,B1 andC1 can independently be a hydrocarbon linker (e.g., NRH-(CH2 )n -COOH), a PEG linker (e.g., NRH-(CH2O )n -COOH, wherein R is H, methyl or ethyl) or one or more amino acid residues, and Z is independently a protecting group. The linker can also incorporate a cleavage site, including a disulfide [NH2- (CH2O )n -SS-(CH2O )n -COOH] or a caspase cleavage site (Val-Cit-PABC).
烃可以是甘氨酸或β-丙氨酸的残基。The hydrocarbon may be the residue of glycine or beta-alanine.
接头可以是二价的并且将cCPP连接到AC。接头可以是二价的并且将cCPP连接到环外肽(EP)。The linker may be bivalent and connects the cCPP to the AC.The linker may be bivalent and connects the cCPP to the exocyclic peptide (EP).
接头可以是三价的并且将cCPP连接到AC和EP。The linker may be trivalent and connects the cCPP to the AC and EP.
接头可以是二价或三价C1-C50亚烷基，其中1-25个亚甲基基团任选地且独立地被-N(H)-、-N(C1-C4烷基)-、-N(环烷基)-、-O-、-C(O)-、-C(O)O-、-S-、-S(O)-、-S(O)2-、-S(O)2N(C1-C4烷基)-、-S(O)2N(环烷基)-、-N(H)C(O)-、-N(C1-C4烷基)C(O)-、-N(环烷基)C(O)-、-C(O)N(H)-、-C(O)N(C1-C4烷基)、-C(O)N(环烷基)、芳基、杂环基、杂芳基、环烷基或环烯基替换。接头可以是二价或三价C1-C50亚烷基，其中1-25个亚甲基基团任选地且独立地被-N(H)-、-O-、-C(O)N(H)-或它们的组合替换。The linker can be a divalent or trivalent C1 -C50 alkylene group in which 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1 -C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2 -, -S(O)2 N(C1 -C4 alkyl)-, -S(O)2 N(cycloalkyl)-, -N(H)C(O)-, -N(C1 -C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1 -C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl or cycloalkenyl. The linker may be a divalent or trivalent C1 -C50 alkylene group, wherein 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.
AC可与环肽的谷氨酸偶联，这将谷氨酸转化为谷氨酰胺。接头(L)可将AC与环肽的谷氨酰胺/谷氨酸偶联。在实施方案中，接头(L)共价结合到AC的主链。AC can be coupled to the glutamic acid of the cyclic peptide, which converts the glutamic acid to glutamine. The linker (L) can couple AC to the glutamine/glutamic acid of the cyclic peptide. In an embodiment, the linker (L) is covalently bound to the backbone of AC.
接头可具有以下结构：The joint can have the following structures:
其中：每个AA独立地是氨基酸残基；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链；x是1-10的整数；y是1-5的整数；并且z是1-10的整数。x可以是1-5的整数。x可以是1-3的整数。x可以是1。y可以是2-4的整数。y可以是4。z可以是1-5的整数。z可以是1-3的整数。z可以是1。每个AA可独立地选自甘氨酸、β-丙氨酸、4-氨基丁酸、5-氨基戊酸和6-氨基己酸。 wherein: each AA is independently an amino acid residue; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of a cCPP; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10. x may be an integer from 1 to 5. x may be an integer from 1 to 3. x may be 1. y may be an integer from 2 to 4. y may be 4. z may be an integer from 1 to 5. z may be an integer from 1 to 3. z may be 1. Each AA may be independently selected from glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, and 6-aminohexanoic acid.
cCPP可通过接头(“L”)附接到AC。接头可通过键合基团(“M”)与AC缀合。The cCPP can be attached to the AC via a linker ("L"). The linker can be conjugated to the AC via a bonding group ("M").
接头可具有以下结构：The joint can have the following structures:
其中：x是1-10的整数；y是1-5的整数；z是1-10的整数；每个AA独立地是氨基酸残基；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链；并且M是本文所定义的键合基团。 wherein: x is an integer from 1 to 10; y is an integer from 1 to 5; z is an integer from 1 to 10; each AA is independently an amino acid residue; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of cCPP; and M is a bonding group as defined herein.
接头可具有以下结构：The joint can have the following structures:
其中：x’是1-23的整数；y是1-5的整数；z’是1-23的整数；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链；并且M是本文所定义的键合基团。wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of cCPP; and M is a bonding group as defined herein.
接头可具有以下结构：The joint can have the following structures:
其中：x’是1-23的整数；y是1-5的整数；并且z’是1-23的整数；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链。wherein: x' is an integer from 1-23; y is an integer from 1-5; and z' is an integer from 1-23; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of cCPP.
x可以是1-10的整数，例如1、2、3、4、5、6、7、8、9或10，包括其间的所有范围和子范围。x can be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including all ranges and subranges therebetween.
x’可以是1-23的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23，包括其间的所有范围和子范围。x’可以是5-15的整数。x’可以是9-13的整数。x’可以是1-5的整数。x’可以是1。x' may be an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. x' may be an integer from 5 to 15. x' may be an integer from 9 to 13. x' may be an integer from 1 to 5. x' may be 1.
y可以是1-5的整数，例如1、2、3、4或5，包括其间的所有范围和子范围。y可以是2-5的整数。y可以是3-5的整数。y可以是3或4。y可以是4或5。y可以是3。y可以是4。y可以是5。y may be an integer from 1 to 5, such as 1, 2, 3, 4, or 5, including all ranges and subranges therebetween. y may be an integer from 2 to 5. y may be an integer from 3 to 5. y may be 3 or 4. y may be 4 or 5. y may be 3. y may be 4. y may be 5.
z可以是1-10的整数，例如1、2、3、4、5、6、7、8、9或10，包括其间的所有范围和子范围。z can be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including all ranges and subranges therebetween.
z’可以是1-23的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23，包括其间的所有范围和子范围。z’可以是5-15的整数。z’可以是9-13的整数。z’可以是11。z' may be an integer from 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. z' may be an integer from 5 to 15. z' may be an integer from 9 to 13. z' may be 11.
如上所讨论，接头或M(其中M是接头的一部分)可共价结合到AC(AC上的任何合适的位置)。接头或M(其中M是接头的一部分)可共价结合到AC的3’端或AC的5’端。接头或M(其中M是接头的一部分)可共价结合到AC的主链。As discussed above, a linker or M (wherein M is part of a linker) can be covalently bound to AC (any suitable position on AC). A linker or M (wherein M is part of a linker) can be covalently bound to the 3' end of AC or the 5' end of AC. A linker or M (wherein M is part of a linker) can be covalently bound to the backbone of AC.
接头可结合到cCPP上的天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺或赖氨酸的侧链，或者谷氨酰胺或天冬酰胺的修饰侧链(例如，具有氨基基团的还原侧链)。接头可结合到cCPP上的赖氨酸的侧链。The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be attached to the side chain of lysine on the cCPP.
接头可具有以下结构：The joint can have the following structures:
其中in
M是将L与AC缀合的基团；M is a group that conjugates L to AC;
AAs是cCPP上的氨基酸的侧链或末端；AAs are the side chains or termini of the amino acids on the cCPP;
每个AAx独立地是氨基酸残基；Each AAx is independently an amino acid residue;
o是0至10的整数；并且o is an integer from 0 to 10; and
p是0至5的整数。p is an integer from 0 to 5.
接头可具有以下结构：The joint can have the following structures:
其中in
M是将L与AC缀合的基团；M is a group that conjugates L to AC;
AAs是cCPP上的氨基酸的侧链或末端；AAs are the side chains or termini of the amino acids on the cCPP;
每个AAx独立地是氨基酸残基；Each AAx is independently an amino acid residue;
o是0至10的整数；并且o is an integer from 0 to 10; and
p是0至5的整数。p is an integer from 0 to 5.
M可包括亚烷基、亚烯基、亚炔基、碳环基或杂环基，它们各自任选地被取代。M可选自：M may include alkylene, alkenylene, alkynylene, carbocyclic or heterocyclic groups, each of which is optionally substituted. M may be selected from:
其中R是烷基、烯基、炔基、碳环基或杂环基。 wherein R is an alkyl, alkenyl, alkynyl, carbocyclic or heterocyclic group.
M可选自：M can be selected from:
其中：R10是亚烷基、环烷基或其中a是0至10。Wherein: R10 is alkylene, cycloalkyl or Where a is 0 to 10.
M可以是R10可以是并且a是0至10。M可以是M can be R10 can be and a is 0 to 10. M can be
M可以是异双官能交联剂，例如其公开于Williams等人Curr.Protoc Nucleic Acid Chem.2010，42，4.41.1-4.41.20中，所述文献的全文以引用方式并入本文。M may be a heterobifunctional cross-linker, such as It is disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-4.41.20, which is incorporated herein by reference in its entirety.
M可以是-C(O)-。M may be -C(O)-.
AAs可以是cCPP上的氨基酸的侧链或末端。AAs的非限制性实例包括天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺或赖氨酸，或谷氨酰胺或天冬酰胺的修饰侧链(例如，具有氨基基团的还原侧链)。AAs可以是如本文所定义的AASC。AAs can be side chains or ends of amino acids on cCPP. Non-limiting examples ofAAs include aspartic acid, glutamic acid, glutamine, asparagine or lysine, or modified side chains of glutamine or asparagine (e.g., reduced side chains with amino groups).AAs can beAAS as defined herein.
每个AAX独立地是天然或非天然氨基酸。一个或多个AAX可以是天然氨基酸。一个或多个AAX可以是非天然氨基酸。一个或多个AAX可以是β-氨基酸。β-氨基酸可以是β-丙氨酸。EachAAX is independently a natural or unnatural amino acid. One or moreAAX can be a natural amino acid. One or moreAAX can be an unnatural amino acid. One or moreAAX can be a β-amino acid. The β-amino acid can be β-alanine.
o可以是0至10的整数，例如0、1、2、3、4、5、6、7、8、9和10。o可以是0、1、2或3。o可以是0。o可以是1。o可以是2。o可以是3。o may be an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. o may be 0, 1, 2, or 3. o may be 0. o may be 1. o may be 2. o may be 3.
p可以是0至5，例如0、1、2、3、4或5。p可以是0。p可以是1。p可以是2。p可以是3。p可以是4。p可以是5。p may be 0 to 5, for example 0, 1, 2, 3, 4 or 5. p may be 0. p may be 1. p may be 2. p may be 3. p may be 4. p may be 5.
接头可具有以下结构：The joint can have the following structures:
其中M、AAs、每个-(R1-J-R2)z”-、o和z”如本文所定义；r可以是0或1。wherein M, AAs , each -(R1- JR2 )z"-, o and z" are as defined herein; r may be 0 or 1.
r可以是0。r可以是1。r can be 0. r can be 1.
接头可具有以下结构：The joint can have the following structures:
其中M、AAs、o、p、q、r和z”各自可如本文所定义。wherein M, AAs , o, p, q, r and z" may each be as defined herein.
z”可以是1至50的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49和50，包括其间的所有范围和值。z”可以是5-20的整数。z”可以是10-15的整数。z" may be an integer from 1 to 50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, including all ranges and values therebetween. z" may be an integer from 5-20. z" may be an integer from 10-15.
接头可具有以下结构：The joint can have the following structures:
其中：in:
M、AAs和o如本文所定义。M,AAs and o are as defined herein.
合适接头的其他非限制性实例包括：Other non-limiting examples of suitable linkers include:
其中M和AAs如本文所定义。wherein M andAAs are as defined herein.
本文提供了包含cCPP和与前mRNA序列中的靶标互补的AC的化合物，所述化合物还包含L，其中接头通过键合基团(M)与AC缀合，其中M是Provided herein are compounds comprising a cCPP and an AC complementary to a target in a pre-mRNA sequence, the compound further comprising L, wherein a linker is conjugated to AC via a bonding group (M), wherein M is
本文提供了包含cCPP和反义化合物(AC)例如反义寡核苷酸的化合物，所述反义化合物与前mRNA序列中的靶标互补，其中所述化合物还包含L，其中接头通过键合基团(M)与AC缀合，其中M选自：Provided herein are compounds comprising a cCPP and an antisense compound (AC), e.g., an antisense oligonucleotide, which is complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein a linker is conjugated to AC via a bonding group (M), wherein M is selected from:
其中：R1是亚烷基、环烷基或其中t’是0至10，其中每个R独立地是烷基、烯基、炔基、碳环基或杂环基，其中R1是并且t’是2。 Wherein:R1 is alkylene, cycloalkyl or wherein t' is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, wherein R1 is And t' is 2.
接头可具有以下结构：The joint can have the following structures:
其中AAs如本文所定义，并且m’是0-10。whereinAAs are as defined herein, and m' is 0-10.
接头可具有下式：The linker may have the formula:
接头可具有下式：其中“碱基”对应于二氨基磷酸酯吗啉代寡聚物的3’端的核碱基。The linker may have the formula: wherein "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.
接头可具有下式：The linker may have the formula:
其中“碱基”对应于二氨基磷酸酯吗啉代寡聚物的3’端的核碱基。 wherein "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.
接头可具有下式：The linker may have the formula:
其中“碱基”对应于货物二氨基磷酸酯吗啉代寡聚物的3’端的核碱基。 where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
接头可具有下式：其中“碱基”对应于货物二氨基磷酸酯吗啉代寡聚物的3’端的核碱基。The linker may have the formula: where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
接头可具有下式：The linker may have the formula:
接头可在AC上的任何合适的位置共价结合到货物。接头可共价结合到货物的3’端或AC的5’端。接头可共价结合到AC的主链。The linker can be covalently bound to the cargo at any suitable position on the AC. The linker can be covalently bound to the 3' end of the cargo or the 5' end of the AC. The linker can be covalently bound to the backbone of the AC.
接头可结合到cCPP上的天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺或赖氨酸的侧链，或者谷氨酰胺或天冬酰胺的修饰侧链(例如，具有氨基基团的还原侧链)。接头可结合到cCPP上的赖氨酸的侧链。The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be attached to the side chain of lysine on the cCPP.
cCPP-接头缀合物cCPP-Linker Conjugate
cCPP可与本文所定义的接头缀合。接头可与如本文所定义的cCPP的AASC缀合。The cCPP may be conjugated to a linker as defined herein. The linker may be conjugated to the AASC of the cCPP as defined herein.
接头可包含-(OCH2CH2)z’-亚基(例如作为间隔区)，其中z’是1至23的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23。“-(OCH2CH2)z’”也称为PEG。cCPP-接头缀合物可具有选自表4的结构：The linker may comprise a -(OCH2 CH2 )z ' -subunit (e.g., as a spacer), wherein z' is an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. "-(OCH2 CH2 )z ' " is also known as PEG. The cCPP-linker conjugate may have a structure selected from Table 4:
表4：cCPP-接头缀合物Table 4: cCPP-linker conjugates
接头可包含-(OCH2CH2)z’-亚基和肽亚基，其中z’是1至23的整数。肽亚基可包含2至10个氨基酸。cCPP-接头缀合物可具有选自表5的结构：The linker may comprise a -(OCH2 CH2 )z' -subunit and a peptide subunit, wherein z' is an integer from 1 to 23. The peptide subunit may comprise 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table 5:
表5：内体逃逸载体(cCPP-接头缀合物)Table 5: Endosomal escape vectors (cCPP-linker conjugates)
cCPP-接头缀合物可以是Ac-PKKKRKV-K(环[FfФGrGrQ])-PEG12-K(N3)-NH2。The cCPP-linker conjugate can be Ac-PKKKRKV-K(cyclo[FfΦGrGrQ])-PEG12-K(N3 )-NH2 .
提供了包含环状细胞穿透肽(cCPP)、接头和环外肽(EP)的EEV。EEV可包含式(B)的结构：Provided are EEVs comprising a cyclic cell penetrating peptide (cCPP), a linker and an exocyclic peptide (EP). The EEV may comprise a structure of formula (B):
或其质子化形式， or its protonated form,
其中：in:
R1、R2和R3各自独立地是H或氨基酸的芳族或杂芳族侧链；R1 , R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
EP是如本文定义的环外肽；EP is an exocyclic peptide as defined herein;
每个m独立地是0-3的整数；Each m is independently an integer from 0 to 3;
n是0-2的整数；n is an integer from 0 to 2;
x’是1-20的整数；x’ is an integer from 1 to 20;
y是1-5的整数；y is an integer from 1 to 5;
q是1-4；并且q is 1-4; and
z’是1-23的整数。z’ is an integer from 1 to 23.
R1、R2、R3、R4、R6、EP、m、q、y、x’、z’如本文所述。R1 , R2 , R3 , R4 , R6 , EP, m, q, y, x′, and z′ are as described herein.
n可以是0。n可以是1。n可以是2。n can be 0. n can be 1. n can be 2.
EEV可包含式(B-a)或(B-b)的结构：The EEV may comprise a structure of formula (B-a) or (B-b):
或其质子化形式，其中EP、R1、R2、R3、R4、m和z’如上文在式(B)中所定义。 or a protonated form thereof, wherein EP, R1 , R2 , R3 , R4 , m and z′ are as defined above in formula (B).
EEV可包含式(B-c)的结构：The EEV may comprise a structure of formula (B-c):
或其质子化形式，其中EP、R1、R2、R3、R4和m如上文在式(B)中所定义；AA是如本文所定义的氨基酸；M如本文所定义；n是0-2的整数；x是1-10的整数；y是1-5的整数；并且z是1-10的整数。or a protonated form thereof, wherein EP, R1 , R2 , R3 , R4 and m are as defined above in formula (B); AA is an amino acid as defined herein; M is as defined herein; n is an integer from 0 to 2; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10.
EEV可具有式(B-1)、(B-2)、(B-3)或(B-4)的结构：The EEV may have a structure of formula (B-1), (B-2), (B-3) or (B-4):
或其质子化形式，其中EP如上文在式(B)中所定义。or a protonated form thereof, wherein EP is as defined above in formula (B).
EEV可包含式(B)并且可具有以下结构：Ac-PKKKRKV-AEEA-K(环[FGFGRGRQ])-PEG12-OH或Ac-PKKKRKV-AEEA-K(环[GfFGrGrQ])-PEG12-OH。The EEV may comprise formula (B) and may have the following structure: Ac-PKKKRKV-AEEA-K(cyclo[FGFGRGRQ])-PEG12 -OH or Ac-PKKKRKV-AEEA-K(cyclo[GfFGrGrQ])-PEG12 -OH.
EEV可包含下式的cCPP：The EEV may comprise a cCPP of the formula:
EEV可包含下式：Ac-PKKKRKV-miniPEG2-Lys(环(FfFGRGRQ)-miniPEG2-K(N3)。The EEV may comprise the formula: Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3).
EEV可以是：An EEV can be:
EEV可以是：Ac-PKKKRKV-K(环(Ff-Nal-GrGrQ)-PEG12-K(N3)-NH2。The EEV may be: Ac-PKKKRKV-K(cyclo(Ff-Nal-GrGrQ)-PEG12 -K(N3 )-NH2 .
EEV可以是EEV can be
EEV可以是Ac-P-K(Tfa)-K(Tfa)-K(Tfa)-R-K(Tfa)-V-AEEA-K(环(Ff-Nal-GrGrQ)-PEG12-OH或Ac-P-K(Tfa)-K(Tfa)-K(Tfa)-R-K(Tfa)-V-AEEA-K(环(FGFGRGRQ)-PEG12-OH。EEV can be Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-AEEA-K(cyclo(Ff-Nal-GrGrQ)-PEG12 -OH or Ac-PK(Tfa )-K(Tfa)-K(Tfa)-RK(Tfa)-V-AEEA-K(cyclo(FGFGRGRQ)-PEG12 -OH.
EEV可以是EEV can be
EEV可以是Ac-PKKKRKV-miniPEG-K(环(Ff-Nal-GrGrQ)-PEG12-OH。The EEV can be Ac-PKKKRKV-miniPEG-K(cyclo(Ff-Nal-GrGrQ)-PEG12-OH.
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是：An EEV can be:
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可选自EEV can be selected from
Ac-rr-miniPEG2-Dap[环(FfФ-Cit-r-Cit-rQ)]-PEG12-OHAc-rr-miniPEG2-Dap[Cycle(FfФ-Cit-r-Cit-rQ)]-PEG12-OH
Ac-frr-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-frr-PEG2-Dap(Cyclic(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-rfr-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-rfr-PEG2-Dap(Cyclic(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-rbfbr-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-rbfbr-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-rrr-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-rrr-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-rbr-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-rbr-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-rbrbr-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-rbrbr-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-hh-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-hh-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-hbh-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-hbh-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-hbhbh-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-hbhbh-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-rbhbh-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-rbhbh-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-hbrbh-PEG2-Dap(环(FfФ-Cit-r-Cit-rQ))-PEG12-OHAc-hbrbh-PEG2-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-PEG12-OH
Ac-rr-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-rr-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-frr-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-frr-Dap(Ring(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-rfr-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-rfr-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-rbfbr-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-rbfbr-Dap(Ring(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-rrr-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-rrr-Dap(Ring(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-rbr-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-rbr-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-rbrbr-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-rbrbr-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-hh-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-hh-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-hbh-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-hbh-Dap(Ring(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-hbhbh-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-hbhbh-Dap(Ring(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-rbhbh-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-rbhbh-Dap(Ring(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-hbrbh-Dap(环(FfФ-Cit-r-Cit-rQ))-b-OHAc-hbrbh-Dap(cyclo(FfФ-Cit-r-Cit-rQ))-b-OH
Ac-KKKK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KKKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KGKK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KGKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KKGK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KKGK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KKK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KGK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KGK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KBK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KBK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KBKBK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KBKBK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KR-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KBR-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KBR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PKKKRKV-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PKKKRKV-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PGKKRKV-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PGKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PKGKRKV-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKGKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PKKGRKV-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKKGRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PKKKGKV-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKKKGKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PKKKRGV-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKKKRGV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-PKKKRKG-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKKKRKG-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KKKRK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-KKKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2
Ac-KKRK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2和Ac-KKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 and
Ac-KRK-miniPEG2-Lys(环(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2。Ac-KRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2.
EEV可选自：EEV can be selected from:
Ac-PKKKRKV-Lys(环[FfФGrGrQ])-PEG12-K(N3)-NH2Ac-PKKKRKV-Lys(cyclo[FfФGrGrQ])-PEG12 -K(N3 )-NH2
Ac-PKKKRKV-miniPEG2-Lys(环[FfФGrGrQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FfФGrGrQ])-miniPEG2 -K(N3 )-NH2
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRGRQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRGRQ])-miniPEG2 -K(N3 )-NH2
Ac-KR-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-KR-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
Ac-PKKKGKV-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-PKKKGKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
Ac-PKKKRKG-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-PKKKRKG-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
Ac-KKKRK-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-KKKRK-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
Ac-PKKKRKV-miniPEG2-Lys(环[FFФGRGRQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FFФGRGRQ])-miniPEG2 -K(N3 )-NH2
Ac-PKKKRKV-miniPEG2-Lys(环[βhFfФGrGrQ])-miniPEG2-K(N3)-NH2和Ac-PKKKRKV-miniPEG2 -Lys(cyclo[βhFfФGrGrQ])-miniPEG2 -K(N3 )-NH2 and
Ac-PKKKRKV-miniPEG2-Lys环[FfΦSrSrQ])-miniPEG2-K(N3)-NH2。Ac-PKKKRKV-miniPEG2 -Lyscyclo[FfΦSrSrQ])-miniPEG2 -K(N3 )-NH2 .
EEV可选自：EEV can be selected from:
Ac-PKKKRKV-miniPEG2-Lys(环(GfFGrGrQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo(GfFGrGrQ])-PEG12 -OH
Ac-PKKKRKV-miniPEG2-Lys(环[FGFKRKRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFKRKRQ])-PEG12 -OH
Ac-PKKKRKV-miniPEG2-Lys(环[FGFRGRGQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFRGRGQ])-PEG12 -OH
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRGRGRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRrRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRrRQ])-PEG12 -OH
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRRRQ])-PEG12-OH和Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRRRQ])-PEG12 -OH and
Ac-PKKKRKV-miniPEG2-Lys(环[FGFRRRRQ])-PEG12-OH。Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FGFRRRRQ])-PEG12 -OH.
EEV可选自：EEV can be selected from:
Ac-K-K-K-R-K-G-miniPEG2-K(环[FGFGRGRQ])-PEG12-OHAc-KKKRKG-miniPEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-K-K-K-R-K-miniPEG2-K(环[FGFGRGRQ])-PEG12-OHAc-KKKRK-miniPEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-K-K-R-K-K-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-KKRKK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-K-R-K-K-K-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-KRKKK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-K-K-K-K-R-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-KKKKR-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-R-K-K-K-K-PEG4-K(环[FGFGRGRQ])-PEG12-OH和Ac-RKKKK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH and
Ac-K-K-K-R-K-PEG4-K(环[FGFGRGRQ])-PEG12-OH。Ac-KKKRK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH.
EEV可选自：EEV can be selected from:
Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-PKKKRKV-PEG2-K(环[GfFGrGrQ])-PEG2-K(N3)-NH2和Ac-PKKKRKV-PEG2 -K(cyclo[GfFGrGrQ])-PEG2 -K(N3 )-NH2 and
Ac-PKKKRKV-PEG2-K(环[GfFGrGrQ])-PEG12-OH。Ac-PKKKRKV-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH.
货物可以是AC并且EEV可选自：Cargo can be AC and EEV can be selected from:
Ac-PKKKRKV-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-PKKKRKV-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-PKKKRKV-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-PKKKRKV-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-PKKKRKV-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
Ac-PKKKRKV-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
Ac-rr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-rr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-rr-PEG2-K(环[FfF-GRGRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FfF-GRGRQ])-PEG12 -OH
Ac-rr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-rr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-rr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
Ac-rr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
Ac-rrr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-rrr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-rrr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
Ac-rrr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-rrr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-rrr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
Ac-rrr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
Ac-rhr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-rhr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-rhr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
Ac-rhr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-rhr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-rhr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
Ac-rhr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
Ac-rbr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-rbr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-rbr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
Ac-rbr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-rbr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-rbr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
Ac-rbr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
Ac-rbrbr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-rbrbr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-rbrbr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
Ac-rbrbr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-rbrbr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-rbrbr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
Ac-rbrbr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
Ac-rbhbr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rbhbr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-rbhbr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rbhbr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-rbhbr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rbhbr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
Ac-rbhbr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rbhbr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
Ac-rbhbr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rbhbr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-rbhbr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rbhbr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
Ac-rbhbr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rbhbr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
Ac-hbrbh-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
Ac-hbrbh-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
Ac-hbrbh-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
Ac-hbrbh-PEG2-K(环]FGFGRGRQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo]FGFGRGRQ])-PEG12 -OH
Ac-hbrbh-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
Ac-hbrbh-PEG2-K(环[FGFGRRRQ])-PEG12-OH和Ac-hbrbh-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH and
Ac-hbrbh-PEG2-K(环[FGFRRRRQ])-PEG12-OH，Ac-hbrbh-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH,
其中b是β-丙氨酸，并且环外序列可以是D或L立体化学。wherein b is β-alanine and the exocyclic sequence can be of D or L stereochemistry.
在实施方案中，与包含单独AC的化合物相比，包含环肽和AC的化合物具有改善的胞质摄取效率。胞质摄取效率可通过将包含环肽和AC的化合物的胞质递送效率与单独AC的胞质递送效率进行比较来测量。In an embodiment, the compound comprising the cyclic peptide and AC has improved cytoplasmic uptake efficiency compared to the compound comprising AC alone. The cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of the compound comprising the cyclic peptide and AC with the cytoplasmic delivery efficiency of AC alone.
反义化合物Antisense compounds
在各种实施方案中，本文公开的化合物包含与反义化合物(AC)缀合的CPP(例如，环肽)。在实施方案中，AC包含针对靶多核苷酸的反义寡核苷酸。术语“反义寡核苷酸”或简称“反义”意在包括与靶向多核苷酸序列互补的寡核苷酸。反义寡核苷酸是与所选序列(例如靶基因mRNA)互补的DNA或RNA单链。In various embodiments, the compounds disclosed herein include a CPP (e.g., a cyclic peptide) conjugated to an antisense compound (AC). In an embodiment, AC includes an antisense oligonucleotide directed against a target polynucleotide. The term "antisense oligonucleotide" or "antisense" for short is intended to include oligonucleotides that are complementary to a targeted polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a selected sequence (e.g., a target gene mRNA).
反义寡核苷酸可调节蛋白质转录、翻译和表达的一个或多个方面。在实施方案中，反义寡核苷酸针对靶前mRNA内的靶序列，来调节前mRNA剪接的一个或多个方面。如本文所用，剪接的调节是指改变前mRNA转录物的处理，使得剪接的mRNA分子由于外显子跳跃或外显子包含、一个或多个外显子的缺失或者剪接的mRNA中通常不存在的序列(例如内含子序列)的缺失或添加而含有不同的外显子的组合。在实施方案中，反义寡核苷酸与前mRNA分子中的靶序列杂交恢复了对突变的前mRNA序列的天然剪接。在实施方案中，反义寡核苷酸杂交导致靶前mRNA的选择性剪接。在实施方案中，反义寡核苷酸杂交导致一个或多个外显子的外显子包含或外显子跳跃。在实施方案中，跳跃的外显子序列包含移码突变、无义突变或错义突变。在实施方案中，跳跃的外显子序列包含核酸缺失、取代或插入。在实施方案中，跳跃的外显子本身不包含序列突变，但邻近的外显子包含导致移码突变或无义突变的突变。在实施方案中，反义寡核苷酸与靶前mRNA内的靶序列杂交防止成熟mRNA分子中包含外显子序列。在实施方案中，反义寡核苷酸与靶前mRNA内的靶序列杂交导致野生型靶蛋白异构体的优先表达。在实施方案中，反义寡核苷酸与靶前mRNA内的靶序列杂交导致包含野生型靶蛋白的活性片段的再剪接靶蛋白的表达。Antisense oligonucleotides can regulate one or more aspects of protein transcription, translation and expression. In an embodiment, antisense oligonucleotides are directed to a target sequence in a target pre-mRNA to regulate one or more aspects of pre-mRNA splicing. As used herein, the regulation of splicing refers to the process of changing the pre-mRNA transcript so that the mRNA molecule of the splicing contains a combination of different exons due to exon skipping or exon inclusion, the deletion of one or more exons, or the deletion or addition of a sequence (e.g., intron sequence) that is not usually present in the mRNA of the splicing. In an embodiment, antisense oligonucleotides hybridize with the target sequence in the pre-mRNA molecule to restore the natural splicing of the pre-mRNA sequence of the mutation. In an embodiment, antisense oligonucleotide hybridization causes the selective splicing of target pre-mRNA. In an embodiment, antisense oligonucleotide hybridization causes the exon inclusion or exon skipping of one or more exons. In an embodiment, the exon sequence of the jump includes a frameshift mutation, a nonsense mutation, or a missense mutation. In an embodiment, the exon sequence of the jump includes a nucleic acid deletion, a substitution, or an insertion. In embodiments, the skipped exon itself does not contain a sequence mutation, but the adjacent exon contains a mutation that results in a frameshift mutation or a nonsense mutation. In embodiments, antisense oligonucleotides hybridize to a target sequence within a target pre-mRNA to prevent the inclusion of exon sequences in mature mRNA molecules. In embodiments, antisense oligonucleotides hybridize to a target sequence within a target pre-mRNA resulting in preferential expression of a wild-type target protein isomer. In embodiments, antisense oligonucleotides hybridize to a target sequence within a target pre-mRNA resulting in expression of a re-splicing target protein comprising an active fragment of a wild-type target protein.
反义机制经由反义寡核苷酸化合物与靶核酸的杂交来发挥作用。在实施方案中，反义寡核苷酸与其靶序列的杂交抑制靶蛋白的表达。在实施方案中，反义寡核苷酸与其靶序列的杂交抑制一种或多种野生型靶蛋白异构体的表达。在实施方案中，反义寡核苷酸与其靶序列的杂交上调靶蛋白的表达。在实施方案中，反义寡核苷酸与其靶序列的杂交增加一种或多种野生型靶蛋白异构体的表达。The antisense mechanism works via hybridization of an antisense oligonucleotide compound with a target nucleic acid. In embodiments, hybridization of an antisense oligonucleotide with its target sequence inhibits the expression of a target protein. In embodiments, hybridization of an antisense oligonucleotide with its target sequence inhibits the expression of one or more wild-type target protein isomers. In embodiments, hybridization of an antisense oligonucleotide with its target sequence upregulates the expression of a target protein. In embodiments, hybridization of an antisense oligonucleotide with its target sequence increases the expression of one or more wild-type target protein isomers.
在实施方案中，反义化合物可通过与互补mRNA结合来抑制基因表达。与靶mRNA的结合可通过阻止互补mRNA链的翻译(通过立体阻断参与翻译的RNA结合蛋白)或通过导致靶mRNA的降解而导致基因表达的抑制。反义DNA可用于靶向特异性互补(编码或非编码)RNA。如果发生结合，所述DNA/RNA杂交体可被RNA酶H降解。在实施方案中，反义寡核苷酸含有约10至约50个核苷酸或约15至约30个核苷酸。在实施方案中，反义寡核苷酸可能不与靶核苷酸序列完全互补。In embodiments, antisense compounds can inhibit gene expression by binding to complementary mRNA. The combination with target mRNA can lead to inhibition of gene expression by preventing the translation of complementary mRNA chain (by three-dimensional blocking RNA binding protein involved in translation) or by causing the degradation of target mRNA. Antisense DNA can be used for targeting specific complementary (coding or non-coding) RNA. If binding occurs, the DNA/RNA hybrid can be degraded by RNase H. In embodiments, antisense oligonucleotide contains about 10 to about 50 nucleotides or about 15 to about 30 nucleotides. In embodiments, antisense oligonucleotide may not be completely complementary to the target nucleotide sequence.
反义寡核苷酸已被证明是蛋白质合成的有效的靶向抑制剂，并且因此可用于通过靶向基因特异性抑制蛋白质合成。反义寡核苷酸抑制蛋白质合成的功效已被充分确定。例如，通过针对其对应的mRNA序列的反义寡核苷酸抑制聚半乳糖醛酸酶(polygalactauronase)和毒蕈碱2型乙酰胆碱受体的合成(美国专利5,739,119和美国专利5,759,829)。此外，已用核蛋白细胞周期蛋白、多重耐药基因(MDG1)、ICAM-1、E-选择素、STK-1、纹状体GABAA受体和人EGF证明了反义抑制的实例(Jaskulski等人，Science.1988年6月10日；240(4858)：1544-6；Vasanthakumar和Ahmed，Cancer Commun.1989；1(4)：225-32；Peris等人，Brain Res Mol Brain Res.1998年6月15日；57(2)：310-20；美国专利5,801,154；美国专利5,789,573；美国专利5,718,709和美国专利5,610,288)。此外，反义构建体也已被描述抑制并且可用于治疗各种异常的细胞增殖，例如癌症(美国专利5,747,470；美国专利5,591,317和美国专利5,783,683)。Antisense oligonucleotides have been shown to be effective targeted inhibitors of protein synthesis, and therefore can be used to inhibit protein synthesis by targeting gene specificity. The efficacy of antisense oligonucleotides in inhibiting protein synthesis has been well established. For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptors has been inhibited by antisense oligonucleotides directed against their corresponding mRNA sequences (U.S. Patent No. 5,739,119 and U.S. Patent No. 5,759,829). In addition, examples of antisense inhibition have been demonstrated with nuclear protein cyclins, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors and human EGF (Jaskulski et al., Science. 1988 Jun 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Additionally, antisense constructs have been described that inhibit and can be used to treat various abnormal cell proliferations, such as cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317; and U.S. Pat. No. 5,783,683).
产生反义寡核苷酸的方法是本领域已知的，并且可以容易地适用于产生靶向任何多核苷酸序列的反义寡核苷酸。对给定靶序列具有特异性的反义寡核苷酸序列的选择是基于对所选靶序列的分析和二级结构、Tm、结合能和相对稳定性的确定。反义寡核苷酸可基于它们相对不能形成将减少或抑制与宿主细胞中的靶mRNA的特异性结合的二聚体、发夹或其他二级结构来选择。mRNA的靶区可包括处于或接近AUG翻译起始密码子的那些区和与mRNA的5’区基本上互补的那些序列。这些二级结构分析和靶位点选择考虑可例如使用第4版的OLIGO引物分析软件(Molecular Biology Insights)和/或BLASTN 2.0.5算法软件来进行(Altschul等人，Nucleic Acids Res.1997，25(17)：3389-402)。Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. The selection of antisense oligonucleotide sequences with specificity for a given target sequence is based on the analysis of the selected target sequence and the determination of secondary structure, Tm, binding energy and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins or other secondary structures that will reduce or inhibit the specific binding to the target mRNA in the host cell. The target region of mRNA may include those regions that are in or near the AUG translation start codon and those sequences that are substantially complementary to the 5' region of mRNA. These secondary structure analysis and target site selection considerations can be performed, for example, using the 4th edition of OLIGO primer analysis software (Molecular Biology Insights) and/or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25 (17): 3389-402).
根据本公开，反义化合物(AC)改变靶基因的剪接、翻译或表达的一个或多个方面，例如通过改变真核靶前mRNA的剪接。根据本公开的AC包含与靶前mRNA序列内(例如，在包括外显子的至少一部分、内含子的至少一部分或两者的序列处)发现的序列互补的核酸序列。使用这些AC提供了一种直接的遗传方法，所述方法能够调节特定致病基因的剪接。反义技术背后的原理是与靶核酸杂交的反义化合物通过多种反义机制中的一种调节基因表达活动诸如剪接或翻译。AC的序列特异性使得所述技术作为选择性地调节参与多种疾病中的任一种的发病机制的前mRNA的剪接的治疗方法而极具吸引力。反义技术是用于改变一种或多种特定基因产物的表达的有效手段并且可因此证明在许多治疗、诊断和研究应用中是有用的。According to the present disclosure, antisense compounds (AC) change one or more aspects of the splicing, translation or expression of the target gene, for example, by changing the splicing of the eukaryotic target pre-mRNA. According to the present disclosure, ACs include nucleic acid sequences complementary to sequences found in the target pre-mRNA sequence (e.g., at a sequence including at least a portion of exons, at least a portion of introns, or both). Using these ACs provides a direct genetic method that can regulate the splicing of specific pathogenic genes. The principle behind antisense technology is that antisense compounds hybridized with target nucleic acids regulate gene expression activities such as splicing or translation through one of a variety of antisense mechanisms. The sequence specificity of AC makes the technology very attractive as a therapeutic method for selectively regulating the splicing of pre-mRNA involved in the pathogenesis of any of a variety of diseases. Antisense technology is an effective means for changing the expression of one or more specific gene products and can therefore prove to be useful in many treatments, diagnosis and research applications.
本文所述的化合物可含有一个或多个不对称中心，并且因此产生对映异构体、非对映异构体和其他立体异构构型，这些构型可根据绝对立体化学定义为(R)或(S)、α或β、或者(D)或(L)。本文提供的反义化合物包括所有这些可能的异构体，以及它们的外消旋和光学纯形式。The compounds described herein may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations which may be defined in terms of absolute stereochemistry as (R) or (S), α or β, or (D) or (L). The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.
反义化合物杂交位点Antisense compound hybridization site
反义机制依赖于反义化合物与靶核酸的杂交。在实施方案中，本公开提供了与靶核酸互补的反义化合物。在实施方案中，靶核酸序列存在于前mRNA分子中。在实施方案中，靶核酸序列存在于前mRNA分子的外显子中。在实施方案中，靶核酸序列存在于前mRNA分子的内含子中。The antisense mechanism relies on the hybridization of antisense compounds with target nucleic acids. In embodiments, the disclosure provides antisense compounds complementary to target nucleic acids. In embodiments, the target nucleic acid sequence is present in the pre-mRNA molecule. In embodiments, the target nucleic acid sequence is present in the exons of the pre-mRNA molecule. In embodiments, the target nucleic acid sequence is present in the introns of the pre-mRNA molecule.
前mRNA分子在细胞核中产生，并在转运到细胞质进行翻译之前或期间进行处理。前mRNA的处理包括在转录物的3’端添加5’甲基化帽和大约200-250个碱基的聚(A)尾。mRNA处理的下一步是前mRNA的剪接，这发生在90-95％的哺乳动物mRNA的成熟过程中。内含子(或介入序列)是初级转录物(或编码它的DNA)的区，不包括在成熟mRNA的编码序列中。外显子是初级转录物的区，当成熟mRNA到达细胞质时，它们仍保留在其中。外显子剪接在一起形成成熟的mRNA序列。剪接连接点也称为剪接位点，其中连接点的5’侧通常称为“5’剪接位点”或“剪接供体位点”，而3’侧称为“3’剪接位点”或“剪接受体位点”。在剪接中，上游外显子的3’端与下游外显子的5’端接合。因此，未剪接的RNA(前mRNA)在内含子的5’端具有外显子/内含子连接点，并且在内含子的3’端具有内含子/外显子连接点。内含子被去除后，外显子在成熟mRNA中有时被称为外显子/外显子连接点处或边界处是邻接的。隐蔽剪接位点是不经常使用但在常用剪接位点被阻塞或不可用时可使用的那些剪接位点。选择性剪接，定义为外显子的不同组合剪接在一起，通常会导致单个基因产生多个mRNA转录物。Pre-mRNA molecules are produced in the nucleus and processed before or during transport to the cytoplasm for translation. Processing of pre-mRNA includes the addition of a 5' methylated cap and a poly (A) tail of approximately 200-250 bases to the 3' end of the transcript. The next step in mRNA processing is the splicing of pre-mRNA, which occurs during the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of the primary transcript (or the DNA encoding it) that are not included in the coding sequence of the mature mRNA. Exons are regions of the primary transcript that remain in the mature mRNA when it reaches the cytoplasm. Exons are spliced together to form a mature mRNA sequence. Splice junctions are also called splice sites, where the 5' side of the junction is often referred to as the "5' splice site" or "splice donor site", and the 3' side is called the "3' splice site" or "splice acceptor site". In splicing, the 3' end of the upstream exon is joined to the 5' end of the downstream exon. Thus, the unspliced RNA (pre-mRNA) has an exon/intron junction at the 5' end of the intron and an intron/exon junction at the 3' end of the intron. After the introns are removed, the exons are adjacent in the mature mRNA at what are sometimes referred to as exon/exon junctions or boundaries. Cryptic splice sites are those splice sites that are not frequently used but can be used when the usual splice sites are blocked or unavailable. Alternative splicing, defined as the splicing together of different combinations of exons, often results in multiple mRNA transcripts from a single gene.
在实施方案中，AC与剪接位点中的序列杂交。在实施方案中，AC与包含剪接位点的一部分的序列杂交。在实施方案中，AC与包含部分或全部剪接位点的序列杂交。在实施方案中，AC与包含部分或全部剪接供体位点的序列杂交。在实施方案中，AC与包含部分或全部剪接受体位点的序列杂交。在实施方案中，AC与包含部分或全部隐蔽剪接位点的序列杂交。在实施方案中，AC与包含外显子/内含子连接点的序列杂交。In an embodiment, AC hybridizes to a sequence in a splice site. In an embodiment, AC hybridizes to a sequence comprising a portion of a splice site. In an embodiment, AC hybridizes to a sequence comprising part or all of a splice site. In an embodiment, AC hybridizes to a sequence comprising part or all of a splice donor site. In an embodiment, AC hybridizes to a sequence comprising part or all of a splice acceptor site. In an embodiment, AC hybridizes to a sequence comprising part or all of a cryptic splice site. In an embodiment, AC hybridizes to a sequence comprising an exon/intron junction.
前mRNA剪接涉及两个连续的生化反应。两种反应都涉及RNA核苷酸之间的剪接体酯交换反应。在第一个反应中，内含子内特定分支点核苷酸(在剪接体组装期间定义)的2’-OH在5’剪接位点处对内含子的第一个核苷酸进行亲核攻击，从而形成套索中间体(lariatintermediate)。在第二个反应中，释放的5’外显子的3’-OH对3’剪接位点处的内含子的最后一个核苷酸进行亲核攻击，从而接合外显子并释放内含子套索。前mRNA剪接由内含子沉默序列(ISS)和末端茎环(TSL)序列调节。如本文所用，术语“内含子沉默序列(ISS)”和“末端茎环(TSL)”分别指内含子和外显子内的序列元件，所述序列元件通过前mRNA内的反式作用蛋白因子的结合来控制选择性剪接，从而导致剪接位点的不同使用。典型地，内含子沉默序列在8至16个核苷酸之间，并且比外显子-内含子连接点处的剪接位点保守性更低。末端茎环序列典型地在12至24个核苷酸之间，并且由于12-24个核苷酸序列内的互补性并因此结合而形成二级环结构。Pre-mRNA splicing involves two consecutive biochemical reactions. Both reactions involve spliceosomal transesterification reactions between RNA nucleotides. In the first reaction, the 2'-OH of a specific branch point nucleotide (defined during spliceosomal assembly) in an intron attacks the first nucleotide of the intron at the 5' splice site with a nucleophilic attack, thereby forming a lariat intermediate. In the second reaction, the 3'-OH of the released 5' exon attacks the last nucleotide of the intron at the 3' splice site with a nucleophilic attack, thereby engaging the exon and releasing the intron lariat. Pre-mRNA splicing is regulated by intron silencing sequences (ISS) and terminal stem loops (TSL) sequences. As used herein, the terms "intron silencing sequence (ISS)" and "terminal stem loop (TSL)" refer to sequence elements in introns and exons, respectively, and the sequence elements control alternative splicing by the combination of trans-acting protein factors in pre-mRNA, thereby resulting in different uses of splice sites. Typically, intronic silencer sequences are between 8 and 16 nucleotides and are less conserved than splice sites at exon-intron junctions. Terminal stem-loop sequences are typically between 12 and 24 nucleotides and form secondary loop structures due to complementarity within the 12-24 nucleotide sequence and thus binding.
在实施方案中，AC与包含部分或全部内含子沉默序列的序列杂交。在实施方案中，AC与包含部分或全部末端茎环的序列杂交。In an embodiment, AC hybridizes to a sequence comprising part or all of an intronic silencing sequence.In an embodiment, AC hybridizes to a sequence comprising part or all of a terminal stem-loop.
至多50％的由点突变引起的人遗传病是由异常剪接引起的。这类点突变可破坏当前剪接位点或创建新的剪接位点，从而导致mRNA转录物包含不同的外显子组合或外显子缺失。点突变还可能导致隐蔽剪接位点的激活或破坏顺式调节元件(即剪接增强子或沉默子)。Up to 50% of human genetic diseases caused by point mutations are caused by abnormal splicing. Such point mutations can destroy current splice sites or create new splice sites, resulting in mRNA transcripts containing different exon combinations or exon deletions. Point mutations may also lead to the activation of cryptic splice sites or the destruction of cis-regulatory elements (i.e., splicing enhancers or silencers).
在实施方案中，AC与包含由靶基因中的突变产生的部分或全部异常剪接位点的序列杂交。在实施方案中，AC与包含部分或全部调节元件的序列杂交。还提供了靶向顺式调节元件的反义化合物。在实施方案中，调节元件在外显子中。在实施方案中，调节元件在内含子中。In an embodiment, AC hybridizes to a sequence comprising part or all of an abnormal splice site produced by a mutation in a target gene. In an embodiment, AC hybridizes to a sequence comprising part or all of a regulatory element. Antisense compounds targeting cis-regulatory elements are also provided. In an embodiment, the regulatory element is in an exon. In an embodiment, the regulatory element is in an intron.
在实施方案中，AC可与翻译起始密码子区、5’帽区、内含子/外显子连接点、编码序列、翻译终止密码子区或者5’-非翻译区或3’-非翻译区中的序列特异性杂交。在实施方案中，AC可与部分或全部前mRNA剪接位点、外显子-外显子连接点或内含子-外显子连接点杂交。在实施方案中，AC可与由于重排或缺失而导致的异常融合连接点杂交。在实施方案中，AC可与选择性剪接的mRNA中的特定外显子杂交。In an embodiment, AC can be specifically hybridized with a sequence in a translation start codon region, a 5' cap region, an intron/exon junction, a coding sequence, a translation stop codon region, or a 5'-untranslated region or a 3'-untranslated region. In an embodiment, AC can be hybridized with part or all of a pre-mRNA splice site, an exon-exon junction, or an intron-exon junction. In an embodiment, AC can be hybridized with an abnormal fusion junction caused by rearrangement or deletion. In an embodiment, AC can be hybridized with a specific exon in an alternatively spliced mRNA.
在实施方案中，AC与长度在5至50个核苷酸之间的序列杂交，所述长度也可称为AC的长度。在实施方案中，AC的长度在5至50个核苷酸之间，例如长度在5至10、10至15、15至20、20至25、25至30、30至35、35至40、40至45之间或45至50个核苷酸之间。在实施方案中，AC的长度是约5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个核苷酸。在实施方案中，AC的长度是至少约5、约6、约7、约8、约9、约10、约11、约12、约13、约14、约15、约16、约17、约18、约19或约20，并且至多约21、约22、约23、约24或约25，并且至多约26、约27、约28、约29、约30、约31、约32、约33、约34、约35、约36、约37、约38、约39或约40，并且至多约41、约42、约43、约44、约45、约46、约47、约48、约49或约50个核苷酸。在一些实施方案中，AC的长度是约10个核苷酸。在一些实施方案中，AC的长度是约15个核苷酸。在一些实施方案中，AC的长度是约16个核苷酸。在一些实施方案中，AC的长度是约17个核苷酸。在一些实施方案中，AC的长度是约18个核苷酸。在一些实施方案中，AC的长度是约19个核苷酸。在一些实施方案中，AC的长度是约20个核苷酸。在一些实施方案中，AC的长度是约21个核苷酸。在一些实施方案中，AC的长度是约22个核苷酸。在一些实施方案中，AC的长度是约23个核苷酸。在一些实施方案中，AC的长度是约24个核苷酸。在一些实施方案中，AC的长度是约25个核苷酸。在一些实施方案中，AC的长度是约26个核苷酸。在一些实施方案中，AC的长度是约27个核苷酸。在一些实施方案中，AC的长度是约28个核苷酸。在一些实施方案中，AC的长度是约29个核苷酸。在一些实施方案中，AC的长度是约30个核苷酸。In an embodiment, AC hybridizes to a sequence between 5 and 50 nucleotides in length, which length may also be referred to as the length of AC. In an embodiment, AC has a length between 5 and 50 nucleotides, such as a length between 5 and 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 nucleotides. In an embodiment, AC has a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In embodiments, the length of AC is at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20, and at most about 21, about 22, about 23, about 24, or about 25, and at most about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40, and at most about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 nucleotides. In some embodiments, the length of AC is about 10 nucleotides. In some embodiments, the length of AC is about 15 nucleotides. In some embodiments, the length of AC is about 16 nucleotides. In some embodiments, the length of AC is about 17 nucleotides. In some embodiments, the length of AC is about 18 nucleotides. In some embodiments, the length of AC is about 19 nucleotides. In some embodiments, the length of AC is about 20 nucleotides. In some embodiments, the length of AC is about 21 nucleotides. In some embodiments, the length of AC is about 22 nucleotides. In some embodiments, the length of AC is about 23 nucleotides. In some embodiments, the length of AC is about 24 nucleotides. In some embodiments, the length of AC is about 25 nucleotides. In some embodiments, the length of AC is about 26 nucleotides. In some embodiments, the length of AC is about 27 nucleotides. In some embodiments, the length of AC is about 28 nucleotides. In some embodiments, the length of AC is about 29 nucleotides. In some embodiments, the length of AC is about 30 nucleotides.
在实施方案中，AC可与靶核酸序列小于100％互补。如本文所用，术语“互补百分比”是指与寡聚化合物或核酸的对应核碱基具有核碱基互补性的AC的核碱基数量除以AC的总长度(核碱基数量)。本领域技术人员认识到，在不消除反义化合物活性的情况下，包含错配是可能的。在实施方案中，AC可含有至多约20％的核苷酸，其破坏AC与靶核酸的碱基配对。在实施方案中，AC含有不超过约15％、不超过约10％、不超过5％的错配或无错配。在实施方案中，AC含有不超过1、2、3、4或5个错配。在实施方案中，AC与靶核酸至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％互补。寡核苷酸的互补性百分比是通过将互补核碱基的数量除以寡核苷酸的核碱基总数来计算。寡核苷酸区的互补性百分比是通过将所述区中互补核碱基的数量除以核碱基区的总数来计算。In an embodiment, AC may be less than 100% complementary to a target nucleic acid sequence. As used herein, the term "complementarity percentage" refers to the number of core bases of AC having core base complementarity with the corresponding core base of an oligomeric compound or nucleic acid divided by the total length (core base number) of AC. Those skilled in the art recognize that it is possible to include mismatches without eliminating the activity of antisense compounds. In an embodiment, AC may contain up to about 20% nucleotides, which destroy the base pairing of AC with a target nucleic acid. In an embodiment, AC contains no more than about 15%, no more than about 10%, no more than 5% mismatches or no mismatches. In an embodiment, AC contains no more than 1, 2, 3, 4 or 5 mismatches. In an embodiment, AC is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid. The complementarity percentage of an oligonucleotide is calculated by dividing the number of complementary core bases by the total number of core bases of an oligonucleotide. The percent complementarity of an oligonucleotide region is calculated by dividing the number of complementary nucleobases in the region by the total number of nucleobases in the region.
在实施方案中，与未修饰的化合物相比，掺入核苷酸亲和力修饰允许更大数量的错配。类似地，某些寡核苷酸序列可比其他寡核苷酸序列更耐受错配。本领域普通技术人员能够确定寡核苷酸之间或寡核苷酸与靶核酸之间的适当数量的错配，诸如通过确定解链温度(Tm)。Tm或ATm可通过本领域普通技术人员熟悉的技术来计算。例如，Freier等人(Nucleic Acids Research，1997，25，22：4429-4443)描述的技术允许本领域普通技术人员评价核苷酸修饰提高RNA：DNA双链体解链温度的能力。In an embodiment, the incorporation of nucleotide affinity modifications allows for a greater number of mismatches compared to unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of ordinary skill in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, such as by determining the melting temperature (Tm). Tm or ATm can be calculated by techniques familiar to one of ordinary skill in the art. For example, the techniques described by Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate the ability of nucleotide modifications to increase the melting temperature of RNA: DNA duplexes.
反义机制Antisense mechanism
根据本公开的AC可调节蛋白质转录、翻译和表达的一个或多个方面。在实施方案中，与靶前mRNA内的靶序列杂交的AC调节前mRNA剪接的一个或多个方面。如本文所用，剪接的调节是指改变前mRNA转录物的处理，使得剪接的mRNA分子由于外显子跳跃或外显子包含、一个或多个外显子的缺失或者剪接的mRNA中通常不存在的序列(例如内含子序列)的缺失或添加而含有不同的外显子的组合。在实施方案中，与前mRNA分子内的靶序列的AC杂交恢复了对突变的前mRNA序列的天然剪接。在实施方案中，AC杂交导致靶前mRNA的选择性剪接。在实施方案中，AC杂交导致一个或多个外显子的外显子包含或外显子跳跃。在实施方案中，跳跃的外显子序列包含移码突变、无义突变或错义突变。在实施方案中，跳跃的外显子序列包含核酸缺失、取代或插入。在实施方案中，跳跃的外显子本身不包含序列突变，但邻近的外显子包含导致移码突变或无义突变的突变。在实施方案中，不包含序列突变的外显子的缺失恢复了成熟mRNA的阅读框。在实施方案中，AC与靶前mRNA内的靶序列杂交导致野生型靶蛋白异构体的优先表达。在实施方案中，AC与靶前mRNA内的靶序列杂交导致包含野生型靶蛋白的活性片段的再剪接靶蛋白的表达。AC according to the present disclosure can regulate one or more aspects of protein transcription, translation and expression. In an embodiment, AC hybridized with a target sequence in a target pre-mRNA regulates one or more aspects of pre-mRNA splicing. As used herein, the regulation of splicing refers to the process of changing the pre-mRNA transcript so that the spliced mRNA molecule contains a combination of different exons due to exon skipping or exon inclusion, deletion of one or more exons, or deletion or addition of sequences (e.g., intron sequences) that are not normally present in the spliced mRNA. In an embodiment, AC hybridization with a target sequence in a pre-mRNA molecule restores the natural splicing of the mutated pre-mRNA sequence. In an embodiment, AC hybridization results in selective splicing of the target pre-mRNA. In an embodiment, AC hybridization results in exon inclusion or exon skipping of one or more exons. In an embodiment, the exon sequence of the jump includes a frameshift mutation, a nonsense mutation, or a missense mutation. In an embodiment, the exon sequence of the jump includes a nucleic acid deletion, substitution, or insertion. In an embodiment, the exon of the jump does not itself include a sequence mutation, but the adjacent exon includes a mutation that causes a frameshift mutation or a nonsense mutation. In embodiments, deletion of exons that do not contain sequence mutations restores the reading frame of mature mRNA. In embodiments, hybridization of AC to target sequences within target pre-mRNA results in preferential expression of wild-type target protein isoforms. In embodiments, hybridization of AC to target sequences within target pre-mRNA results in expression of re-spliced target proteins comprising active fragments of wild-type target proteins.
反义机制经由反义化合物与靶核酸的杂交来发挥作用。在实施方案中，AC与其靶序列杂交抑制靶蛋白的表达。在实施方案中，AC与其靶序列杂交抑制一种或多种野生型靶蛋白异构体的表达。在实施方案中，AC与其靶序列杂交上调靶蛋白的表达。在实施方案中，AC与其靶序列杂交增加一种或多种野生型靶蛋白异构体的表达。The antisense mechanism works via hybridization of antisense compounds to target nucleic acids. In embodiments, AC hybridizes to its target sequence to inhibit the expression of a target protein. In embodiments, AC hybridizes to its target sequence to inhibit the expression of one or more wild-type target protein isomers. In embodiments, AC hybridizes to its target sequence to upregulate the expression of a target protein. In embodiments, AC hybridizes to its target sequence to increase the expression of one or more wild-type target protein isomers.
本公开的AC的功效可通过评价由其施用所影响的反义活性来评估。如本文所用，术语“反义活性”是指可归因于反义化合物与其靶核酸杂交的任何可检测和/或可测量的活性。此种检测和/或测量可以是直接或间接的。在实施方案中，反义活性是通过检测和/或测量靶蛋白的量来评估。在实施方案中，反义活性是通过检测和/或测量再剪接靶蛋白的量来评估。在实施方案中，反义活性是通过检测和/或测量靶核酸和/或切割的靶核酸和/或选择性剪接的靶核酸的量来评估。The efficacy of the AC of the present disclosure can be evaluated by evaluating the antisense activity affected by its administration. As used herein, the term "antisense activity" refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound with its target nucleic acid. Such detection and/or measurement can be direct or indirect. In an embodiment, the antisense activity is evaluated by detecting and/or measuring the amount of the target protein. In an embodiment, the antisense activity is evaluated by detecting and/or measuring the amount of the re-splicing target protein. In an embodiment, the antisense activity is evaluated by detecting and/or measuring the amount of the target nucleic acid and/or the cut target nucleic acid and/or the selectively spliced target nucleic acid.
反义化合物设计Antisense compound design
根据本公开的AC的设计将取决于所靶向的序列。将AC靶向特定的靶核酸分子可以是多步骤过程。所述过程通常从靶核酸的鉴定开始，所述靶核酸的表达将被调节。如本文所用，术语“靶核酸”和“编码靶基因的核酸”涵盖编码所选择的靶基因的DNA、从此种DNA转录的RNA(包括前mRNA和mRNA)以及从此种RNA衍生的cDNA。例如，靶核酸可以是表达与特定病症或疾病病况相关的细胞基因(或由所述基因转录的mRNA)，或感染原的核酸分子。The design of AC according to the present disclosure will depend on the sequence being targeted. Targeting AC to a specific target nucleic acid molecule can be a multi-step process. The process generally starts with the identification of the target nucleic acid, the expression of which will be regulated. As used herein, the terms "target nucleic acid" and "nucleic acid encoding a target gene" encompass DNA encoding a selected target gene, RNA transcribed from such DNA (including pre-mRNA and mRNA), and cDNA derived from such RNA. For example, a target nucleic acid can be a cellular gene (or mRNA transcribed from such a gene) expressing a particular disorder or disease condition, or a nucleic acid molecule of an infectious agent.
本领域技术人员将能够设计、合成和筛选不同核碱基序列的反义化合物，以鉴定产生反义活性的序列。例如，可设计改变靶前mRNA的剪接或抑制靶蛋白的表达的反义化合物。用于设计、合成和筛选针对预选靶核酸的反义活性的反义化合物的方法可见于例如由Stanley T.Crooke编辑的“Antisense Drug Technology，Principles，Strategies，andApplications”，CRC Press，Boca Raton，Florida，其全文以引用方式并入以用于任何目的。Those skilled in the art will be able to design, synthesize and screen antisense compounds of different nucleobase sequences to identify sequences that produce antisense activity. For example, antisense compounds that alter the splicing of target pre-mRNA or inhibit the expression of target protein can be designed. Methods for designing, synthesizing and screening antisense compounds for antisense activity against preselected target nucleic acids can be found in, for example, "Antisense Drug Technology, Principles, Strategies, and Applications," edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida, which is incorporated by reference in its entirety for any purpose.
在实施方案中，反义化合物包含修饰的核苷、修饰的核苷间键联和/或缀合基团。In an embodiment, the antisense compounds comprise modified nucleosides, modified internucleoside linkages and/or conjugated groups.
在实施方案中，反义化合物是“三环-DNA(tc-DNA)”，其是指一类受约束的DNA类似物，其中每个核苷酸通过引入环丙烷环而被修饰以限制主链的构象柔性并且优化扭转角γ的主链几何形状。含同碱基腺嘌呤和胸腺嘧啶的tc-DNA与互补的RNA形成非常稳定的A-T碱基对。In an embodiment, the antisense compound is "tricyclic-DNA (tc-DNA)", which refers to a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and optimize the backbone geometry of the torsion angle γ. tc-DNA containing the same bases adenine and thymine forms very stable A-T base pairs with complementary RNA.
核苷Nucleosides
在实施方案中，提供了包含连接核苷的反义化合物。在实施方案中，一些或所有的核苷是修饰的核苷。在实施方案中，一个或多个核苷包含修饰的核碱基。在实施方案中，一个或多个核苷包含修饰的糖。化学修饰的核苷通常用于掺入反义化合物中以增强一种或多种特性，诸如核酸酶抗性、药代动力学或对靶RNA的亲和力。In embodiments, antisense compounds comprising linked nucleosides are provided. In embodiments, some or all of the nucleosides are modified nucleosides. In embodiments, one or more nucleosides comprise modified core bases. In embodiments, one or more nucleosides comprise modified sugars. Chemically modified nucleosides are typically used for incorporation into antisense compounds to enhance one or more characteristics, such as nuclease resistance, pharmacokinetics or affinity to target RNA.
通常，核碱基是含有能够与另一核酸的碱基氢键合的一个或多个原子或原子团的任何基团。除了“未修饰的”或“天然的”核碱基诸如嘌呤核碱基腺嘌呤(A)和鸟嘌呤(G)以及嘧啶核碱基胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)外，本领域技术人员已知的许多修饰的核碱基或核碱基模拟物也适用于本文所述的化合物。术语修饰的核碱基和核碱基模拟物可重叠，但通常修饰的核碱基是指在结构上与亲本核碱基类似的核碱基，例如像7-脱氮嘌呤、5-甲基胞嘧啶或G-钳，而核碱基模拟物将包括更复杂的结构，例如像三环吩噁嗪核碱基模拟物。用于制备上述修饰的核碱基的方法是本领域技术人员熟知的。Typically, a nucleobase is any group containing one or more atoms or groups of atoms that can hydrogen bond with the base of another nucleic acid. In addition to "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimetics known to those skilled in the art are also suitable for use in the compounds described herein. The terms modified nucleobase and nucleobase mimetics can overlap, but generally modified nucleobases refer to nucleobases that are structurally similar to the parent nucleobase, such as 7-deazapurines, 5-methylcytosine or G-clamps, while nucleobase mimetics will include more complex structures, such as tricyclic phenoxazine nucleobase mimetics. Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.
在实施方案中，本文提供的AC包含一个或多个具有修饰的糖部分的核苷。在实施方案中，天然核苷的呋喃糖基糖环可以多种方式被修饰，包括但不限于添加取代基、桥接两个非偕环原子以形成双环核酸(BNA)以及用原子或基团诸如-S-、-N(R)-C(R1)(R2)取代4’-位的环氧。修饰的糖部分是众所周知的，并且可用于改变(典型地是增加)反义化合物对其靶标的亲和力和/或增加核酸酶抗性。修饰的糖的代表性列表包括但不限于非双环取代的糖，尤其是具有2’-F、2’-OCH3或2’-O(CH2)2-OCH3取代基的非双环2’-取代的糖；和4’-硫代修饰的糖。糖也可用糖模拟基团等替换，例如，呋喃糖环可用吗啉环替换。用于制备修饰的糖的方法是本领域技术人员熟知的。一些教导制备此类修饰的糖的代表性专利和出版物包括但不限于美国专利：4,981,957；5,118,800；5,319,080；5,359,044；5,393,878；5,446,137；5,466,786；5,514,785；5,519,134；5,567,811；5,576,427；5,591,722；5,597,909；5,610,300；5,627,053；5,639,873；5,646,265；5,658,873；5,670,633；5,792,747；5,700,920；和6,600,032；以及WO 2005/121371。In an embodiment, AC provided herein comprises one or more nucleosides with modified sugar moieties. In an embodiment, the furanosyl sugar ring of a natural nucleoside can be modified in a variety of ways, including but not limited to adding substituents, bridging two non-geminal ring atoms to form a bicyclic nucleic acid (BNA), and replacing the 4'-position epoxide with an atom or group such as -S-, -N(R)-C(R1 )(R2 ). Modified sugar moieties are well known and can be used to change (typically increase) the affinity of antisense compounds to their targets and/or increase nuclease resistance. A representative list of modified sugars includes but is not limited to non-bicyclic substituted sugars, especially non-bicyclic 2'-substituted sugars with 2'-F, 2'-OCH3 or 2'-O(CH2 )2 -OCH3 substituents; and 4'-thio modified sugars. Sugars can also be replaced with sugar mimetic groups, for example, the furanose ring can be replaced with a morpholine ring. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Patents: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and 6,600,032; and WO 2005/121371.
在实施方案中，核苷包含双环修饰的糖(BNA)，包括LNA(4’-(CH2)-O-2’桥)、2’-硫代-LNA(4’-(CH2)-S-2’桥)、2’-氨基-LNA(4’-(CH2)-NR-2’桥)、ENA(4’-(CH2)2-O-2’桥)、4’-(CH2)3-2’桥接的BNA、4’-(CH2CH(CH3))-2’桥接的BNA″cEt(4’-(CH(CH3)-O-2’桥)和cMOEBNA(4’-(CH(CH2OCH3)-O-2’桥)。在专利文献以及科学文献中已制备并公开了某些此类BNA(参见例如Srivastava等人J.Am.Chem.Soc.2007，ACS Advanced online publication，10.1021/ja071106y，Albaek等人J.Org.Chem.，2006，71，7731-7740，Fluiter等人Chembiochem 2005，6，1104-1109，Singh等人，Chem.Commun.，1998，4，455-456；Koshkin等人，Tetrahedron，1998，54，3607-3630；Wahlestedt等人，Proc.Natl.Acad.Sci.U.S.A.，2000，97，5633-5638；Kumar等人，Bioorg.Med.Chem.Lett.，1998，8，2219-2222；WO 94/14226；WO 2005/021570；Singh等人，J.Org.Chem.，1998，63，10035-10039，WO 2007/090071；公开BNA的已发布美国专利和已公布申请的实例包括例如美国专利号7,053,207；6,268,490；6,770,748；6,794,499；7,034,133；和6,525,191；以及美国授权前公开号2004-0171570；2004-0219565；2004-0014959；2003-0207841；2004-0143114；和20030082807。In an embodiment, the nucleoside comprises a bicyclic modified sugar (BNA), including LNA (4'-(CH2 )-0-2' bridge), 2'-thio-LNA (4'-(CH2 )-S-2' bridge), 2'-amino-LNA (4'-(CH2 )-NR-2' bridge), ENA (4'-(CH2 )2-0-2' bridge), 4'-(CH2 )3-2 ' bridged BNA, 4'-(CH2CH (CH3 ))-2' bridged BNA"cEt (4'-(CH(CH3 )-0-2' bridge), and cMOEBNA (4'-(CH(CH2OCH3 )-0-2 ' bridge). Certain such BNAs have been prepared and disclosed in the patent literature as well as in the scientific literature (see, e.g., Srivastava et al. J. Am. Chem. Soc. 2007, ACS Advanced Online publication, 10.1021/ja071106y, Albaek et al. J. Org. Chem., 2006, 71, 7731-7740, Fluiter et al. Chembiochem 2005, 6, 1104-1109, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94/14226; WO 2005/021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039, WO 2007/090071; Examples of issued U.S. patents and published applications disclosing BNAs include, for example, U.S. Patent Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.
本文还提供了“锁核酸”(LNA)，其中核糖基糖环的2’-羟基连接到糖环的4’碳原子，从而形成2’-C，4’-C-氧亚甲基键联以形成双环糖部分(综述于Elayadi等人，Curr.Opinion Invens.Drugs，2001，2，558-561；Braasch等人，Chem.Biol.，2001，81-7；和Orum等人，Curr.Opinion Mol.Ther.，2001，3，239-243；还参见美国专利：6,268,490和6,670,461)。所述键联可以是桥接2’氧原子和4’碳原子的亚甲基(-CH2-)基团，为此术语LNA用于双环部分；在所述位置上为亚乙基的情况下，使用术语ENATM(Singh等人，Chem.Commun.，1998，4，455-456；ENATM：Morita等人，Bioorganic Medicinal Chemistry，2003，11，2211-2226)。LNA和其他双环糖类似物显示出与互补DNA和RNA的非常高的双链体热稳定性(Tm＝+3至+10℃)、对3’-核酸外切降解的稳定性和良好的溶解性。含有LNA的有效并且无毒的反义寡核苷酸已有描述(Wahlestedt等人，Proc.Natl.Acad.Sci.U.S.A.，2000，97，5633-5638)。Also provided herein are "locked nucleic acids" (LNAs) in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage to form a bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 81-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Patents: 6,268,490 and 6,670,461). The linkage may be a methylene (-CH2- ) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic portion; in the case of an ethylene group at said position, the term ENA™ is used (Singh et al., Chem. Commun., 1998, 4, 455-456; ENA™ : Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogs show very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability to 3'-exonucleolytic degradation and good solubility. Effective and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).
也已研究的LNA异构体是α-L-LNA，其已显示对3’-外切核酸酶具有改善的稳定性。将α-L-LNA掺入显示有效反义活性的反义间隔体和嵌合体中(Frieden等人，Nucleic AcidsResearch，2003，21，6365-6372)。An LNA isomer that has also been studied is α-L-LNA, which has been shown to have improved stability against 3'-exonucleases. α-L-LNA has been incorporated into antisense spacers and chimeras that have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
LNA单体腺嘌呤、胞嘧啶、鸟嘌呤、5-甲基-胞嘧啶、胸腺嘧啶和尿嘧啶的合成和制备以及它们的寡聚化和核酸识别特性已有描述(Koshkin等人，Tetrahedron，1998，54，3607-3630)。LNA及其制备也描述于WO 98/39352和WO 99/14226中。The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil as well as their oligomerization and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNA and its preparation are also described in WO 98/39352 and WO 99/14226.
还制备了LNA的类似物，硫代磷酸-LNA和2’-硫代-LNA(Kumar等人，Bioorg.Med.Chem.Lett.，1998，8，2219-2222)。含有寡脱氧核糖核苷酸双链体作为核酸聚合酶底物的锁核苷类似物的制备也已有描述(Wengel等人，WO 99/14226)。一种新的构象受限的高亲和力寡核苷酸类似物2’-氨基-LNA的合成已在本领域中描述(Singh等人，J.Org.Chem.，1998，63，10035-10039)。另外，已制备了2’-氨基-LNA和2’-甲基氨基-LNA，并且先前已报道了它们与互补RNA和DNA链的双链体的热稳定性。Analogs of LNA, phosphorothioate-LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as nucleic acid polymerase substrates has also been described (Wengel et al., WO 99/14226). The synthesis of a new conformationally restricted high affinity oligonucleotide analog 2'-amino-LNA has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino-LNA and 2'-methylamino-LNA have been prepared, and their thermal stability in duplexes with complementary RNA and DNA chains has been previously reported.
核苷间键联Internucleoside linkage
本文描述了核苷间连接基团，其将核苷或另外修饰的单体单元连接在一起，从而形成反义化合物。核苷间连接基团的两个主要类别是通过磷原子的存在或不存在来定义。代表性含磷核苷间键联包括但不限于磷酸二酯、磷酸三酯、甲基磷酸酯、氨基磷酸酯(包括二氨基磷酸酯)和硫代磷酸酯。代表性不含磷的核苷间连接基团包括但不限于亚甲基甲基亚氨基(-CH2-N(CH3)-O-CH2-)、硫代二酯(-O-C(O)-S-)、硫代氨基甲酸酯(-O-C(O)(NH)-S-)；硅氧烷(-O-Si(H)2-O-)；和N，N’-二甲肼(-CH2-N(CH3)-N(CH3)-)。具有非磷核苷间连接基团的反义化合物称为寡核苷。与天然磷酸二酯键联相比，修饰的核苷间键联可用于改变(典型地是增加)反义化合物的核酸酶抗性。具有手性原子的核苷间键联可被制备成外消旋的、手性的或作为混合物。代表性手性核苷间键联包括但不限于烷基膦酸酯和硫代磷酸酯。含磷和不含磷的键联的制备方法是本领域技术人员熟知的。Described herein is an internucleoside linking group that links nucleosides or additionally modified monomeric units together to form antisense compounds. The two main categories of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate (including diaminophosphorate) and thiophosphate. Representative phosphorus-free internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2 -N(CH3 )-O-CH2 -), thiodiester (-OC(O)-S-), thiocarbamate (-OC(O)(NH)-S-); Siloxane (-O-Si(H)2 -O-); and N, N'-dimethylhydrazine (-CH2 -N(CH3 )-N(CH3 )-). Antisense compounds with non-phosphorus internucleoside linking groups are referred to as oligonucleosides. Compared to natural phosphodiester linkages, modified internucleoside linkages can be used to change (typically increase) the nuclease resistance of antisense compounds. Internucleoside linkages with chiral atoms can be prepared as racemic, chiral or as mixtures. Representative chiral internucleoside linkages include, but are not limited to, alkyl phosphonates and thiophosphates. The preparation methods of phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.
在实施方案中，磷酸基团可连接到糖的2’、3’或5’羟基部分。在形成寡核苷酸中，磷酸基团共价连接彼此相邻的核苷以形成线性聚合化合物。在寡核苷酸内，磷酸基团通常被称为形成寡核苷酸的核苷间主链。RNA和DNA的正常键联或主链是3’至5’磷酸二酯键联。In embodiments, the phosphate group may be connected to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming an oligonucleotide, the phosphate group covalently links adjacent nucleosides to form a linear polymeric compound. In an oligonucleotide, the phosphate group is generally referred to as forming the internucleoside backbone of an oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
缀合基团Conjugated Group
在实施方案中，通过一个或多个缀合基团的共价附接来修饰AC。通常，缀合基团修饰所附接AC的一种或多种特性，包括但不限于药效学、药代动力学、结合、吸收、细胞分布、细胞摄取、电荷和清除。缀合基团在化学领域中是常规使用的，并且直接地或经由任选的连接部分或连接基团连接到亲本化合物，诸如AC。缀合基团包括但不限于嵌入剂、报道分子、聚胺、聚酰胺、聚乙二醇、硫醚、聚醚、胆固醇、硫胆固醇、胆酸部分、叶酸、脂质、磷脂、生物素、吩嗪、菲啶、蒽醌、金刚烷、吖啶、荧光素、罗丹明、香豆素和染料。在实施方案中，缀合基团是聚乙二醇(PEG)，并且PEG与AC或环肽缀合。In an embodiment, AC is modified by covalent attachment of one or more conjugated groups. Typically, the conjugated group modifies one or more properties of the attached AC, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cellular uptake, charge and clearance. Conjugated groups are conventionally used in the chemical field and are directly or via an optional linking moiety or linking group connected to a parent compound such as AC. Conjugated groups include but are not limited to intercalators, reporters, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dyes. In an embodiment, the conjugated group is polyethylene glycol (PEG), and PEG is conjugated to AC or cyclic peptides.
缀合基团包括脂质部分，诸如胆固醇部分(Letsinger等人，Proc.Natl.Acad.Sci.USA，1989，86，6553)；胆酸(Manoharan等人，Bioorg.Med.Chem.Lett.，1994，4，1053)；硫醚，例如己基-S-三苯甲基硫醇(Manoharan等人，Ann.N.Y.Acad.Sci.，1992，660，306；Manoharan等人，Bioorg.Med.Chem.Let.，1993，3，2765)；硫胆固醇(Oberhauser等人，Nucl.Acids Res.，1992，20，533)；脂族链，例如十二烷二醇或十一烷基残基(Saison-Behmoaras等人，EMBO J.，1991，10，111；Kabanov等人，FEBSLett.，1990，259，327；Svinarchuk等人，Biochimie，1993，75，49)；磷脂，例如二-十六烷基外消旋甘油或三乙基铵-1，2-二-O-十六烷基外消旋甘油-3-H-膦酸酯(Manoharan等人，Tetrahedron Lett.，1995，36，3651；Shea等人，Nucl.Acids Res.，1990，18，3777)；聚胺或聚乙二醇链(Manoharan等人，Nucleosides&Nucleotides，1995，14，969)；金刚烷乙酸(Manoharan等人，Tetrahedron Lett.，1995，36，3651)；棕榈基部分(Mishra等人，Biochim.Biophys.Acta，1995，1264，229)；或十八胺或己基氨基-羰基-氧胆固醇部分(Crooke等人，J.Pharmacol.Exp.Ther.，1996，277，923)。Conjugated groups include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids, such as di-hexadecyl racemic glycerol or triethylammonium-1,2-di-O-hexadecyl racemic glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); Lett., 1995, 36, 3651); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).
连接基团或双官能连接部分诸如本领域已知的那些可包括在本文提供的化合物中。连接基团可用于将化学官能团、缀合基团、报道基团和其他基团附接到亲本化合物(例如像AC)的选择性位点。在实施方案中，双官能连接部分包含具有两个官能团的烃基部分。在实施方案中，选择官能团中的一者与感兴趣的亲本分子或化合物结合，并且选择另一者与基本上任何选择的基团诸如化学官能团或缀合基团结合。可使用本文所述的任何接头。在实施方案中，接头包含重复单元诸如乙二醇或氨基酸单元的链结构或寡聚物。在双官能连接部分中使用的官能团的实例包括但不限于用于与亲核基团反应的亲电体和用于与亲电基团反应的亲核体。在实施方案中，双官能连接部分可包括氨基、羟基、羧酸、硫醇、不饱和度(例如双键或三键)等。双官能连接部分的一些非限制性实例包括8-氨基-3，6-二氧杂辛酸(ADO)、琥珀酰亚胺基4-(N-马来酰亚胺基甲基)环己烷-1-羧酸酯(SMCC)和6-氨基己酸(AHEX或AHA)。其他连接基团包括但不限于取代的C1-C10烷基、取代或未取代的C2-C10烯基或取代或未取代的C2-C10炔基，其中取代基基团的非限制性列表包括羟基、氨基、烷氧基、羧基、苄基、苯基、硝基、硫醇、硫代烷氧基、卤素、烷基、芳基、烯基和炔基。Linking groups or bifunctional linking moieties such as those known in the art may be included in the compounds provided herein. Linking groups may be used to attach chemical functional groups, conjugated groups, reporter groups and other groups to the selective sites of parent compounds (e.g., such as AC). In embodiments, bifunctional linking moieties include alkyl moieties with two functional groups. In embodiments, one of the functional groups is selected to be combined with a parent molecule or compound of interest, and another is selected to be combined with substantially any selected group such as a chemical functional group or a conjugated group. Any joint described herein may be used. In embodiments, joints include chain structures or oligomers of repeating units such as ethylene glycol or amino acid units. Examples of the functional groups used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In embodiments, bifunctional linking moieties may include amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double bonds or triple bonds), etc. Some non-limiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1 -C10 alkyl, substituted or unsubstituted C2 -C10 alkenyl, or substituted or unsubstituted C2 -C10 alkynyl, wherein a non-limiting list of substituent groups includes hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.
在实施方案中，AC可连接到10精氨酸-丝氨酸二肽重复序列。与10精氨酸-丝氨酸二肽重复序列连接的用于人工募集剪接增强因子的AC已在体外应用，以诱导包含突变的BRCA1和SMN2外显子，否则这些外显子将被跳跃。参见Cartegni和Krainer 2003，通过引用并入本文。In embodiments, the AC may be linked to 10 arginine-serine dipeptide repeats. AC linked to 10 arginine-serine dipeptide repeats for artificial recruitment of splicing enhancers has been used in vitro to induce BRCA1 and SMN2 exons containing mutations that would otherwise be skipped. See Cartegni and Krainer 2003, incorporated herein by reference.
在实施方案中，AC的长度可以是5至50个核苷酸(例如，5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50，包括其中的所有值和范围)。在实施方案中，AC的长度可以是5-10个核苷酸。在实施方案中，AC的长度可以是10-15个核苷酸。在实施方案中，AC的长度可以是15-20个核苷酸。在实施方案中，AC的长度可以是20-25个核苷酸。在实施方案中，AC的长度可以是25-30个核苷酸。在实施方案中，AC的长度可以是30-35个核苷酸。在实施方案中，AC的长度可以是35-40个核苷酸。在实施方案中，AC的长度可以是40-45个核苷酸。在实施方案中，AC的长度可以是45-50个核苷酸。In an embodiment, the length of AC can be 5 to 50 nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, including all values and ranges therein). In an embodiment, the length of AC can be 5-10 nucleotides. In an embodiment, the length of AC can be 10-15 nucleotides. In an embodiment, the length of AC can be 15-20 nucleotides. In an embodiment, the length of AC can be 20-25 nucleotides. In an embodiment, the length of AC can be 25-30 nucleotides. In an embodiment, the length of AC can be 30-35 nucleotides. In an embodiment, AC may be 35-40 nucleotides in length. In an embodiment, AC may be 40-45 nucleotides in length. In an embodiment, AC may be 45-50 nucleotides in length.
在实施方案中，AC结合到编码抗肌萎缩蛋白的人DMD基因。在实施方案中，AC结合到DMD的外显子44的至少一部分。在实施方案中，AC结合到DMD的外显子44的3’侧翼的至少一部分。在实施方案中，AC结合到DMD的外显子44的5’侧翼内含子的至少一部分。在实施方案中，结合到DMD的外显子44的AC的长度为约18至约30个核酸，例如长度为约18、约19、约20、约21、约22、约23、约24、约25、约26、约27、约28、约29或约30个核酸。In embodiments, AC is bound to the human DMD gene encoding dystrophin. In embodiments, AC is bound to at least a portion of exon 44 of DMD. In embodiments, AC is bound to at least a portion of the 3' flank of exon 44 of DMD. In embodiments, AC is bound to at least a portion of the 5' flank intron of exon 44 of DMD. In embodiments, the length of AC bound to exon 44 of DMD is about 18 to about 30 nucleic acids, for example, a length of about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 or about 30 nucleic acids.
在实施方案中，DMD的外显子44从5’至3’的核酸序列是：In an embodiment, the nucleic acid sequence from 5' to 3' of exon 44 of DMD is:
在实施方案中，DMD的外显子44从5’至3’的核酸序列是：GGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTITCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAG(SEQ ID NO：2)。在实施方案中，外显子44的序列在SEQ ID NO：1的5’端包含1、2、3、4或5个核苷酸或更多。在实施方案中，外显子44的序列在SEQ ID NO：2的5’端包含1、2、3、4或5个核苷酸或更多。在实施方案中，外显子44的序列在SEQ ID NO：1的3’端包含1、2、3、4或5个核苷酸或更多。在实施方案中，外显子44的序列在SEQ ID NO：2的3’端包含1、2、3、4或5个核苷酸或更多。In an embodiment, the nucleic acid sequence of exon 44 of DMD from 5' to 3' is: GGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTITCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAG (SEQ ID NO: 2). In an embodiment, the sequence of exon 44 comprises 1, 2, 3, 4 or 5 nucleotides or more at the 5' end of SEQ ID NO: 1. In an embodiment, the sequence of exon 44 comprises 1, 2, 3, 4 or 5 nucleotides or more at the 5' end of SEQ ID NO: 2. In an embodiment, the sequence of exon 44 comprises 1, 2, 3, 4 or 5 nucleotides or more at the 3' end of SEQ ID NO: 1. In an embodiment, the sequence of exon 44 comprises 1, 2, 3, 4 or 5 nucleotides or more at the 3' end of SEQ ID NO: 2.
在实施方案中，AC包含SEQ ID NO：1的18个连续核苷酸(例如，AC是18-mer)，其中18-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130或131处。在实施方案中，AC包含SEQ ID NO：2的18个连续核苷酸(例如，AC是18-mer)，其中18-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131或132处。In an embodiment, AC comprises 18 consecutive nucleotides of SEQ ID NO: 1 (eg, AC is an 18-mer), wherein the first nucleotide of the 18-mer starts at SEQ ID NO: 4,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 2, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 or 131. In an embodiment, AC comprises 18 consecutive nucleotides of SEQ ID NO: 2 (eg, AC is an 18-mer), wherein the first nucleotide of the 18-mer starts at SEQ ID NO: 4,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 or 132.
在实施方案中，AC包含SEQ ID NO：1的19个连续核苷酸(例如，AC是19-mer)，其中19-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129或130处。在实施方案中，AC包含SEQ ID NO：2的19个连续核苷酸(例如，AC是19-mer)，其中19-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130或131处。In an embodiment, AC comprises 19 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 19-mer), wherein the first nucleotide of the 19-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130. In an embodiment, AC comprises 19 consecutive nucleotides of SEQ ID NO: 2 (eg, AC is a 19-mer), wherein the first nucleotide of the 19-mer starts at SEQ ID NO: NO: 2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 2, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 or 131.
在实施方案中，AC包含SEQ ID NO：1的20个连续核苷酸(例如，AC是20-mer)，其中20-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128或129处。在实施方案中，AC包含SEQ ID NO：2的20个连续核苷酸(例如，AC是20-mer)，其中20-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129或130处。41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 129. In an embodiment, AC comprises 20 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 20-mer), wherein the first nucleotide of the 20-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130.
在实施方案中，AC包含SEQ ID NO：1的21个连续核苷酸(例如，AC是21-mer)，其中21-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127或128处。在实施方案中，AC包含SEQ ID NO：2的21个连续核苷酸(例如，AC是21-mer)，其中21-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128或129处。In an embodiment, AC comprises 21 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 21-mer), wherein the first nucleotide of the 21-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 124, 125, 126, 127, or 128. In an embodiment, AC comprises 21 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 21-mer), wherein the first nucleotide of the 21-mer starts at SEQ ID NO: 2. 4,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 129.
在实施方案中，AC包含SEQ ID NO：1的22个连续核苷酸(例如，AC是22-mer)，其中22-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126或127处。在实施方案中，AC包含SEQ ID NO：2的22个连续核苷酸(例如，AC是22-mer)，其中22-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127或128处。In an embodiment, AC comprises 22 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 22-mer), wherein the first nucleotide of the 22-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, or 127. In an embodiment, AC comprises 22 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 22-mer), wherein the first nucleotide of the 22-mer starts at SEQ ID NO: 4,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 or 128.
在实施方案中，AC包含SEQ ID NO：1的23个连续核苷酸(例如，AC是23-mer)，其中23-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125或126处。在实施方案中，AC包含SEQ ID NO：2的23个连续核苷酸(例如，AC是23-mer)，其中23-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126或127处。43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105 124, 125 or 126. In an embodiment, AC comprises 23 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 23-mer), wherein the first nucleotide of the 23-mer starts at SEQ ID NO: 2. NO: 2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or 127.
在实施方案中，AC包含SEQ ID NO：1的24个连续核苷酸(例如，AC是24-mer)，其中24-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124或125处。在实施方案中，AC包含SEQ ID NO：2的24个连续核苷酸(例如，AC是24-mer)，其中24-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125或126处。In an embodiment, AC comprises 24 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 24-mer), wherein the first nucleotide of the 24-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 8, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125. In an embodiment, AC comprises 24 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 24-mer), wherein the first nucleotide of the 24-mer starts at SEQ ID NO: 45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 or 126.
在实施方案中，AC包含SEQ ID NO：1的25个连续核苷酸(例如，AC是25-mer)，其中25-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123或124处。在实施方案中，AC包含SEQID NO：2的25个连续核苷酸(例如，AC是25-mer)，其中25-mer的第一核苷酸起始于SEQ IDNO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124或125处。In an embodiment, AC comprises 25 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 25-mer), wherein the first nucleotide of the 25-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124. In an embodiment, AC comprises 25 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 25-mer), wherein the first nucleotide of the 25-mer starts at SEQ ID NO: IDNO:2 position 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125.
在实施方案中，AC包含SEQ ID NO：1的26个连续核苷酸(例如，AC是26-mer)，其中26-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122或123处。在实施方案中，AC包含SEQ IDNO：2的26个连续核苷酸(例如，AC是26-mer)，其中26-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123或124处。43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 17, 18, 19, 20, 21, 22, 23 or 24. In an embodiment, AC comprises 26 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 26-mer), wherein the first nucleotide of the 26-mer starts at SEQ ID NO: 2. NO: 2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 or 124.
在实施方案中，AC包含SEQ ID NO：1的27个连续核苷酸(例如，AC是27-mer)，其中27-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121或122处。在实施方案中，AC包含SEQ ID NO：2的27个连续核苷酸(例如，AC是27-mer)，其中27-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122或123处。In an embodiment, AC comprises 27 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 27-mer), wherein the first nucleotide of the 27-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, or 122. In an embodiment, AC comprises 27 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 27-mer), wherein the first nucleotide of the 27-mer starts at SEQ ID NO: NO: 2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 123.
在实施方案中，AC包含SEQ ID NO：1的28个连续核苷酸(例如，AC是28-mer)，其中28-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120或121处。在实施方案中，AC包含SEQ ID NO：2的28个连续核苷酸(例如，AC是28-mer)，其中28-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121或122处。In an embodiment, AC comprises 28 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 28-mer), wherein the first nucleotide of the 28-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121. In an embodiment, AC comprises 28 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 28-mer), wherein the first nucleotide of the 28-mer starts at SEQ ID NO: NO: 2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122.
在实施方案中，AC包含SEQ ID NO：1的29个连续核苷酸(例如，AC是29-mer)，其中29-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119或120处。在实施方案中，AC包含SEQ ID NO：2的29个连续核苷酸(例如，AC是29-mer)，其中29-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120或121处。41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120. In an embodiment, AC comprises 29 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 29-mer), wherein the first nucleotide of the 29-mer starts at SEQ ID NO: 2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 or 121.
在实施方案中，AC包含SEQ ID NO：1的30个连续核苷酸(例如，AC是30-mer)，其中30-mer的第一核苷酸起始于SEQ ID NO：1的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118或119处。在实施方案中，AC包含SEQ ID NO：2的30个连续核苷酸(例如，AC是30-mer)，其中30-mer的第一核苷酸起始于SEQ ID NO：2的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119或120处。In an embodiment, AC comprises 30 consecutive nucleotides of SEQ ID NO: 1 (e.g., AC is a 30-mer), wherein the first nucleotide of the 30-mer starts at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 114, 115, 116, 117, 118, or 119. In an embodiment, AC comprises 30 consecutive nucleotides of SEQ ID NO: 2 (e.g., AC is a 30-mer), wherein the first nucleotide of the 30-mer starts at SEQ ID NO: 2. NO: 2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120.
在实施方案中，结合到DMD的外显子44的AC选自表6A-6M中所示的核酸序列中的任一种、其反向互补序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核酸序列同一性的序列。In an embodiment, AC that binds to exon 44 of DMD is selected from any one of the nucleic acid sequences shown in Tables 6A-6M, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto.
在实施方案中，结合到DMD的外显子44的序列的AC选自表6A-6M中所示的核酸序列中的任一种。在实施方案中，结合到DMD的外显子44的AC选自表6A-6M内的核酸序列中的任一种、其反向互补序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核酸序列同一性的序列。在实施方案中，结合到DMD的外显子44的AC包含一种或多种修饰的核酸、一种或多种修饰的核苷酸键联或它们的组合。在实施方案中，结合到外显子44的AC包含一个或多个吗啉环、一个或多个二氨基磷酸酯键联或它们的组合。在实施方案中，结合到DMD的外显子44的AC是具有选自表6A-6M内的核酸序列中的任一种的序列、其反向互补序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核酸序列同一性的序列的反义二氨基磷酸酯吗啉代寡聚物(PMO)。In embodiments, the AC of the sequence of exon 44 that is attached to DMD is selected from any one of the nucleic acid sequences shown in Table 6A-6M. In embodiments, the AC of the exon 44 that is attached to DMD is selected from any one of the nucleic acid sequences in Table 6A-6M, its reverse complementary sequence or with it there is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of nucleic acid sequence. In embodiments, the AC of the exon 44 that is attached to DMD comprises one or more modified nucleic acids, one or more modified nucleotide linkages or their combinations. In embodiments, the AC that is attached to exon 44 comprises one or more morpholine rings, one or more diaminophosphoryl linkages or their combinations. In an embodiment, the AC that binds to exon 44 of DMD is an antisense phosphorodiamidate morpholino oligomer (PMO) having a sequence selected from any one of the nucleic acid sequences within Tables 6A-6M, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto.
在实施方案中，结合到DMD的外显子44的AC是5’-TGAAAACGCCGCCATTTCTCAACAG-3’。在实施方案中，结合到DMD的外显子44的AC是5’-ACTGTTCAGCTTCTGTTAGCCACTG-3’。在实施方案中，结合到DMD的外显子44的AC包含一种或多种修饰的核酸、一种或多种修饰的核苷酸键联或它们的组合。在实施方案中，结合到外显子44的AC包含一个或多个吗啉环、一个或多个二氨基磷酸酯键联或它们的组合。在实施方案中，结合到DMD的外显子44的AC是具有序列5’-TGAAAACGCCGCCATITCTCAACAG-3’的反义二氨基磷酸酯吗啉代寡聚物(PMO)。在实施方案中，结合到DMD的外显子44的AC是具有序列5’-ACTGTTCAGCTTCTGTTAGCCACTG-3’的反义二氨基磷酸酯吗啉代寡聚物(PMO)。In an embodiment, the AC that is bound to exon 44 of DMD is 5'-TGAAAACGCCGCCATTTCTCAACAG-3'. In an embodiment, the AC that is bound to exon 44 of DMD is 5'-ACTGTTCAGCTTCTGTTAGCCACTG-3'. In an embodiment, the AC that is bound to exon 44 of DMD comprises one or more modified nucleic acids, one or more modified nucleotide linkages, or a combination thereof. In an embodiment, the AC that is bound to exon 44 of DMD comprises one or more morpholine rings, one or more phosphorodiamidate linkages, or a combination thereof. In an embodiment, the AC that is bound to exon 44 of DMD is an antisense phosphorodiamidate morpholino oligomer (PMO) with the sequence 5'-TGAAAACGCCGCCATITCTCAACAG-3'. In an embodiment, the AC that is bound to exon 44 of DMD is an antisense phosphorodiamidate morpholino oligomer (PMO) with the sequence 5'-ACTGTTCAGCTTCTGTTAGCCACTG-3'.
表6A.与DMD的外显子44结合的18-merACTable 6A. 18-mer ACs that bind to exon 44 of DMD
表6B.与DMD的外显子44结合的19-mer ACTable 6B. 19-mer ACs that bind to exon 44 of DMD
表6C.与DMD的外显子44结合的20-mer ACTable 6C. 20-mer ACs that bind to exon 44 of DMD
表6D.与DMD的外显子44结合的21-mer ACTable 6D. 21-mer ACs that bind to exon 44 of DMD
表6E.与DMD的外显子44结合的22-mer ACTable 6E. 22-mer ACs that bind to exon 44 of DMD
表6F.与DMD的外显子44结合的23-mer ACTable 6F. 23-mer ACs that bind to exon 44 of DMD
表6G.与DMD的外显子44结合的24-mer ACTable 6G. 24-mer ACs that bind to exon 44 of DMD
表6H.与DMD的外显子44结合的25-mer ACTable 6H. 25-mer ACs that bind to exon 44 of DMD
表6I.与DMD的外显子44结合的26-mer ACTable 6I. 26-mer ACs that bind to exon 44 of DMD
表6J.与DMD的外显子44结合的27-mer ACTable 6J. 27-mer ACs that bind to exon 44 of DMD
表6K.与DMD的外显子44结合的28-mer ACTable 6K. 28-mer ACs that bind to exon 44 of DMD
表6L.与DMD的外显子44结合的29-mer dACTable 6L. 29-mer dACs that bind to exon 44 of DMD
表6M.与DMD的外显子44结合的30-mer ACTable 6M. 30-mer ACs that bind to exon 44 of DMD
在实施方案中，本文描述的任何AC，包括表6A-6M中的AC、其反向互补序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核酸序列同一性的序列，包含选自以下的至少一种修饰的核苷酸或核酸：硫代磷酸酯(PS)核苷酸、二氨基磷酸酯吗啉代(PMO)核苷酸、锁核酸(LNA)、肽核酸(PNA)、包含2’-O-甲基(2’-OMe)修饰的主链的核苷酸、2’O-甲氧基-乙基(2’-MOE)核苷酸、2’，4’约束乙基(cEt)核苷酸和2’-脱氧-2’-氟-β-D-阿糖核酸(2’F-ANA)。在实施方案中，AC与靶序列的杂交促进或诱导外显子44的剪接。在实施方案中，AC包含至少一种二氨基磷酸酯吗啉代(PMO)核苷酸。在实施方案中，1、2、3、4、5、6、7、8、9、10、11、12、13、1，4、15、16、17、18、19、20、21、22、23、24、25、26、2，7、28、29、30个或更多个核苷酸被修饰。在实施方案中，AC中的每个核苷酸是二氨基磷酸酯吗啉代(PMO)核苷酸。In an embodiment, any AC described herein, including AC in Tables 6A-6M, a reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto, comprises at least one modified nucleotide or nucleic acid selected from the group consisting of phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino (PMO) nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), nucleotides comprising a 2'-O-methyl (2'-OMe) modified backbone, 2'O-methoxy-ethyl (2'-MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides, and 2'-deoxy-2'-fluoro-β-D-arabino nucleic acid (2'F-ANA). In embodiments, hybridization of AC to a target sequence promotes or induces splicing of exon 44. In embodiments, AC comprises at least one phosphorodiamidate morpholino (PMO) nucleotide. In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2, 7, 28, 29, 30 or more nucleotides are modified. In embodiments, each nucleotide in AC is a phosphorodiamidate morpholino (PMO) nucleotide.
在实施方案中，化合物具有以下结构：In an embodiment, the compound has the following structure:
其中：in:
CPP是本文所述的环肽(也称为细胞穿透肽)；CPP is a cyclic peptide (also called a cell-penetrating peptide) as described herein;
L是接头；L is the connector;
B各自独立地是与靶序列中的碱基互补的核碱基；和B are each independently a nucleobase that is complementary to a base in the target sequence; and
n是1至50的整数。在实施方案中，B和n的和对应于表6A-6M中所示的序列、其反向互补序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核酸序列同一性的序列。n is an integer from 1 to 50. In an embodiment, the sum of B and n corresponds to a sequence shown in Tables 6A-6M, a reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto.
与AC缀合的环状细胞穿透肽(cCPP)Cyclic cell penetrating peptide (cCPP) conjugated to AC
环状细胞穿透肽(cCPP)可与AC缀合。Circular cell penetrating peptides (cCPPs) can be conjugated to AC.
AC可通过接头与cCPP缀合。货物部分可在末端羰基处与接头缀合以提供以下结构：AC can be conjugated to cCPP via a linker. The cargo moiety can be conjugated to the linker at the terminal carbonyl group to provide the following structure:
其中： in:
EP是环外肽，并且M、AASC、AC、x’、y和z’如上文所定义，*是与AASC的附接点。x’可以是1。y可以是4。z’可以是11。-(OCH2CH-2)x’-和/或-(OCH2CH-2)z’-可独立地被一个或多个氨基酸替换，包括例如甘氨酸、β-丙氨酸、4-氨基丁酸、5-氨基戊酸、6-氨基己酸或它们的组合。EP is an exocyclic peptide, and M, AASC , AC, x', y and z' are as defined above, * is the point of attachment to AASC . x' may be 1. y may be 4. z' may be 11. -(OCH2 CH-2 )x' - and/or -(OCH2 CH-2 )z' - may be independently replaced by one or more amino acids, including, for example, glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or a combination thereof.
内体逃逸载体(EEV)可包含环状细胞穿透肽(cCPP)、环外肽(EP)和接头，并且可与AC缀合以形成包含式(C)的结构的EEV-缀合物：The endosomal escape vector (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP), and a linker, and may be conjugated with AC to form an EEV-conjugate comprising a structure of formula (C):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
EP是如本文定义的环外肽；EP is an exocyclic peptide as defined herein;
AC如本文所定义；AC is as defined herein;
每个m独立地是0-3的整数；Each m is independently an integer from 0 to 3;
n是0-2的整数；n is an integer from 0 to 2;
x’是2-20的整数；x’ is an integer from 2 to 20;
y是1-5的整数；y is an integer from 1 to 5;
q是1-4的整数；并且q is an integer from 1 to 4; and
z’是2-20的整数。z’ is an integer from 2 to 20.
R1、R2、R3、R4、EP、AC、m、n、x’、y、q和z’如本文所定义。R1 , R2 , R3 , R4 , EP, AC, m, n, x′, y, q and z′ are as defined herein.
EEV可与AC缀合，并且EEV-缀合物可包含式(C-a)或(C-b)的结构：EEV can be conjugated to AC, and the EEV-conjugate can comprise a structure of formula (C-a) or (C-b):
或其质子化形式，其中EP、m和z如上文在式(C)中所定义。 or a protonated form thereof, wherein EP, m and z are as defined above in formula (C).
EEV可与AC缀合，并且EEV-缀合物可包含式(C-c)的结构：EEV can be conjugated to AC, and the EEV-conjugate can comprise a structure of formula (C-c):
或其质子化形式，其中EP、R1、R2、R3、R4和m如上文在式(III)中所定义；AA可以是如本文所定义的氨基酸；n可以是0-2的整数；x可以是1-10的整数；y可以是1-5的整数；并且z可以是1-10的整数。or a protonated form thereof, wherein EP, R1 , R2 , R3 , R4 and m are as defined above in formula (III); AA may be an amino acid as defined herein; n may be an integer from 0 to 2; x may be an integer from 1 to 10; y may be an integer from 1 to 5; and z may be an integer from 1 to 10.
EEV可与寡核苷酸AC缀合，并且EEV-寡核苷酸缀合物可包含式(C-1)、(C-2)、(C-3)或(C-4)的结构：EEV can be conjugated to oligonucleotide AC, and the EEV-oligonucleotide conjugate can comprise a structure of formula (C-1), (C-2), (C-3), or (C-4):
在上式中，EP是环外肽并且AC可具有15-30个核苷酸的序列，其与包含前mRNA序列中的DMD基因的外显子44的至少一部分的靶序列互补。在实施方案中，AC可选自表6A-6M中所示的寡核苷酸、其反向互补序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核酸序列同一性的序列。In the above formula, EP is an exocyclic peptide and AC may have a sequence of 15-30 nucleotides that is complementary to a target sequence comprising at least a portion of exon 44 of a DMD gene in a pre-mRNA sequence. In an embodiment, AC may be selected from the oligonucleotides shown in Tables 6A-6M, their reverse complementary sequences, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto.
在实施方案中，本文所述的化合物形成多聚体。在实施方案中，多聚化经由非共价相互作用发生，例如通过疏水相互作用、离子相互作用、氢键合或偶极-偶极相互作用。在实施方案中，化合物形成二聚体、三聚体、四聚体、五聚体、六聚体、七聚体、八聚体或九聚体。在实施方案中，化合物包含1、2、3、4、5、6、7、8、9或10个环肽。在实施方案中，化合物包含1、2、3、4、5、6、7、8、9或10个AC。在实施方案中，化合物包含1、2、3、4、5、6、7、8、9或10个EP。在实施方案中，化合物包含1至10个环肽和1至10个AC。在实施方案中，化合物包含1至10个环肽、1至10个AC或1至10个EP。In embodiments, the compounds described herein form polymers. In embodiments, multimerization occurs via non-covalent interactions, for example, by hydrophobic interactions, ionic interactions, hydrogen bonding or dipole-dipole interactions. In embodiments, the compounds form dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers or nonamers. In embodiments, the compounds comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cyclic peptides. In embodiments, the compounds comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ACs. In embodiments, the compounds comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 EPs. In embodiments, the compounds comprise 1 to 10 cyclic peptides and 1 to 10 ACs. In embodiments, the compounds comprise 1 to 10 cyclic peptides, 1 to 10 ACs or 1 to 10 EPs.
在实施方案中，本公开的化合物包含以下结构中的任一种。以下化合物仅是说明性的，并且以下结构中的任一种中的环肽、接头和AC中的任一种可被本文所述的环肽、接头或AC中的任一种替换。In an embodiment, a compound of the present disclosure comprises any of the following structures. The following compounds are illustrative only, and any of the cyclic peptides, linkers, and ACs in any of the following structures may be replaced with any of the cyclic peptides, linkers, or ACs described herein.
胞质递送效率Cytoplasmic delivery efficiency
对环状细胞穿透肽(cCPP)的修饰可提高胞质递送效率。通过将具有修饰的序列的cCPP的胞质递送效率与对照序列进行比较，可测量改善的胞质摄取效率。对照序列不包括修饰的序列中的特定置换氨基酸残基(包括但不限于精氨酸、苯丙氨酸和/或甘氨酸)，但在其他方面是相同的。Modification of cyclic cell penetrating peptides (cCPPs) can improve cytoplasmic delivery efficiency. By comparing the cytoplasmic delivery efficiency of cCPPs with modified sequences with control sequences, improved cytoplasmic uptake efficiency can be measured. The control sequence does not include specific replacement amino acid residues (including but not limited to arginine, phenylalanine and/or glycine) in the modified sequence, but is otherwise identical.
在实施方案中，与包含单独AC的化合物相比，包含环肽和AC的化合物具有改善的胞质摄取效率。胞质摄取效率可通过将包含环肽和AC的化合物的胞质递送效率与单独AC的胞质递送效率进行比较来测量。In an embodiment, the compound comprising the cyclic peptide and AC has improved cytoplasmic uptake efficiency compared to the compound comprising AC alone. The cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of the compound comprising the cyclic peptide and AC with the cytoplasmic delivery efficiency of AC alone.
如本文所用，胞质递送效率是指cCPP穿过细胞膜并进入细胞的胞质的能力。cCPP的胞质递送效率不一定依赖于受体或细胞类型。胞质递送效率可指绝对胞质递送效率或相对胞质递送效率。As used herein, cytoplasmic delivery efficiency refers to the ability of a cCPP to cross the cell membrane and enter the cytoplasm of a cell. The cytoplasmic delivery efficiency of a cCPP does not necessarily depend on receptors or cell types. The cytoplasmic delivery efficiency may refer to an absolute cytoplasmic delivery efficiency or a relative cytoplasmic delivery efficiency.
绝对胞质递送效率是生长培养基中cCPP(或cCPP-AC缀合物)的胞质浓度与cCPP(或cCPP-AC缀合物)的浓度的比率。相对胞质递送效率是指与对照cCPP在胞质中的浓度相比cCPP在胞质中的浓度。定量可通过荧光标记cCPP(例如，用FITC染料)并使用本领域熟知的技术测量荧光强度来实现。The absolute cytoplasmic delivery efficiency is the ratio of the cytoplasmic concentration of the cCPP (or cCPP-AC conjugate) to the concentration of the cCPP (or cCPP-AC conjugate) in the growth medium. The relative cytoplasmic delivery efficiency refers to the concentration of the cCPP in the cytoplasm compared to the concentration of the control cCPP in the cytoplasm. Quantification can be achieved by fluorescently labeling the cCPP (e.g., with a FITC dye) and measuring the fluorescence intensity using techniques well known in the art.
通过比较(i)被某种细胞类型(例如，HeLa细胞)内化的本发明cCPP的量与(ii)被相同细胞类型内化的对照cCPP的量来确定相对胞质递送效率。为了测量相对胞质递送效率，可将所述细胞类型在cCPP的存在下孵育指定的时间段(例如，30分钟、1小时、2小时等)，之后使用本领域已知的方法例如荧光显微术定量被细胞内化的cCPP的量。单独地，将相同浓度的对照cCPP在所述细胞类型的存在下孵育相同的时间段，并且对被细胞内化的对照cCPP的量进行定量。The relative cytoplasmic delivery efficiency is determined by comparing (i) the amount of the cCPP of the invention internalized by a certain cell type (e.g., HeLa cells) with (ii) the amount of the control cCPP internalized by the same cell type. To measure the relative cytoplasmic delivery efficiency, the cell type can be incubated in the presence of the cCPP for a specified period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), followed by quantification of the amount of the cCPP internalized by the cell using methods known in the art, such as fluorescence microscopy. Separately, the same concentration of the control cCPP is incubated in the presence of the cell type for the same period of time, and the amount of the control cCPP internalized by the cell is quantified.
相对胞质递送效率可通过测量具有修饰的序列的cCPP对胞内靶标的IC50并将具有修饰的序列的cCPP对对照序列的IC50(如本文所述)进行比较来确定。Relative cytoplasmic delivery efficiency can be determined by measuring theIC50 of a cCPP with a modified sequence for an intracellular target and comparing the cCPP with a modified sequence to theIC50 of a control sequence (as described herein).
与环(FfΦRrRrQ)相比，cCPP的相对胞质递送效率可在约50％至约450％的范围内，例如约60％、约70％、约80％、约90％、约100％、约110％、约120％、约130％、约140％、约150％、约160％、约170％、约180％、约190％、约200％、约210％、约220％、约230％、约240％、约250％、约260％、约270％、约280％、约290％、约300％、约310％、约320％、约330％、约340％、约350％、约360％、约370％、约380％、约390％、约400％、约410％、约420％、约430％、约440％、约450％、约460％、约470％、约480％、约490％、约500％、约510％、约520％、约530％、约540％、约550％、约560％、约570％、约580％或约590％，包括其间的所有值和子范围。与包含环(FfΦRrRrQ)的环肽相比，cCPP的相对胞质递送效率可提高大于约600％。Compared to ring (FfΦRrRrQ), the relative cytoplasmic delivery efficiency of cCPP can range from about 50% to about 450%, such as about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 600%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about 770%, about 780%, about 790%, about 800%, about 810%, about 820%, about 830%, about 84 %, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, or about 590%, including all values and subranges therebetween. The relative cytoplasmic delivery efficiency of cCPP can be increased by greater than about 600% compared to the cyclic peptide comprising the ring (FfΦRrRrQ).
绝对胞质递送效力为约40％至约100％，例如约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％，包括其间的所有值和子范围。The absolute cytoplasmic delivery efficacy is about 40% to about 100%, for example, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, including all values and subranges therebetween.
与在其他方面相同的序列相比，本公开的cCPP可将胞质递送效率提高约1.1倍至约30倍，例如，约1.2、约1.3、约1.4、约1.5、约1.6、约1.7、约1.8、约1.9、约2.0、约2.5、约3.0、约3.5、约4.0、约4.5、约5.0、约5.5、约6.0、约6.5、约7.0、约7.5、约8.0、约8.5、约9.0、约10、约10.5、约11.0、约11.5、约12.0、约12.5、约13.0、约13.5、约14.0、约14.5、约15.0、约15.5、约16.0、约16.5、约17.0、约17.5、约18.0、约18.5、约19.0、约19.5、约20、约20.5、约21.0、约21.5、约22.0、约22.5、约23.0、约23.5、约24.0、约24.5、约25.0、约25.5、约26.0、约26.5、约27.0、约27.5、约28.0、约28.5、约29.0或约29.5倍，包括其间的所有值和子范围。The cCPPs of the present disclosure can increase the efficiency of cytoplasmic delivery by about 1.1-fold to about 30-fold, e.g., about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10, about 10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0, about 20. 5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0, about 19.5, about 20, about 20.5, about 21.0, about 21.5, about 22.0, about 22.5, about 23.0, about 23.5, about 24.0, about 24.5, about 25.0, about 25.5, about 26.0, about 26.5, about 27.0, about 27.5, about 28.0, about 28.5, about 29.0, or about 29.5 times, including all values and subranges therebetween.
再剪接靶蛋白Resplicing target protein
“靶蛋白”是由靶基因的转录和翻译产生的氨基酸序列。如本文所用，“再剪接靶蛋白”是指由于AC与从靶基因转录的靶前mRNA结合而编码的蛋白质。“野生型靶蛋白”是指由野生型靶基因编码的靶前mRNA的正确剪接产生的天然存在的、正确翻译的蛋白质异构体。本发明化合物和方法可产生与野生型靶蛋白相比含有一个或多个氨基酸取代、缺失和/或插入的再剪接靶蛋白。在实施方案中，再剪接靶蛋白保留一些野生型靶蛋白活性。在实施方案中，通过施用本发明化合物产生的再剪接靶蛋白与野生型靶蛋白同源。在实施方案中，再剪接靶蛋白具有与野生型靶蛋白至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％并且至多100％同一的氨基酸序列。在实施方案中，再剪接靶蛋白与野生型靶蛋白基本上相同。在实施方案中，再剪接靶蛋白的氨基酸序列与野生型靶蛋白的氨基酸序列至少50％同一。在实施方案中，再剪接靶蛋白的氨基酸序列与野生型靶蛋白的氨基酸序列至少75％同一。在实施方案中，再剪接靶蛋白的氨基酸序列与野生型靶蛋白的氨基酸序列至少90％同一。在实施方案中，再剪接靶蛋白是野生型靶蛋白的缩短形式。"Target protein" is an amino acid sequence produced by transcription and translation of a target gene. As used herein, "re-splicing target protein" refers to a protein encoded by AC binding to a target pre-mRNA transcribed from a target gene. "Wild-type target protein" refers to a naturally occurring, correctly translated protein isomer produced by the correct splicing of a target pre-mRNA encoded by a wild-type target gene. The compounds and methods of the present invention can produce re-splicing target proteins containing one or more amino acid substitutions, deletions and/or insertions compared to the wild-type target protein. In an embodiment, the re-splicing target protein retains some wild-type target protein activity. In an embodiment, the re-splicing target protein produced by administering the compounds of the present invention is homologous to the wild-type target protein. In embodiments, the re-splicing target protein has an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% and up to 100% identical to the wild-type target protein. In embodiments, the re-splicing target protein is substantially identical to the wild-type target protein. In embodiments, the amino acid sequence of the re-splicing target protein is at least 50% identical to the amino acid sequence of the wild-type target protein. In embodiments, the amino acid sequence of the re-splicing target protein is at least 75% identical to the amino acid sequence of the wild-type target protein. In embodiments, the amino acid sequence of the re-splicing target protein is at least 90% identical to the amino acid sequence of the wild-type target protein. In embodiments, the re-splicing target protein is a shortened form of the wild-type target protein.
在实施方案中，再剪接靶蛋白可挽救与靶基因的转录和翻译相关的疾病的一种或多种表型或症状。在实施方案中，再剪接靶蛋白可挽救与靶蛋白的表达相关的疾病的一种或多种表型或症状。在实施方案中，再剪接靶蛋白是野生型靶蛋白的活性片段。在实施方案中，再剪接靶蛋白以与野生型靶蛋白基本相似的方式起作用。在实施方案中，再剪接靶蛋白使得细胞与表达野生型靶蛋白的相似细胞基本上相似地起作用。在实施方案中，再剪接靶蛋白并不治愈与靶基因或靶蛋白相关的疾病，但改善疾病的一种或多种症状。在实施方案中，再剪接靶蛋白导致靶蛋白功能提高至少约1％、约2％、约3％、约4％、约5％、约6％、约7％、约8％、约9％、约10％、约15％、约205、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％或约95％，并且至多约100％。In an embodiment, the re-splicing target protein can rescue one or more phenotypes or symptoms of a disease associated with the transcription and translation of the target gene. In an embodiment, the re-splicing target protein can rescue one or more phenotypes or symptoms of a disease associated with the expression of the target protein. In an embodiment, the re-splicing target protein is an active fragment of a wild-type target protein. In an embodiment, the re-splicing target protein acts in a manner substantially similar to the wild-type target protein. In an embodiment, the re-splicing target protein causes a cell to act substantially similarly to a similar cell expressing a wild-type target protein. In an embodiment, the re-splicing target protein does not cure a disease associated with a target gene or target protein, but improves one or more symptoms of the disease. In embodiments, resplicing of the target protein results in an increase in target protein function by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 205, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%, and at most about 100%.
在实施方案中，再剪接靶蛋白可具有比野生型靶蛋白的大小减少约1个或更多个氨基酸的氨基酸序列，例如减少约5、约10、约15、约20、约25、约30、约35、约40、约45、约50、约55、约60、约65、约70、约75、约80、约90、约95、约100、约105、约110、约115、约120、约125、约130、约135、约140、约145、约150、约155、约160、约165、约170、约175或约180个或更多个氨基酸。In an embodiment, the re-splicing target protein may have an amino acid sequence that is about 1 or more amino acids smaller than the size of the wild-type target protein, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, or about 180 or more amino acids smaller.
在实施方案中，再剪接靶蛋白可具有相对于靶蛋白改进的一种或多种特性。在实施方案中，再剪接靶蛋白可具有相对于野生型靶蛋白改进的一种或多种特性。在实施方案中，可通过促进靶前mRNA的不同剪接来增强酶活性或稳定性。在实施方案中，再剪接靶蛋白可具有与野生型靶蛋白异构体同一或基本上相似的序列，与另一种野生型靶蛋白异构体相比具有改进的特性。In embodiments, the re-splicing target protein may have one or more properties that are improved relative to the target protein. In embodiments, the re-splicing target protein may have one or more properties that are improved relative to the wild-type target protein. In embodiments, the enzyme activity or stability may be enhanced by promoting different splicing of the target pre-mRNA. In embodiments, the re-splicing target protein may have a sequence that is identical or substantially similar to a wild-type target protein isoform, and has improved properties compared to another wild-type target protein isoform.
在实施方案中，靶蛋白的一种或多种特性在再剪接靶蛋白中不存在(消除)或减少。在实施方案中，野生型靶蛋白的一种或多种特性在再剪接靶蛋白中不存在(消除)或减少。可减少或消除的特性的非限制性实例包括免疫原性、血管生成性、血栓形成性、聚集性和配体结合活性。In an embodiment, one or more properties of the target protein are absent (eliminated) or reduced in the re-splicing target protein. In an embodiment, one or more properties of the wild-type target protein are absent (eliminated) or reduced in the re-splicing target protein. Non-limiting examples of properties that can be reduced or eliminated include immunogenicity, angiogenicity, thrombogenicity, aggregation, and ligand binding activity.
在实施方案中，与野生型靶蛋白相比，再剪接靶蛋白含有一个或多个氨基酸取代。在实施方案中，取代可以是保守取代或非保守取代。保守氨基酸取代的实例包括用一种氨基酸取代以下组中的一者中的另一种氨基酸：碱性氨基酸(精氨酸、赖氨酸和组氨酸)、酸性氨基酸(谷氨酸和天冬氨酸)、极性氨基酸(谷氨酰胺和天冬酰胺)、疏水氨基酸(亮氨酸、异亮氨酸和缬氨酸)、芳族氨基酸(苯丙氨酸、色氨酸和酪氨酸)和小氨基酸(甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。在实施方案中，结构上相似的氨基酸被取代以反转残基的电荷(例如，谷氨酰胺取代谷氨酸，或反之亦然，天冬氨酸取代天冬酰胺，或反之亦然)。在实施方案中，酪氨酸取代苯丙氨酸，或反之亦然。氨基酸取代的其他非限制性实例例如由H.Neurath和R.L.Hill，1979在The Proteins，Academic Press，New York中描述。常见取代是Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu和Asp/Gly。In an embodiment, the re-splicing target protein contains one or more amino acid substitutions compared to the wild-type target protein. In an embodiment, the substitution may be a conservative substitution or a non-conservative substitution. Examples of conservative amino acid substitutions include substitution of one amino acid for another amino acid in one of the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). In an embodiment, structurally similar amino acids are substituted to reverse the charge of the residue (e.g., glutamine replaces glutamic acid, or vice versa, aspartic acid replaces asparagine, or vice versa). In an embodiment, tyrosine replaces phenylalanine, or vice versa. Other non-limiting examples of amino acid substitutions are described, for example, by H.Neurath and R.L.Hill, 1979 in The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
在实施方案中，与野生型靶蛋白相比，再剪接靶蛋白可在一个或多个(例如，几个)位置处包含取代、缺失和/或插入。在实施方案中，再剪接靶蛋白氨基酸序列中的氨基酸取代、缺失和/或插入的数量不超过200个、不超过150个、不超过100个、不超过50个、不超过40个、不超过30个、不超过20个或不超过10个，例如1、2、3、4、5、6、7、8、9或10个。In an embodiment, the re-splicing target protein may comprise a substitution, deletion and/or insertion at one or more (e.g., several) positions compared to the wild-type target protein. In an embodiment, the number of amino acid substitutions, deletions and/or insertions in the re-splicing target protein amino acid sequence is no more than 200, no more than 150, no more than 100, no more than 50, no more than 40, no more than 30, no more than 20 or no more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
治疗方法Treatment
在实施方案中，以约0.1mg/kg至约1000mg/kg，例如，约0.1mg/kg、约0.2mg/kg、约0.3mg/kg、约0.4mg/kg、约0.5mg/kg、约0.6mg/kg、约0.7mg/kg、约0.8mg/kg、约0.9mg/kg、约1mg/kg、约2mg/kg、约3mg/kg、约4mg/kg、约5mg/kg、约6mg/kg、约7mg/kg、约8mg/kg、约9mg/kg、约10mg/kg、约11mg/kg、约12mg/kg、约13mg/kg、约14mg/kg、约15mg/kg、约16mg/kg、约17mg/kg、约18mg/kg、约19mg/kg、约20mg/kg、约21mg/kg、约22mg/kg、约23mg/kg、约24mg/kg、约25mg/kg、约26mg/kg、约27mg/kg、约28mg/kg、约29mg/kg、约30mg/kg、约31mg/kg、约32mg/kg、约33mg/kg、约34mg/kg、约35mg/kg、约36mg/kg、约37mg/kg、约38mg/kg、约39mg/kg、约40mg/kg、约41mg/kg、约42mg/kg、约43mg/kg、约44mg/kg、约45mg/kg、约46mg/kg、约47mg/kg、约48mg/kg、约49mg/kg、约50mg/kg、约51mg/kg、约52mg/kg、约53mg/kg、约54mg/kg、约55mg/kg、约56mg/kg、约57mg/kg、约58mg/kg、约59mg/kg、约60mg/kg、约61mg/kg、约62mg/kg、约63mg/kg、约64mg/kg、约65mg/kg、约66mg/kg、约67mg/kg、约68mg/kg、约69mg/kg、约70mg/kg、约71mg/kg、约72mg/kg、约73mg/kg、约74mg/kg、约75mg/kg、约76mg/kg、约77mg/kg、约78mg/kg、约79mg/kg、约80mg/kg、约81mg/kg、约82mg/kg、约83mg/kg、约84mg/kg、约85mg/kg、约86mg/kg、约87mg/kg、约88mg/kg、约89mg/kg、约90mg/kg、约91mg/kg、约92mg/kg、约93mg/kg、约94mg/kg、约95mg/kg、约96mg/kg、约97mg/kg、约98mg/kg、约99mg/kg、约100mg/kg、约110mg/kg、约120mg/kg、约130mg/kg、约140mg/kg、约150mg/kg、约160mg/kg、约170mg/kg、约180mg/kg、约190mg/kg、约200mg/kg、约210mg/kg、约220mg/kg、约230mg/kg、约240mg/kg、约250mg/kg、约260mg/kg、约270mg/kg、约280mg/kg、约290mg/kg、约300mg/kg、约310mg/kg、约320mg/kg、约330mg/kg、约340mg/kg、约350mg/kg、约360mg/kg、约370mg/kg、约380mg/kg、约390mg/kg、约400mg/kg、约410mg/kg、约420mg/kg、约430mg/kg、约440mg/kg、约450mg/kg、约460mg/kg、约470mg/kg、约480mg/kg、约490mg/kg、约500mg/kg、约510mg/kg、约520mg/kg、约530mg/kg、约540mg/kg、约550mg/kg、约560mg/kg、约570mg/kg、约580mg/kg、约590mg/kg、约600mg/kg、约610mg/kg、约620mg/kg、约630mg/kg、约640mg/kg、约650mg/kg、约660mg/kg、约670mg/kg、约680mg/kg、约690mg/kg、约700mg/kg、约710mg/kg、约720mg/kg、约730mg/kg、约740mg/kg、约750mg/kg、约760mg/kg、约770mg/kg、约780mg/kg、约790mg/kg、约800mg/kg、约810mg/kg、约820mg/kg、约830mg/kg、约840mg/kg、约850mg/kg、约860mg/kg、约870mg/kg、约880mg/kg、约890mg/kg、约900mg/kg、约910mg/kg、约920mg/kg、约930mg/kg、约940mg/kg、约950mg/kg、约960mg/kg、约970mg/kg、约980mg/kg、约990mg/kg或约1000mg/kg(包括其中和之间的所有值和范围)的剂量向诊断患有杜兴氏肌营养不良(DMD)的患者施用本公开的AC。In embodiments, the dosage form is about 0.1 mg/kg to about 1000 mg/kg, for example, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg. kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg, about 33mg/kg, about 34mg/kg, about 35mg/kg, about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg , about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg, about 60mg/kg, about 61mg/kg, about 62mg/kg, about 63mg/kg, about 64mg/kg, about 65mg/kg, about 66mg/kg, about 67mg/kg, about 68mg/kg, about 69mg/kg, about 70mg/kg, about 71mg/kg, about 72mg/kg, about 73mg/kg, about 74mg/kg, about 75mg/kg, about 76mg/kg, about 77mg/kg, about 78mg/kg, about 79mg/kg, about 80mg/kg, about 81mg/kg, about 82mg/kg, about 83mg/kg, about 84mg/kg, about 85mg/kg, about 86mg/kg, about 87mg/kg, about 88mg/kg, about 89mg/kg, about 90mg/kg, about 91mg/kg, about 92mg/kg, about 93mg/kg, about 94mg/kg, about 95mg/kg, about 96mg/kg, about 97mg/kg g/kg, about 98mg/kg, about 99mg/kg, about 100mg/kg, about 110mg/kg, about 120mg/kg, about 130mg/kg, about 140mg/kg, about 150mg/kg, about 160mg/kg, about 170mg/kg, about 180mg/kg, about 190mg/kg, about 200mg/kg, about 210mg/kg, about 220mg/kg, about 230mg/kg, about 240mg/kg, about 250mg/kg, about 260mg/kg, about 270mg/kg, about 280mg/kg, about 290mg/kg, about 300mg/kg, about 310mg/kg, about 3 20mg/kg, about 330mg/kg, about 340mg/kg, about 350mg/kg, about 360mg/kg, about 370mg/kg, about 380mg/kg, about 390mg/kg, about 400mg/kg, about 410mg/kg, about 420mg/kg, about 430mg/kg, about 440mg/kg, about 450mg/kg, about 460mg/kg, about 470mg/kg, about 480mg/kg, about 490mg/kg, about 500mg/kg, about 510mg/kg, about 520mg/kg, about 530mg/kg, about 540mg/kg, about 550mg/kg, about 560mg/kg g, about 570 mg/kg, about 580 mg/kg, about 590 mg/kg, about 600 mg/kg, about 610 mg/kg, about 620 mg/kg, about 630 mg/kg, about 640 mg/kg, about 650 mg/kg, about 660 mg/kg, about 670 mg/kg, about 680 mg/kg, about 690 mg/kg, about 700 mg/kg, about 710 mg/kg, about 720 mg/kg, about 730 mg/kg, about 740 mg/kg, about 750 mg/kg, about 760 mg/kg, about 770 mg/kg, about 780 mg/kg, about 790 mg/kg, about 800 mg/kg, about 810 mg/kg The AC of the present disclosure is administered to a patient diagnosed with Duchenne muscular dystrophy (DMD) at a dosage of about 10 mg/kg, about 820 mg/kg, about 830 mg/kg, about 840 mg/kg, about 850 mg/kg, about 860 mg/kg, about 870 mg/kg, about 880 mg/kg, about 890 mg/kg, about 900 mg/kg, about 910 mg/kg, about 920 mg/kg, about 930 mg/kg, about 940 mg/kg, about 950 mg/kg, about 960 mg/kg, about 970 mg/kg, about 980 mg/kg, about 990 mg/kg, or about 1000 mg/kg, including all values and ranges therein and therebetween.
本公开提供了一种治疗有需要的受试者的杜兴氏肌营养不良(DMD)的方法，所述方法包括施用本文所公开的化合物。在一些实施方案中，靶基因是DMD。在实施方案中，靶序列包括DMD的外显子44的至少一部分、DMD的3’内含子侧翼外显子44的至少一部分、DMD的5’内含子侧翼外显子44的至少一部分或它们的组合。The present disclosure provides a method for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising administering a compound disclosed herein. In some embodiments, the target gene is DMD. In embodiments, the target sequence comprises at least a portion of exon 44 of DMD, at least a portion of 3' intron flanking exon 44 of DMD, at least a portion of 5' intron flanking exon 44 of DMD, or a combination thereof.
在各种实施方案中，治疗是指受试者中一种或多种症状的部分或完全缓和、改善、缓解、抑制、发作延迟、严重程度和/或发生率降低。In various embodiments, treatment refers to partial or complete alleviation, amelioration, alleviation, suppression, delayed onset, reduction in severity and/or incidence of one or more symptoms in a subject.
在实施方案中，提供了一种用于改变有需要的受试者中靶基因的表达的方法，所述方法包括施用本文所公开的化合物。在实施方案中，治疗导致靶蛋白的表达降低。在实施方案中，治疗导致再剪接靶蛋白的表达。在实施方案中，治疗导致野生型靶蛋白异构体的优先表达。In an embodiment, a method for altering the expression of a target gene in a subject in need thereof is provided, the method comprising administering a compound disclosed herein. In an embodiment, the treatment results in reduced expression of the target protein. In an embodiment, the treatment results in expression of a re-splicing target protein. In an embodiment, the treatment results in preferential expression of a wild-type target protein isoform.
在实施方案中，与治疗前受试者中或患有类似疾病的无治疗的一个或多个对照个体中的靶蛋白的平均水平相比，根据本公开的治疗导致受试者中靶蛋白的表达减少超过约5％，例如约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％和约100％。在实施方案中，与治疗前受试者中或患有类似疾病的无治疗的一个或多个对照个体中的靶蛋白的平均水平相比，根据本公开的治疗导致受试者中再剪接靶蛋白的表达增加超过约5％，例如约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％和约100％。在实施方案中，与治疗前受试者中或患有类似疾病的无治疗的一个或多个对照个体中的靶蛋白的平均水平相比，根据本公开的治疗导致受试者中野生型靶蛋白异构体的表达增加或减少超过约5％，例如约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％和约100％。In embodiments, treatment according to the present disclosure results in a decrease in expression of a target protein in a subject by greater than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the target protein in the subject before treatment or in one or more control individuals with a similar disease who have not been treated. In embodiments, treatment according to the present disclosure results in an increase in expression of a re-splicing target protein in a subject by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the target protein in the subject before treatment or in one or more control individuals with a similar disease without treatment. In embodiments, treatment according to the present disclosure results in an increase or decrease in expression of a wild-type target protein isoform in a subject by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the target protein in the subject before treatment or in one or more control individuals with a similar disease who have not been treated.
如本文所用，术语“改进”、“增加”、“降低”、“减少”等指示相对于对照的值。在实施方案中，合适的对照是基线测量结果，诸如在起始本文所述的治疗之前在同一个体中的测量结果，或在不存在本文所述的治疗的情况下对照个体(或多个对照个体)中的测量结果。“对照个体”是罹患相同疾病的个体，其年龄和/或性别与所治疗的个体大致相同(以确保所治疗个体和对照个体的疾病阶段具有可比性)。As used herein, the terms "improve", "increase", "decrease", "reduced", etc. indicate a value relative to a control. In embodiments, a suitable control is a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein. A "control individual" is an individual suffering from the same disease who is about the same age and/or sex as the individual being treated (to ensure that the disease stage of the treated individual and the control individual is comparable).
所治疗的个体(也称为“患者”或“受试者”)是患有疾病或具有形成疾病的潜力的个体(胎儿、婴儿、儿童、青少年或成年人)。个体可能患有由异常基因表达或异常基因剪接介导的疾病。在各种实施方案中，患有疾病的个体可具有野生型靶蛋白表达或活性水平，所述野生型靶蛋白表达或活性水平为未患有所述疾病的个体中正常蛋白表达或活性水平的约1％至99％。在实施方案中，所述范围包括但不限于正常胸苷磷酸化酶表达或活性水平的约80-99％、约65-80％、约50-65％、约30-50％、约25-30％、约20-25％、约15-20％、约10-15％、约5-10％或约1-5％。在实施方案中，个体可具有比正常野生型靶蛋白表达或活性水平高约1％至约500％的靶蛋白表达或活性水平。在实施方案中，所述范围包括但不限于约1-10％、约10-50％、约50-100％、约100-200％、约200-300％、约300-400％、约400-500％或约500-1000％更高的靶蛋白表达或活性水平。The treated individual (also referred to as "patient" or "subject") is an individual (fetus, infant, child, adolescent or adult) suffering from a disease or having the potential to form a disease. An individual may suffer from a disease mediated by abnormal gene expression or abnormal gene splicing. In various embodiments, an individual suffering from a disease may have a wild-type target protein expression or activity level, and the wild-type target protein expression or activity level is about 1% to 99% of the normal protein expression or activity level in an individual not suffering from the disease. In embodiments, the range includes but is not limited to about 80-99%, about 65-80%, about 50-65%, about 30-50%, about 25-30%, about 20-25%, about 15-20%, about 10-15%, about 5-10% or about 1-5% of normal thymidine phosphorylase expression or activity level. In embodiments, an individual may have a target protein expression or activity level that is about 1% to about 500% higher than the normal wild-type target protein expression or activity level. In embodiments, the range includes, but is not limited to, about 1-10%, about 10-50%, about 50-100%, about 100-200%, about 200-300%, about 300-400%, about 400-500%, or about 500-1000% higher target protein expression or activity levels.
在实施方案中，个体是最近被诊断患有所述疾病的个体。典型地，早期治疗(诊断后尽快开始治疗)对于最大限度地减少疾病的影响并最大限度地提高治疗益处非常重要。In embodiments, the individual is one who has recently been diagnosed with the disease. Typically, early treatment (starting treatment as soon as possible after diagnosis) is very important to minimize the effects of the disease and maximize the benefit of treatment.
在实施方案中，在DMD动物模型中评价本公开的化合物和AC对DMD的功效。动物模型是用于研究疾病发病机制的宝贵资源，并提供了测试抗肌萎缩蛋白相关活性的手段。在实施方案中，使用mdx小鼠和肌营养不良金毛猎犬(GRMD)犬(两者都是抗肌萎缩蛋白阴性的)(参见例如Collins&Morgan，Int J Exp Pathol 84：165-172，2003)来评价本公开的化合物。在实施方案中，利用C57BL/10ScSn-Dmdmdx/J(Bl10/mdx)或D2.B10-Dmdmdx/J(D2/mdx)小鼠模型来评价本公开的化合物。在实施方案中，使用携带人DMD基因并且缺乏小鼠Dmd基因的转基因小鼠(无hDMD/Dmd小鼠)来评价本公开的化合物。这种小鼠可通过雄性hDMD小鼠(可从Jackson Laboratory，Bar Harbor，ME获得)与雌性无DMD小鼠杂交产生。以下参考文献中的每一者描述了这些模型并全文以引用方式并入本文：J NeuromusculDis.2018；5(4)：407-417.；Proc Natl Acad Sci U S A.1984；81(4)：1189-92.；Am JPathol.2010；176(5)：2414-24.；J Clin Invest.2009；119(12)：3703-12；国际公开号WO2019014772。这些和其他动物模型可用于测量各种抗肌萎缩蛋白的功能活性。In an embodiment, the efficacy of the compounds of the present disclosure and AC to DMD is evaluated in a DMD animal model. Animal models are valuable resources for studying the pathogenesis of the disease and provide a means for testing the activity associated with dystrophin. In an embodiment, mdx mice and golden retriever muscular dystrophy (GRMD) dogs (both of which are dystrophin negative) are used (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003) to evaluate the compounds of the present disclosure. In an embodiment, C57BL / 10ScSn-Dmdmdx / J (Bl10 / mdx) or D2.B10-Dmdmdx / J (D2 / mdx) mouse models are used to evaluate the compounds of the present disclosure. In an embodiment, transgenic mice carrying human DMD genes and lacking mouse Dmd genes (no hDMD / Dmd mice) are used to evaluate the compounds of the present disclosure. Such mice can be generated by crossing male hDMD mice (available from Jackson Laboratory, Bar Harbor, ME) with female DMD-free mice. Each of the following references describes these models and is incorporated herein by reference in its entirety: J Neuromuscul Dis. 2018; 5(4): 407-417.; Proc Natl Acad Sci U S A. 1984; 81(4): 1189-92.; Am J Pathol. 2010; 176(5): 2414-24.; J Clin Invest. 2009; 119(12): 3703-12; International Publication No. WO2019014772. These and other animal models can be used to measure the functional activity of various dystrophin proteins.
在实施方案中，使用体外模型来评价本公开的组合物的功效。在实施方案中，体外模型是永生化肌细胞模型。所述模型在以下文章中进行了描述，所述文章的全文以引用方式并入本文：Nguyen等人J Pers Med.2017年12月；7(4)：13。In an embodiment, an in vitro model is used to evaluate the efficacy of the disclosed compositions. In an embodiment, the in vitro model is an immortalized myocyte model. The model is described in the following article, the entire text of which is incorporated herein by reference: Nguyen et al. J Pers Med. 2017 Dec; 7(4): 13.
制备方法Preparation method
本文所述的化合物可以有机合成领域技术人员已知的多种方式或本领域技术人员所理解的其变化形式制备。本文所述的化合物可由容易获得的起始材料制备。最佳反应条件可随所用的特定反应物或溶剂而变化，但此类条件可由本领域技术人员确定。The compounds described herein can be prepared in a variety of ways known to those skilled in the art of organic synthesis or variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.
本文所述化合物的变化形式包括如针对每种化合物所述的各种成分的添加、减去或移动。类似地，当分子中存在一个或多个手性中心时，分子的手性可改变。另外，化合物合成可涉及各种化学基团的保护和脱保护。保护和脱保护的使用以及适当的保护基团的选择可由本领域技术人员确定。保护基团的化学性质可见于例如Wuts和Greene，ProtectiveGroups in Organic Synthesis，第4版，Wiley&Sons，2006，其全文以引用方式并入本文。The variation of compounds described herein includes the addition, subtraction or movement of various components as described for each compound. Similarly, when there are one or more chiral centers in a molecule, the chirality of the molecule can be changed. In addition, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection and the selection of appropriate protecting groups can be determined by those skilled in the art. The chemical properties of protecting groups can be found in, for example, Wuts and Greene, Protective Groups in Organic Synthesis, 4th edition, Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.
用于制备所公开的化合物和组合物的起始材料和试剂可购自商业供应商诸如Aldrich Chemical Co.，(Milwaukee，WI)、Acros Organics(Morris Plains，NJ)、FisherScientific(Pittsburgh，PA)、Sigma(St.Louis，MO)、Pfizer(New York，NY)、GlaxoSmithKline(Raleigh，NC)、Merck(Whitehouse Station，NJ)、Johnson&Johnson(NewBrunswick，NJ)、Aventis(Bridgewater，NJ)、AstraZeneca(Wilmington，DE)、Novartis(Basel，Switzerland)、Wyeth(Madison，NJ)、Bristol-Myers-Squibb(New York，NY)、Roche(Basel，Switzerland)、Lilly(Indianapolis，IN)、Abbott(Abbott Park，IL)、ScheringPlough(Kenilworth，NJ)或Boehringer Ingelheim(Ingelheim，Germany)，或者通过本领域技术人员已知的方法按照参考文献中所阐述的程序制备，所述参考文献诸如Fieser andFieser’s Reagents for Organic Synthesis，第1-17卷(John Wiley and Sons，1991)；Rodd’s Chemistry of Carbon Compounds，第1-5卷和补充版(Elsevier SciencePublishers，1989)；Organic Reactions，第1-40卷(John Wiley and Sons，1991)；March’sAdvanced Organic Chemistry，(John Wiley and Sons，第4版)；以及Larock’sComprehensive Organic Transformations(VCH Publishers Inc.，1989)。其他材料，诸如本文所公开的药物载剂可从商业来源获得。The starting materials and reagents used to prepare the disclosed compounds and compositions can be purchased from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or prepared by methods known to those skilled in the art according to the procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementary Editions (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, can be obtained from commercial sources.
产生本文所述化合物的反应可在溶剂中进行，所述溶剂可由有机合成领域的技术人员选择。在进行反应的条件(即，温度和压力)下，溶剂可基本上不与起始材料(反应物)、中间体或产物反应。反应可在一种溶剂或多于一种溶剂的混合物中进行。产物或中间体形成可根据本领域已知的任何合适的方法进行监测。例如，产物形成可通过光谱手段诸如核磁共振光谱法(例如，1H或13C)、红外光谱法、分光光度测定法(例如，、UV-可见光)或质谱法，或通过色谱法诸如高效液相色谱法(HPLC)或薄层色谱法来监测。The reaction that produces the compound described herein can be carried out in a solvent, and the solvent can be selected by a technician in the field of organic synthesis. Under the conditions (i.e., temperature and pressure) of the reaction, the solvent may not react substantially with the starting material (reactant), intermediate or product. The reaction can be carried out in a solvent or a mixture of more than one solvent. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectral means such as nuclear magnetic resonance spectroscopy (e.g.,1 H or13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible light) or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
所公开的化合物可通过固相肽合成来制备，其中氨基酸α-N-末端被酸或碱保护基团保护。此类保护基团应具有对肽键联形成条件稳定的特性，同时可容易地去除而不破坏生长的肽链或使其中所含的任何手性中心外消旋化。合适的保护基团是9-芴基甲氧羰基(Fmoc)、叔丁氧羰基(Boc)、苄氧羰基(Cbz)、联苯异丙氧羰基、叔戊氧羰基、异冰片基氧羰基、α，α-二甲基-3，5-二甲氧基苄氧羰基、邻硝基苯基亚磺酰基、2-氰基-叔丁氧羰基等。9-芴基甲氧羰基(Fmoc)保护基团对于所公开化合物的合成是特别优选的。对于侧链氨基基团如赖氨酸和精氨酸，其他优选的侧链保护基团是2，2，5，7，8-五甲基色满-6-磺酰基(pmc)、硝基、对甲苯磺酰基、4-甲氧基苯-磺酰基、Cbz、Boc和金刚烷氧羰基；对于酪氨酸是苄基、邻溴苄氧基-羰基、2，6-二氯苄基、异丙基、叔丁基(t-Bu)、环己基、环戊基和乙酰基(Ac)；对于丝氨酸是叔丁基、苄基和四氢吡喃基；对于组氨酸是三苯甲基、苄基、Cbz、对甲苯磺酰基和2，4-二硝基苯基；对于色氨酸是甲酰基；对于天冬氨酸和谷氨酸是苄基和叔丁基，并且对于半胱氨酸是三苯基甲基(三苯甲基)。在固相肽合成方法中，将α-C-末端氨基酸附接到合适的固体支持物或树脂上。可用于上述合成的合适固体支持物是对逐步缩合-脱保护反应的试剂和反应条件呈惰性以及不溶于所用介质的那些材料。用于合成α-C-末端羧基肽的固体支持物是可购自Applied Biosystems(Foster City，Calif.)的4-羟甲基苯氧基甲基-共聚(苯乙烯-1％二乙烯基苯)或4-(2’，4’-二甲氧基苯基-Fmoc-氨基甲基)苯氧基乙酰胺基乙基树脂。α-C-末端氨基酸通过N，N’-二环己基碳二亚胺(DCC)、N，N’-二异丙基碳二亚胺(DIC)或O-苯并三唑-1-基-N，N，N’，N’-四甲基脲六氟磷酸盐(HBTU)，在有或没有4-二甲基氨基吡啶(DMAP)、1-羟基苯并三唑(HOBT)、苯并三唑-1-基氧基-三(二甲基氨基)鏻六氟磷酸盐(BOP)或双(2-氧代-3-噁唑烷基)氯化膦(BOPCl)的情况下，在10℃至50℃之间的温度下在溶剂诸如二氯甲烷或DMF中介导偶联约1至约24小时而偶联到树脂上。当固体支持物是4-(2’，4’-二甲氧基苯基-Fmoc-氨基甲基)苯氧基-乙酰氨基乙基树脂时，在如上所述与α-C-末端氨基酸偶联之前，用仲胺(优选地哌啶)切割Fmoc基团。一种用于与脱保护的4-(2’，4’-二甲氧基苯基-Fmoc-氨基甲基)苯氧基-乙酰氨基乙基树脂偶联的方法是在DMF中的O-苯并三唑-1-基-N，N，N’，N’-四甲基脲六氟磷酸盐(HBTU，1当量)和1-羟基苯并三唑(HOBT，1当量)。可在自动多肽合成仪中进行连续的受保护氨基酸的偶联。在一个实例中，用Fmoc保护生长的肽链的氨基酸中的α-N-末端。从生长的肽的α-N-末端侧去除Fmoc保护基团是通过用仲胺(优选地哌啶)处理完成的。然后以约3倍摩尔过量引入每个受保护的氨基酸，并且优选地在DMF中进行偶联。偶联剂可以是O-苯并三唑-1-基-N，N，N’，N’-四甲基脲六氟磷酸盐(HBTU，1当量)和1-羟基苯并三唑(HOBT，1当量)。在固相合成结束时，将多肽从树脂上去除并脱保护，连续地或在单一操作中进行。通过用包含茴香硫醚、水、乙二硫醇和三氟乙酸的切割试剂处理树脂结合的多肽，可在单一操作中完成多肽的去除和脱保护。在多肽的α-C-末端是烷基酰胺的情况下，通过用烷基胺氨解来切割树脂。另选地，可通过酯交换(例如，用甲醇)、接着氨解或通过直接转酰胺基来去除肽。受保护的肽可在此时纯化或直接用于下一步骤。侧链保护基团的去除可使用上述切割混合物来完成。完全脱保护的肽可通过使用任何或所有以下类型的一系列色谱步骤来纯化：在弱碱性树脂(乙酸盐形式)上的离子交换；在未衍生的聚苯乙烯-二乙烯基苯(例如，Amberlite XAD)上的疏水吸附色谱法；硅胶吸附色谱法；羧甲基纤维素上的离子交换色谱法；分配色谱法(例如，在Sephadex G-25、LH-20上或逆流分布)；高效液相色谱法(HPLC)，特别是在辛基-二氧化硅或十八烷基甲硅烷基-二氧化硅键合相柱填料上的反相HPLC。The disclosed compounds can be prepared by solid phase peptide synthesis, wherein the amino acid α-N-terminus is protected by an acid or base protecting group. Such protecting groups should have the property of being stable to the peptide linkage forming conditions, while being easily removable without destroying the growing peptide chain or racemizing any chiral center contained therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, tert-pentyloxycarbonyl, isobornyloxycarbonyl, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-tert-butyloxycarbonyl, and the like. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. For side chain amino groups such as lysine and arginine, other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for aspartic acid and glutamic acid, benzyl and t-butyl, and for cysteine, triphenylmethyl (trityl). In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and are insoluble in the medium used. The solid support used for the synthesis of α-C-terminal carboxyl peptides is 4-hydroxymethylphenoxymethyl-co-poly(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin, which can be purchased from Applied Biosystems (Foster City, Calif.). The α-C-terminal amino acid is coupled to the resin by N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC), or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl) at temperatures between 10°C and 50°C in a solvent such as dichloromethane or DMF for about 1 to about 24 hours. When the solid support is 4-(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine (preferably piperidine) before coupling with the α-C-terminal amino acid as described above. A method for coupling with a deprotected 4-(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. The coupling of consecutive protected amino acids can be performed in an automatic peptide synthesizer. In one example, the α-N-terminus in the amino acid of the growing peptide chain is protected with Fmoc. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine (preferably piperidine). Each protected amino acid is then introduced in about 3 times molar excess, and coupling is preferably performed in DMF. The coupling agent can be O-benzotriazole-1-yl-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). At the end of solid phase synthesis, the polypeptide is removed from the resin and deprotected, continuously or in a single operation. By treating the resin-bound polypeptide with a cutting agent comprising thioanisole, water, ethanedithiol and trifluoroacetic acid, the removal and deprotection of the polypeptide can be completed in a single operation. In the case where the α-C-terminus of the polypeptide is an alkylamide, the resin is cut by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification (e.g., with methanol), followed by aminolysis or by direct transamidation. The protected peptide can be purified at this time or used directly in the next step. The removal of the side chain protecting group can be completed using the above-mentioned cutting mixture. The fully deprotected peptide can be purified by a series of chromatographic steps using any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography (e.g., on Sephadex G-25, LH-20 or countercurrent distribution); high performance liquid chromatography (HPLC), particularly reversed phase HPLC on octyl-silica or octadecylsilyl-silica bonded phase column packings.
上述聚合物，诸如PEG基团，可在用于使蛋白质与活化的聚合物分子反应的任何合适的条件下附接到AC。可使用本领域已知的任何方法，包括经由酰化、还原烷基化、迈克尔加成、硫醇烷基化或通过PEG部分上的反应性基团(例如醛、氨基、酯、硫醇、α-卤代乙酰基、马来酰亚胺基或肼基)与AC上的反应性基团(例如醛、氨基、酯、硫醇、α-卤代乙酰基、马来酰亚胺基或肼基)的其他化学选择性缀合/连接方法。可用于将水溶性聚合物连接到一种或多种蛋白质的活化基团包括但不限于砜、马来酰亚胺、巯基、硫醇、三氟甲磺酸酯、三氟乙磺酸酯(tresylate)、氮杂环丙烷(azidirine)、环氧乙烷、5-吡啶基和α-卤代酰基(例如，α-碘乙酸、α-溴乙酸、α-氯乙酸)。如果通过还原烷基化附接到AC，则所选聚合物应具有单一反应性醛，以便控制聚合度。参见例如Kinstler等人，Adv.Drug.Delivery Rev.54：477-485(2002)；Roberts等人，Adv.Drug Delivery Rev.54：459-476(2002)；和Zalipsky等人，Adv.Drug Delivery Rev.16：157-182(1995)。The above polymers, such as PEG groups, can be attached to AC under any suitable conditions for reacting proteins with activated polymer molecules. Any method known in the art can be used, including other chemical selective conjugation/linking methods via acylation, reductive alkylation, Michael addition, thiol alkylation, or through reactive groups on the PEG portion (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazine) and reactive groups on AC (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazine). Activated groups that can be used to attach water-soluble polymers to one or more proteins include, but are not limited to, sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane, 5-pyridyl, and α-haloacyl (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). If attached to AC by reductive alkylation, the selected polymer should have a single reactive aldehyde in order to control the degree of polymerization. See, e.g., Kinstler et al., Adv. Drug. Delivery Rev. 54:477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54:459-476 (2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16:157-182 (1995).
为了将AC直接共价连接到CPP，CPP的适当氨基酸残基可与有机衍生剂反应，所述有机衍生剂能够与氨基酸的选定侧链或N-末端或C-末端反应。肽或缀合物部分上的反应性基团包括例如醛、氨基、酯、硫醇、α-卤代乙酰基、马来酰亚胺基或肼基。衍生剂包括例如马来酰亚胺基苯甲酰基磺基琥珀酰亚胺酯(通过半胱氨酸残基缀合)、N-羟基琥珀酰亚胺(通过赖氨酸残基)、戊二醛、琥珀酸酐或本领域已知的其他剂。To covalently link AC directly to CPP, the appropriate amino acid residues of the CPP can be reacted with an organic derivatizing agent that is capable of reacting with selected side chains or the N-terminus or C-terminus of the amino acid. Reactive groups on the peptide or conjugate moiety include, for example, aldehyde, amino, ester, thiol, α-haloacetyl, maleimido or hydrazine groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide esters (conjugated via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.
合成寡聚反义化合物的方法是本领域已知的。本公开不限于合成AC的方法。在实施方案中，本文提供了具有可用于形成核苷间键联(包括例如磷酸二酯和硫代磷酸酯核苷间键联)的反应性磷基团的化合物。前体或反义化合物的制备和/或纯化方法并不限制本文提供的组合物或方法。用于DNA、RNA和反义化合物的合成和纯化的方法是本领域技术人员熟知的。Methods for synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited to methods for synthesizing AC. In embodiments, compounds with reactive phosphorus groups that can be used to form internucleoside linkages (including, for example, phosphodiester and thiophosphate internucleoside linkages) are provided herein. The preparation and/or purification methods of precursors or antisense compounds do not limit the compositions or methods provided herein. Methods for the synthesis and purification of DNA, RNA, and antisense compounds are well known to those skilled in the art.
修饰和未修饰的核苷的寡聚化可根据关于DNA(Protocols forOligonucleotides and Analogs，Agrawal编(1993)，Humana Press)和/或RNA(Scaringe，Methods(2001)，23，206-217.Gait等人，Applications of Chemically synthesized RNAin RNA：Protein Interactions，Smith编(1998)，1-36.Gallo等人，Tetrahedron(2001)，57，5707-5713)的文献程序常规进行。Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Agrawal ed. (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Smith ed. (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
本文提供的反义化合物可通过熟知的固相合成技术方便并且常规地制备。用于此类合成的设备由包括例如Applied Biosystems(Foster City，CA)的几家供应商出售。可另外或替代地使用本领域已知的用于此类合成的任何其他方法。使用类似技术来制备寡核苷酸(诸如硫代磷酸酯和烷基化衍生物)为人所熟知。本发明不受反义化合物合成方法的限制。Antisense compounds provided herein can be conveniently and routinely prepared by well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several suppliers including, for example, Applied Biosystems (Foster City, CA). Any other method known in the art for such synthesis can be used in addition or alternatively. It is well known to prepare oligonucleotides (such as phosphorothioates and alkylated derivatives) using similar techniques. The present invention is not limited by the method for synthesizing antisense compounds.
寡核苷酸纯化和分析的方法是本领域技术人员已知的。分析方法包括毛细管电泳(CE)和电喷雾质谱法。此类合成和分析方法可在多孔板中进行。本发明的方法不限于寡聚物纯化方法。Methods for oligonucleotide purification and analysis are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. Such synthesis and analytical methods can be performed in multiwell plates. The methods of the present invention are not limited to oligomer purification methods.
施用方法Application method
所公开的化合物和含有它们的组合物的体内应用可通过本领域技术人员目前或预期已知的任何合适的方法和技术来实现。例如，所公开的化合物可被配制成生理学上或药学上可接受的形式，并通过本领域已知的任何合适的途径施用，包括例如口服和肠胃外施用途径。如本文所用，术语肠胃外包括皮下、皮内、静脉内、肌内、腹膜内、胸骨内和鞘内施用，诸如通过注射。所公开的化合物或组合物的施用可以是单次施用，或以连续或不同的间隔施用，如本领域技术人员可容易地确定的。The in vivo application of the disclosed compounds and compositions containing them can be achieved by any suitable method and technology currently or expectedly known to those skilled in the art. For example, the disclosed compounds can be formulated into physiologically or pharmaceutically acceptable forms and administered by any suitable route known in the art, including, for example, oral and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal and intrathecal administration, such as by injection. The administration of the disclosed compounds or compositions can be a single administration, or administered at continuous or different intervals, as can be easily determined by those skilled in the art.
本文公开的化合物和包含它们的组合物也可利用脂质体技术、缓释胶囊、可植入泵和可生物降解容器来施用。这些递送方法可有利地在延长的时间段内提供均匀的剂量。化合物还可以其盐衍生物形式或结晶形式施用。The compounds disclosed herein and compositions comprising them can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide uniform dosages over extended time periods. The compounds can also be administered in the form of their salt derivatives or in crystalline form.
本文公开的化合物可根据用于制备药学上可接受的组合物的已知方法配制。在本领域技术人员熟知且容易获得的许多来源中详细描述了制剂。例如，Remington’sPharmaceutical Science，E.W.Martin(1995)描述了可与所公开的方法结合使用的制剂。通常，本文公开的化合物可被配制成使得有效量的化合物与合适的载剂组合，以便促进化合物的有效施用。所用的组合物还可以呈各种形式。这些形式包括例如固体、半固体和液体剂型，诸如片剂、丸剂、粉剂、液体溶液或悬浮液、栓剂、可注射和可输注溶液以及喷雾剂。优选的形式取决于预期的施用方式和治疗应用。组合物还优选地包含本领域技术人员已知的常规药学上可接受的载剂和稀释剂。与化合物一起使用的载剂或稀释剂的实例包括乙醇、二甲亚砜、甘油、氧化铝、淀粉、盐水和等效载剂和稀释剂。为了提供用于期望的治疗性治疗的此类剂量的施用，基于包含载剂或稀释剂的总组合物的重量，本文所公开的组合物可有利地包含总共约0.1重量％至100重量％之间的主题化合物中的一种或多种。The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in many sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science, E.W.Martin (1995) describes formulations that can be used in combination with the disclosed methods. Generally, the compounds disclosed herein can be formulated so that an effective amount of the compound is combined with a suitable carrier to facilitate the effective administration of the compound. The composition used can also be in various forms. These forms include, for example, solid, semisolid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The composition also preferably contains conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents used with the compound include ethanol, dimethyl sulfoxide, glycerol, aluminum oxide, starch, saline, and equivalent carriers and diluents. To provide for administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein may advantageously contain a total of between about 0.1 wt % to 100 wt % of one or more of the subject compounds, based on the weight of the total composition including carrier or diluent.
适于施用的制剂包括例如无菌注射水溶液，其可含有抗氧化剂、缓冲剂、抑菌剂和使制剂与预期接受者的血液等渗的溶质；以及水性和非水性无菌悬浮液，其可包含悬浮剂和增稠剂。制剂可存在于单位剂量或多剂量容器例如密封的安瓿和小瓶中，并且可储存在冷冻干燥(冻干)条件下，在使用前仅需要无菌液体载剂(例如注射用水)的条件。临时注射溶液和悬浮液可由无菌粉末、颗粒、片剂等制备。应当理解，除了以上特别提及的成分外，本文所公开的组合物还可包含本领域中关于所讨论的制剂类型的其他常规剂。Preparations suitable for administration include, for example, sterile injection aqueous solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The preparation may be present in unit dose or multi-dose containers such as sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions, requiring only sterile liquid carriers (e.g., water for injection) before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, and the like. It should be understood that, in addition to the ingredients specifically mentioned above, the compositions disclosed herein may also include other conventional agents in the art for the type of preparation discussed.
本文所公开的化合物和包含它们的组合物可通过与细胞直接接触或经由载剂手段递送至细胞。用于将化合物和组合物递送至细胞的载剂手段是本领域已知的，并且包括例如将组合物包封在脂质体部分中。用于将本文所公开的化合物和组合物递送至细胞的另一手段包括将化合物附接到靶向递送至靶细胞的蛋白质或核酸。美国专利号6,960,648以及美国申请公开号20030032594和20020120100公开了可与另一种组合物偶联并允许所述组合物跨生物膜易位的氨基酸序列。美国申请公开号20020035243也描述了用于跨细胞膜转运生物部分以用于细胞内递送的组合物。也可将化合物掺入聚合物中，所述聚合物的实例包括用于颅内肿瘤的聚(D-L丙交酯-共-乙交酯)聚合物；摩尔比为20∶80的聚[双(对羧基苯氧基)丙烷∶癸二酸](如GLIADEL中所用)；软骨素；甲壳质；和壳聚糖。Compounds disclosed herein and compositions comprising them can be delivered to cells by direct contact with cells or via carrier means. Carrier means for delivering compounds and compositions to cells are known in the art, and include, for example, encapsulating the composition in a liposome portion. Another means for delivering compounds disclosed herein and compositions to cells includes attaching the compound to a protein or nucleic acid targeted for delivery to a target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow the composition to translocate across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transcellular membrane transport of biological parts for intracellular delivery. The compounds may also be incorporated into polymers, examples of which include poly(D-L lactide-co-glycolide) polymers for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] at a molar ratio of 20:80 (as used in GLIADEL); chondroitin; chitin; and chitosan.
本文所公开的化合物和组合物，包括其药学上可接受的盐或前药，可通过输注或注射静脉内、肌内或腹膜内施用。活性剂或其盐的溶液可在水中制备，任选地与无毒表面活性剂混合。分散体也可在甘油、液体聚乙二醇、三醋精以及它们的混合物中以及在油中制备。在普通的储存和使用条件下，这些制剂可含有防腐剂以防止微生物的生长。The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, can be administered intravenously, intramuscularly or intraperitoneally by infusion or injection. Solutions of the active agent or its salt can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof, and in oil. Under ordinary storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms.
适于注射或输注的药物剂型可包括包含活性成分的无菌水溶液或分散体或无菌粉末，其适于临时制备任选地包封在脂质体中的无菌可注射或可输注溶液或分散体。最终剂型在制造和储存条件下应当是无菌的、流体的和稳定的。液体载剂或媒介物可以是溶剂或液体分散介质，包括例如水、乙醇、多元醇(例如，甘油、丙二醇、液体聚乙二醇等)、植物油、无毒甘油酯以及它们的合适混合物。适当的流动性可例如通过形成脂质体、在分散体的情况下通过维持所需的粒度或通过使用表面活性剂来维持。任选地，可通过各种其他抗细菌剂和抗真菌剂，例如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等来防止微生物的作用。在许多情况下，将优选包含等渗剂，例如糖、缓冲剂或氯化钠。通过包含延迟吸收的剂，例如单硬脂酸铝和明胶，可实现可注射组合物的延长吸收。The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or sterile powder containing active ingredients, which is suitable for temporary preparation of sterile injectable or infusible solutions or dispersions optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under manufacturing and storage conditions. The liquid carrier or vehicle can be a solvent or liquid dispersion medium, including, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and their suitable mixtures. Suitable fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using a surfactant. Optionally, the effect of microorganisms can be prevented by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, buffers or sodium chloride, will preferably be included. By including an agent that delays absorption, such as aluminum monostearate and gelatin, the extended absorption of injectable compositions can be achieved.
通过将所需量的本文所公开的化合物和/或剂与上文列举的各种其他成分掺入适当溶剂中，根据需要，随后过滤灭菌来制备无菌可注射溶液。在用于制备无菌可注射溶液的无菌粉末的情况下，优选的制备方法是真空干燥和冷冻干燥技术，其产生活性成分加上存在于先前无菌过滤的溶液中的任何附加的期望成分的粉末。Sterile injectable solutions are prepared by incorporating the desired amount of the compounds and/or agents disclosed herein with the various other ingredients listed above in an appropriate solvent, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients present in a previously sterile-filtered solution.
本文所公开的化合物和剂以及药物组合物的有用剂量可通过比较它们在动物模型中的体外活性和体内活性来确定。将小鼠和其他动物中的有效剂量外推至人的方法是本领域已知的。Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.
组合物施用的剂量范围是大到足以产生影响症状或病症的期望效果的剂量范围。剂量不应大到引起不良副作用，诸如不希望的交叉反应、过敏反应等。通常，剂量将随患者的年龄、状况、性别和疾病程度而变化，并且可由本领域技术人员确定。在任何禁忌症的情况下，剂量可由个别医师调整。剂量可变化，并且可每天一次或多次剂量施用，持续一天或几天。The dosage range of the composition administration is a dosage range large enough to produce the desired effect affecting the symptom or condition. The dosage should not be large enough to cause adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Usually, the dosage will vary with the age, condition, sex and degree of disease of the patient, and can be determined by those skilled in the art. In the case of any contraindications, the dosage can be adjusted by individual physicians. The dosage can vary and can be administered once or multiple times a day for one or more days.
还公开了包含本文所公开的化合物与药学上可接受的载剂的组合的药物组合物。包含一定量化合物的适于口服、局部或肠胃外施用的药物组合物构成优选的方面。施用于患者(特别是人)的剂量应当足以在合理的时间范围内在患者中实现治疗反应，而没有致死毒性，并且优选地引起不超过可接受水平的副作用或发病率。本领域技术人员将认识到，剂量将取决于多种因素，包括受试者的状况(健康)、受试者的体重、同时治疗的种类(如果有的话)、治疗频率、治疗比率以及病理状况的严重程度和阶段。Also disclosed are pharmaceutical compositions comprising a combination of a compound disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions suitable for oral, topical or parenteral administration comprising a certain amount of compound constitute preferred aspects. The dosage applied to a patient (particularly a human) should be sufficient to achieve a therapeutic response in the patient within a reasonable time frame without lethal toxicity, and preferably cause no more than an acceptable level of side effects or morbidity. Those skilled in the art will recognize that dosage will depend on a variety of factors, including the subject's condition (health), the subject's weight, the type of simultaneous treatment (if any), the frequency of treatment, the treatment ratio, and the severity and stage of the pathological condition.
还公开了在一个或多个容器中包含本文所公开的化合物的试剂盒。所公开的试剂盒可任选地包括药学上可接受的载剂和/或稀释剂。在一个实施方案中，试剂盒包括如本文所述的一种或多种其他组分、助剂或佐剂。在另一个实施方案中，试剂盒包括一种或多种抗癌剂，诸如本文所述的那些剂。在一个实施方案中，试剂盒包括描述如何施用试剂盒的化合物或组合物的说明书或包装材料。试剂盒的容器可以是任何合适的材料，例如玻璃、塑料、金属等，并且可以是任何合适的大小、形状或配置。在一个实施方案中，本文所公开的化合物和/或剂作为固体(诸如片剂、丸剂或粉末形式)提供在试剂盒中。在另一个实施方案中，本文所公开的化合物和/或剂作为液体或溶液提供在试剂盒中。在一个实施方案中，试剂盒包括含有液体或溶液形式的本文所公开的化合物和/或剂的安瓿或注射器。Also disclosed is a kit comprising a compound disclosed herein in one or more containers. The disclosed kit may optionally include a pharmaceutically acceptable carrier and/or diluent. In one embodiment, the kit includes one or more other components, auxiliary agents or adjuvants as described herein. In another embodiment, the kit includes one or more anticancer agents, such as those described herein. In one embodiment, the kit includes instructions or packaging materials describing how to administer the compound or composition of the kit. The container of the kit can be any suitable material, such as glass, plastic, metal, etc., and can be any suitable size, shape or configuration. In one embodiment, the compound and/or agent disclosed herein are provided in the kit as a solid (such as a tablet, pill or powder form). In another embodiment, the compound and/or agent disclosed herein are provided in the kit as a liquid or solution. In one embodiment, the kit includes an ampoule or syringe containing a compound and/or agent disclosed herein in liquid or solution form.
已描述了本发明的多个实施方案。然而，应当理解，在不背离本发明的精神和范围的情况下，可进行各种修改。因此，其他实施方案也在以下权利要求的范围内。Several embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the following claims.
某些定义Some definitions
如在说明书和所附权利要求书中所使用，单数形式“一个/种(a/an)”和“所述”包括多个指示物，除非上下文另外明确指出。因此，例如，提及“一种组合物”包括两种或更多种此类组合物的混合物，提及“一种剂”包括两种或更多种此类剂的混合物，提及“所述组分”包括两种或更多种此类组分的混合物等。As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an agent" includes mixtures of two or more such agents, reference to "the component" includes mixtures of two or more such components, etc.
术语“约”当紧接在数值之前时意指范围(例如，所述值的加或减10％)。例如，“约50”可意指45至55，“约25,000”可意指22,500至27,500等，除非本公开的上下文另外指出，或者与此类解释不一致。例如，在诸如“约49、约50、约55、...”的数值列表中，“约50”意指延伸至小于前后值之间一半间隔的范围，例如，大于49.5至小于52.5。此外，短语“小于约”值或“大于约”值应当根据本文提供的术语“约”的定义来理解。类似地，术语“约”当在一系列数值或数值范围(例如，“约10、20、30”或“约10-30”)之前时分别指所述系列中的所有值或所述范围的端点。The term "about" means a range (e.g., plus or minus 10% of the value) when it is immediately before a numerical value. For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc., unless otherwise indicated by the context of the present disclosure, or inconsistent with such an explanation. For example, in a numerical list such as "about 49, about 50, about 55, ...", "about 50" means a range extending to less than half the interval between the preceding and following values, for example, greater than 49.5 to less than 52.5. In addition, the phrase "less than about" value or "greater than about" value should be understood according to the definition of the term "about" provided herein. Similarly, the term "about" refers to all values in the series or the endpoints of the range when it is before a series of numerical values or numerical ranges (e.g., "about 10, 20, 30" or "about 10-30"), respectively.
如本文所用，术语“环状细胞穿透肽”或“cCPP”是指促进反义化合物递送的肽。As used herein, the term "cyclic cell penetrating peptide" or "cCPP" refers to a peptide that facilitates the delivery of an antisense compound.
术语“miniPEG”、“PEG2”和“AEEA”在本文中可互换使用，是指2-[2-[2-氨基乙氧基]乙氧基]乙酸。The terms "miniPEG," "PEG2," and "AEEA" are used interchangeably herein to refer to 2-[2-[2-aminoethoxy]ethoxy]acetic acid.
如本文所用，术语“内体逃逸载体”(EEV)是指通过化学键联(即，共价键或非共价相互作用)与接头和/或环外肽(EP)缀合的cCPP。EEV可以是式(B)的EEV。As used herein, the term "endosomal escape vector" (EEV) refers to a cCPP conjugated to a linker and/or an exocyclic peptide (EP) by chemical bonding (ie, covalent bond or non-covalent interaction). The EEV may be an EEV of formula (B).
如本文所用，术语“EEV-缀合物”是指通过化学键联(即，共价键或非共价相互作用)与AC缀合的本文所定义的内体逃逸载体。AC可通过EEV递送至细胞中。EEV-缀合物可以是式(C)的EEV-缀合物。As used herein, the term "EEV-conjugate" refers to an endosomal escape vector as defined herein conjugated to AC by chemical bonding (i.e., covalent bonding or non-covalent interaction). AC can be delivered to cells by EEV. The EEV-conjugate can be an EEV-conjugate of formula (C).
如本文所用，术语“环外肽”(EP)和“调节肽”(MP)可互换使用，是指通过肽键连接的两个或更多个氨基酸残基，其可与本文所公开的环状细胞穿透肽(cCPP)缀合。当与本文所公开的环肽缀合时，EP可改变化合物的组织分布和/或保留。典型地，EP包含至少一个带正电荷的氨基酸残基，例如至少一个赖氨酸残基和/或至少一个精氨酸残基。本文描述了EP的非限制性实例。EP可以是在本领域中被鉴定为“核定位序列”(NLS)的肽。核定位序列的非限制性实例包括SV40病毒大T抗原的核定位序列，其最小功能单元是七个氨基酸序列PKKKRKV、具有序列NLSKRPAAIKKAGQAKKKK的双分型核质蛋白NLS、具有氨基酸序列PAAKRVKLD或RQRRNELKRSF的c-myc核定位序列、来自输入蛋白-α的IBB域的序列RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV、肌瘤T蛋白的序列VSRKRPRP和PPKKARED、人p53的序列PQPKKKPL、小鼠c-ablIV的序列SALIKKKKKMAP、流感病毒NS1的序列DRLRR和PKQKKRK、肝炎病毒δ抗原的序列RKLKKKIKKL和小鼠Mxl蛋白的序列REKKKFLKRR、人聚(ADP-核糖)聚合酶的序列KRKGDEVDGVDEVAKKKSKK和类固醇激素受体(人)糖皮质激素的序列RKCLQAGMNLEARKTKK。国际公开号2001/038547描述了NLS的附加实例并且全文以引用方式并入本文。As used herein, the terms "exocyclic peptide" (EP) and "modulatory peptide" (MP) are used interchangeably and refer to two or more amino acid residues connected by a peptide bond, which can be conjugated to the cyclic cell penetrating peptide (cCPP) disclosed herein. When conjugated to the cyclic peptide disclosed herein, the EP can change the tissue distribution and/or retention of the compound. Typically, the EP comprises at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one arginine residue. Non-limiting examples of EP are described herein. The EP can be a peptide identified in the art as a "nuclear localization sequence" (NLS). Non-limiting examples of nuclear localization sequences include the nuclear localization sequence of the SV40 virus large T antigen, whose minimum functional unit is the seven amino acid sequence PKKKRKV, the bipartite nucleoplasmic protein NLS having the sequence NLSKRPAAIKKAGQAKKKK, the c-myc nuclear localization sequence having the amino acid sequence PAAKRVKLD or RQRRNELKRSF, the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of importin-α, the sequence V of the myoma T protein. SRKRPRP and PPKKARED, the sequence PQPKKKPL of human p53, the sequence SALIKKKKKMAP of mouse c-ablIV, the sequences DRLRR and PKQKKRK of influenza virus NS1, the sequence RKLKKKIKKL of hepatitis virus delta antigen and the sequence REKKKFLKRR of mouse Mxl protein, the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase and the sequence RKCLQAGMNLEARKTKK of steroid hormone receptor (human) glucocorticoid. International Publication No. 2001/038547 describes additional examples of NLSs and is incorporated herein by reference in its entirety.
如本文所用，“接头”或“L”是指将一个或多个部分(例如，环外肽(EP)和AC)与环状细胞穿透肽(cCPP)共价键合的部分。接头可包含天然或非天然氨基酸或多肽。接头可以是含有两个或更多个适于将cCPP结合到AC从而形成本文所公开的化合物的适当官能团的合成化合物。接头可包含聚乙二醇(PEG)部分。接头可包含一个或多个氨基酸。cCPP可经由接头与AC共价结合。As used herein, "linker" or "L" refers to a moiety that covalently bonds one or more moieties (e.g., exocyclic peptide (EP) and AC) to a cyclic cell penetrating peptide (cCPP). The linker may comprise a natural or non-natural amino acid or polypeptide. The linker may be a synthetic compound containing two or more suitable functional groups suitable for binding cCPP to AC to form a compound disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. The cCPP may be covalently bound to AC via a linker.
如本文所用，术语“寡核苷酸”是指包含多个连接的核苷酸或核苷的寡聚化合物。寡核苷酸的一个或多个核苷酸可被修饰。寡核苷酸可包括核糖核酸(RNA)或脱氧核糖核酸(DNA)。寡核苷酸可由天然和/或修饰的核碱基、糖和共价核苷间键联构成，并且可进一步包括非核酸缀合物。As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of connected nucleotides or nucleosides. One or more nucleotides of an oligonucleotide may be modified. An oligonucleotide may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). An oligonucleotide may be composed of natural and/or modified nucleobases, sugars, and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.
术语“肽”、“蛋白质”和“多肽”可互换使用，是指包含通过一个氨基酸的羧基基团与另一个氨基酸的α氨基基团连接的两个或更多个氨基酸的天然或合成分子。两个或更多个氨基酸残基可通过一个氨基酸的羧基基团与α氨基基团连接。多肽的两个或更多个氨基酸可通过肽键接合。多肽可包括肽主链修饰，其中两个或更多个氨基酸通过除肽键以外的键共价附接。多肽可包括一种或多种非天然氨基酸、氨基酸类似物或能够整合到多肽中的其他合成分子。术语多肽包括天然存在的和人工存在的氨基酸。术语多肽包括例如包含约2至约100个氨基酸残基的肽以及包含超过约100个氨基酸残基或超过约1000个氨基酸残基的蛋白质，包括但不限于治疗性蛋白质，诸如抗体、酶、受体、可溶性蛋白质等。The terms "peptide", "protein" and "polypeptide" are used interchangeably and refer to natural or synthetic molecules comprising two or more amino acids connected by the carboxyl group of one amino acid to the alpha amino group of another amino acid. Two or more amino acid residues can be connected to the alpha amino group through the carboxyl group of one amino acid. Two or more amino acids of a polypeptide can be joined by peptide bonds. A polypeptide may include a peptide backbone modification in which two or more amino acids are covalently attached by a bond other than a peptide bond. A polypeptide may include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide. The term polypeptide includes naturally occurring and artificially occurring amino acids. The term polypeptide includes, for example, peptides comprising about 2 to about 100 amino acid residues and proteins comprising more than about 100 amino acid residues or more than about 1000 amino acid residues, including but not limited to therapeutic proteins, such as antibodies, enzymes, receptors, soluble proteins, etc.
术语“治疗性多肽”指具有治疗、预防或其他生物活性的多肽。治疗性多肽可以任何合适的方式产生。例如，治疗性多肽可从天然存在的环境中分离或纯化，可化学合成，可重组产生，或它们的组合。The term "therapeutic polypeptide" refers to a polypeptide having therapeutic, preventive or other biological activity. Therapeutic polypeptides can be produced in any suitable manner. For example, therapeutic polypeptides can be isolated or purified from naturally occurring environments, can be chemically synthesized, can be recombinantly produced, or a combination thereof.
术语“小分子”是指具有药理学活性且分子量小于约2000道尔顿、或小于约1000道尔顿、或小于约500道尔顿的有机化合物。小分子治疗剂典型地通过化学合成来制造。The term "small molecule" refers to an organic compound that has pharmacological activity and has a molecular weight of less than about 2000 Daltons, or less than about 1000 Daltons, or less than about 500 Daltons. Small molecule therapeutics are typically made by chemical synthesis.
如本文所用，术语“邻接”是指通过共价键连接的两个氨基酸。例如，在代表性环状细胞穿透肽(cCPP)诸如AA1/AA2、AA2/AA3、AA3/AA4，和AA5/AA1的情况下举例说明了邻接氨基酸对。As used herein, the term "adjacent" refers to two amino acids linked by a covalent bond. For example, in representative cyclic cell penetrating peptides (cCPPs) such as Contiguous amino acid pairs are exemplified in the cases of AA1 /AA2 , AA2 /AA3 , AA3 /AA4 , and AA5 /AA1 .
如本文所用，化学物质的残基是指存在于特定产物中的化学物质的衍生物。为了形成产物，所述物质的至少一个原子被与另一部分的键替换，使得产物含有所述化学物质的衍生物或残基。例如，本文所述的环状细胞穿透肽(cCPP)具有通过形成一个或多个肽键而掺入其中的氨基酸(例如，精氨酸)。掺入cCPP中的氨基酸可称为残基，或简单地称为氨基酸。因此，精氨酸或精氨酸残基是指As used herein, a residue of a chemical substance refers to a derivative of a chemical substance present in a particular product. To form a product, at least one atom of the substance is replaced by a bond to another moiety, such that the product contains a derivative or residue of the chemical substance. For example, the cyclic cell penetrating peptides (cCPPs) described herein have an amino acid (e.g., arginine) incorporated therein by forming one or more peptide bonds. The amino acid incorporated into the cCPP can be referred to as a residue, or simply an amino acid. Thus, arginine or an arginine residue refers to
术语“其质子化形式”是指氨基酸的质子化形式。例如，精氨酸侧链上的胍基可被质子化以形成胍鎓基团。质子化形式的精氨酸的结构是The term "a protonated form thereof" refers to the protonated form of an amino acid. For example, the guanidinium group on the side chain of arginine can be protonated to form a guanidinium group. The structure of the protonated form of arginine is
如本文所用，术语“手性”是指具有多于一种在原子的三维空间排列上不同的立体异构体的分子，其中一种立体异构体是另一种立体异构体的不可重叠的镜像。除了甘氨酸外，氨基酸具有与羧基基团相邻的手性碳原子。术语“对映体”是指手性的立体异构体。手性分子可以是具有“D”和“L”对映体的氨基酸残基。没有手性中心的分子，诸如甘氨酸，可被称为“非手性的”。As used herein, the term "chiral" refers to molecules having more than one stereoisomer that differs in the three-dimensional spatial arrangement of the atoms, wherein one stereoisomer is a non-superimposable mirror image of another stereoisomer. With the exception of glycine, amino acids have chiral carbon atoms adjacent to the carboxyl group. The term "enantiomer" refers to a chiral stereoisomer. A chiral molecule can be an amino acid residue having "D" and "L" enantiomers. Molecules without chiral centers, such as glycine, can be referred to as "achiral".
如本文所用，术语“疏水的”是指不溶于水或在水中溶解度极小的部分。通常，中性部分和/或非极性部分，或者主要是中性和/或非极性的部分是疏水的。疏水性可通过本文以下公开的方法之一来测量。As used herein, the term "hydrophobic" refers to a portion that is insoluble or has minimal solubility in water. Typically, a neutral portion and/or a non-polar portion, or a predominantly neutral and/or non-polar portion is hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein below.
如本文所用，“芳族”是指具有4n+2个π电子的不饱和环分子，其中n是任何整数。术语“非芳族”是指不属于芳族定义的任何不饱和环分子。As used herein, "aromatic" refers to an unsaturated ring molecule having 4n+2 π electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated ring molecule that does not fall within the definition of aromatic.
“烷基”、“烷基链”或“烷基基团”是指具有一至四十个碳原子并且通过单键与分子的其余部分附接的完全饱和的直链或支链烃链基团。包括包含1至40的任何数量的碳原子的烷基。包含至多40个碳原子的烷基是C1-C40烷基，包含至多10个碳原子的烷基是C1-C10烷基，包含至多6个碳原子的烷基是C1-C6烷基，并且包含至多5个碳原子的烷基是C1-C5烷基。C1-C5烷基包括C5烷基、C4烷基、C3烷基、C2烷基和C1烷基(即，甲基)。C1-C6烷基包括上文对于C1-C5烷基所述的所有部分，但还包括C6烷基。C1-C10烷基包括上文对于C1-C5烷基和C1-C6烷基所述的所有部分，但还包括C7、C8、C9和C10烷基。类似地，C1-C12烷基包括所有前述部分，但还包括C11和C12烷基。C1-C12烷基的非限制性实例包括甲基、乙基、正丙基、异丙基、仲丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、叔戊基、正己基、正庚基、正辛基、正壬基、正癸基、正十一烷基和正十二烷基。除非在说明书中另有具体说明，否则烷基基团可任选地被取代。"Alkyl,""alkylchain," or "alkyl group" refers to a fully saturated straight or branched hydrocarbon chain radical having from one to forty carbon atoms and attached to the rest of the molecule by a single bond. Alkyl groups containing any number of carbon atoms from 1 to 40 are included. Alkyl groups containing up to 40 carbon atoms are C1 -C40 alkyl groups, alkyl groups containing up to 10 carbon atoms are C1 -C10 alkyl groups, alkyl groups containing up to 6 carbon atoms are C1 -C6 alkyl groups, and alkyl groups containing up to 5 carbon atoms are C1 -C5 alkyl groups. C1 -C5 alkyl groups include C5 alkyl groups, C4 alkyl groups, C3 alkyl groups, C2 alkyl groups, and C1 alkyl groups (i.e., methyl groups). C1 -C6 alkyl groups include all of the moieties described above for C1 -C5 alkyl groups, but also include C6 alkyl groups. C1 -C10 alkyl includes all the moieties described above for C1 -C5 alkyl and C1 -C6 alkyl, but also includes C7 , C8 , C9 and C10 alkyl. Similarly, C1 -C12 alkyl includes all the aforementioned moieties, but also includes C11 and C12 alkyl. Non-limiting examples of C1 -C12 alkyl include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless otherwise specifically stated in the specification, the alkyl group may be optionally substituted.
“亚烷基”、“亚烷基链”或“亚烷基基团”是指具有一至四十个碳原子的完全饱和的直链或支链二价烃链基团。C2-C40亚烷基的非限制性实例包括亚乙基、亚丙基、正亚丁基、亚乙烯基、亚丙烯基、正亚丁烯基、亚丙炔基、正亚丁炔基等。除非在说明书中另有具体说明，否则亚烷基链可任选地被取代。"Alkylene", "alkylene chain" or "alkylene group" refers to a fully saturated straight or branched divalent hydrocarbon chain group having one to forty carbon atoms. Non-limiting examples ofC2 -C40 alkylene include ethylene, propylene, n-butylene, vinylene, propenylene, n-butylene, propynylene, n-butylene, etc. Unless otherwise specifically stated in the specification, the alkylene chain may be optionally substituted.
“烯基”、“烯基链”或“烯基基团”是指具有二至四十个碳原子并具有一个或多个碳-碳双键的直链或支链烃链基团。每个烯基基团通过单键与分子的其余部分附接。包括包含2至40的任何数量的碳原子的烯基基团。包含至多40个碳原子的烯基是C2-C40烯基，包含至多10个碳原子的烯基是C2-C10烯基，包含至多6个碳原子的烯基是C2-C6烯基，并且包含至多5个碳原子的烯基是C2-C5烯基。C2-C5烯基包括C5烯基、C4烯基、C3烯基和C2烯基。C2-C6烯基包括上文关于C2-C5烯基所述的所有部分，但还包括C6烯基。C2-C10烯基包括上文对于C2-C5烯基和C2-C6烯基所述的所有部分，但还包括C7、C8、C9和C10烯基。类似地，C2-C12烯基包括所有前述部分，但还包括C11和C12烯基。C2-C12烯基的非限制性实例包括乙烯基(ethenyl/vinyl)、1-丙烯基、2-丙烯基(烯丙基)、异丙烯基、2-甲基-1-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、1-戊烯基、2-戊烯基、3-戊烯基、4-戊烯基、1-己烯基、2-己烯基、3-己烯基、4-己烯基、5-己烯基、1-庚烯基、2-庚烯基、3-庚烯基、4-庚烯基、5-庚烯基、6-庚烯基、1-辛烯基、2-辛烯基、3-辛烯基、4-辛烯基、5-辛烯基、6-辛烯基、7-辛烯基、1-壬烯基、2-壬烯基、3-壬烯基、4-壬烯基、5-壬烯基、6-壬烯基、7-壬烯基、8-壬烯基、1-癸烯基、2-癸烯基、3-癸烯基、4-癸烯基、5-癸烯基、6-癸烯基、7-癸烯基、8-癸烯基、9-癸烯基、1-十一烯基、2-十一烯基、3-十一烯基、4-十一烯基、5-十一烯基、6-十一烯基、7-十一烯基、8-十一烯基、9-十一烯基、10-十一烯基、1-十二烯基、2-十二烯基、3-十二烯基、4-十二烯基、5-十二烯基、6-十二烯基、7-十二烯基、8-十二烯基、9-十二烯基、10-十二烯基和11-十二烯基。除非在说明书中另有具体说明，否则烷基基团可任选地被取代。"Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain group having from two to forty carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups containing any number of carbon atoms from 2 to 40 are included. Alkenyl groups containing up to 40 carbon atoms areC2 -C40 alkenyl, alkenyl groups containing up to 10 carbon atoms areC2 -C10 alkenyl, alkenyl groups containing up to 6 carbon atoms areC2 -C6 alkenyl, and alkenyl groups containing up to 5 carbon atoms areC2 -C5 alkenyl.C2 -C5 alkenyl includesC5 alkenyl,C4 alkenyl,C3 alkenyl andC2 alkenyl.C2 -C6 alkenyl includes all of the moieties described above forC2 -C5 alkenyl, but also includesC6 alkenyl.C2 -C10 alkenyl includes all of the moieties described above for C2-C5 alkenyl andC2 -C6 alkenyl, but also includesC7 ,C8 ,C9 , andC10 alkenyl. Similarly,C2 -C12 alkenyl includes all of the aforementioned moieties, but also includesC11 andC12 alkenyl. Non-limiting examples ofalkenyl groups include ethenyl/vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless otherwise specifically stated in the specification, the alkyl group may be optionally substituted.
“亚烯基”、“亚烯基链”或“亚烯基基团”是指具有二至四十个碳原子并且具有一个或多个碳-碳双键的直链或支链二价烃链基团。C2-C40亚烯基的非限制性实例包括乙烯、丙烯、丁烯等。除非在说明书中另有具体说明，否则亚烯基链可以是任选的。"Alkenylene", "alkenylene chain" or "alkenylene group" refers to a straight or branched divalent hydrocarbon chain group having two to forty carbon atoms and having one or more carbon-carbon double bonds. Non-limiting examples ofC2 -C40 alkenylene include ethylene, propylene, butene, etc. Unless specifically stated otherwise in the specification, the alkenylene chain may be optional.
“烷氧基”或“烷氧基基团”是指基团-OR，其中R是如本文所定义的烷基、烯基、炔基、环烷基或杂环基。除非在说明书中另有具体说明，否则烷氧基基团可任选地被取代。"Alkoxy" or "alkoxy group" refers to a group -OR, wherein R is alkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl as defined herein. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted.
“酰基”或“酰基基团”是指基团-C(O)R，其中R是氢、烷基、烯基、炔基、碳环基或杂环基，如本文所定义。除非在说明书中另有具体说明，否则酰基可任选地被取代。"Acyl" or "acyl group" refers to the group -C(O)R, wherein R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, as defined herein. Unless stated otherwise specifically in the specification, an acyl group may be optionally substituted.
“烷基氨基甲酰基”或“烷基氨基甲酰基基团”是指基团-O-C(O)-NRaRb，其中Ra和Rb相同或不同并且独立地是如本文所定义的烷基、烯基、炔基、芳基、杂芳基，或RaRb可合一起形成如本文所定义的环烷基基团或杂环基基团。除非在说明书中另有具体说明，否则烷基氨基甲酰基基团可任选地被取代。"Alkylcarbamoyl" or "alkylcarbamoyl group" refers to the group -OC(O)-NRaRb, wherein Ra and Rbarethesame or different and are independently alkyl,alkenyl , alkynyl, aryl, heteroaryl as defined herein, orRaRb may be taken together to form a cycloalkyl group or a heterocyclyl group as defined herein. Unless stated otherwise specifically in the specification, an alkylcarbamoyl group may be optionally substituted.
“烷基羧酰胺基”或“烷基羧酰胺基基团”是指基团-C(O)-NRaRb，其中Ra和Rb相同或不同并且独立地是如本文所定义的烷基、烯基、炔基、芳基、杂芳基、环烷基、环烯基、环炔基或杂环基基团，或RaRb可合一起形成如本文所定义的环烷基基团。除非在说明书中另有具体说明，否则烷基羧酰胺基基团可任选地被取代。"Alkylcarboxamido" or "alkylcarboxamido group" refers to the group -C(O)-NRaRb , whereinRa andRb are the same or different and are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl or heterocyclyl groups as defined herein, orRaRb may be takentogether to form acycloalkyl group as defined herein. Unless specifically stated otherwise in the specification, an alkylcarboxamido group may be optionally substituted.
“芳基”是指包含氢、6至18个碳原子和至少一个芳环的烃环体系基团。为了本发明的目的，芳基基团可以是单环、双环、三环或四环环体系，其可包括稠环或桥环体系。芳基基团包括但不限于衍生自醋蒽烯(aceanthrylene)、苊烯(acenaphthylene)、醋菲烯(acephenanthrylene)、蒽、薁(azulene)、苯、屈(chrysene)、荧蒽(fluoranthene)、芴、不对称引达省(as-indacene)、对称引达省(s-indacene)、茚满、茚、萘、非那烯(phenalene)、菲、七曜烯(pleiadene)、芘和苯并菲的芳基基团。除非在说明书中另有具体说明，否则术语“芳基”意在包括任选取代的芳基基团。"Aryl" refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of the present invention, an aryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused ring or bridged ring system. Aryl groups include, but are not limited to, aryl groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene. Unless otherwise specifically stated in the specification, the term "aryl" is intended to include optionally substituted aryl groups.
“杂芳基”是指包含氢原子、一至十三个碳原子、一至六个选自氮、氧和硫的杂原子和至少一个芳环的5至20元环体系基团。为了本发明的目的，杂芳基基团可以是单环、双环、三环或四环环体系，其可包括稠环或桥环体系；并且杂芳基基团中的氮、碳或硫原子可任选地被氧化；氮原子可任选地被季铵化。实例包括但不限于氮杂环庚烯基、吖啶基、苯并咪唑基、苯并噻唑基、苯并吲哚基、苯并二氧杂环戊烯基、苯并呋喃基、苯并噁唑基、苯并噻唑基、苯并噻二唑基、苯并[b][1，4]二氧杂环庚烯基、1，4-苯并二噁烷基、苯并萘并呋喃基、苯并噁唑基、苯并二氧杂环戊烯基、苯并二氧杂环己烯基、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基(苯并苯硫基)、苯并三唑基、苯并[4，6]咪唑并[1，2-a]吡啶基、咔唑基、噌啉基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、异噻唑基、咪唑基、吲唑基、吲哚基、吲唑基、异吲哚基、吲哚啉基、异吲哚啉基、异喹啉基、吲哚嗪基、异噁唑基、萘啶基、噁二唑基、2-氧代氮杂环庚烯基、噁唑基、环氧乙烷基、1-氧吡啶基、1-氧化嘧啶基、1-氧化吡嗪基、1-氧化哒嗪基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基、蝶啶基、嘌呤基、吡咯基、吡唑基、吡啶基、吡嗪基、嘧啶基、哒嗪基、喹唑啉基、喹喔啉基、喹啉基、奎宁环基、异喹啉基、四氢喹啉基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基和苯硫基(即噻吩基)。除非在说明书中另有具体说明，否则杂芳基基团可任选地被取代。"Heteroaryl" refers to a 5- to 20-membered ring system radical containing hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepanyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl (benzophenylthio), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, oxazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxypyridinyl, 1-oxypyrimidinyl, 1-oxypyrazinyl, 1-oxypyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.
本文所用的术语“取代的”意指其中至少一个原子被非氢原子替换的任何上述基团(即，烷基、烯基、炔基、环烷基、环烯基、环炔基、杂环基、芳基、杂芳基、烷氧基、芳氧基、酰基、烷基氨基甲酰基、烷基羧酰胺基、烷氧羰基、烷硫基或芳硫基)，所述非氢原子诸如但不限于：卤素原子，诸如F、Cl、Br和I；诸如羟基基团、烷氧基基团和酯基团的基团中的氧原子；诸如硫醇基团、硫代烷基基团、砜基团、磺酰基基团和亚砜基团的基团中的硫原子；诸如胺、酰胺、烷基胺、二烷基胺、芳基胺、烷基芳基胺、二芳基胺、N-氧化物、酰亚胺和烯胺的基团中的氮原子；诸如三烷基甲硅烷基基团、二烷基芳基甲硅烷基基团、烷基二芳基甲硅烷基基团和三芳基甲硅烷基基团的基团中的硅原子；以及各种其他基团中的其他杂原子。“取代的”还意指其中一个或多个原子被与杂原子(诸如氧代、羰基、羧基和酯基团中的氧；以及诸如亚胺、肟、腙和腈的基团中的氮)的高阶键(例如双键或三键)替换的任何上述基团。例如，“取代的”包括其中一个或多个原子被-NRgRh、-NRgC(＝O)Rh、-NRgC(＝O)NRgRh、-NRgC(＝O)ORh、-NRgSO2Rh、-OC(＝O)NRgRh、-ORg、-SRg、-SORg、-SO2Rg、-OSO2Rg、-SO2ORg、＝NSO2Rg和-SO2NRgRh替换的任何上述基团。“取代的”还意指其中一个或多个氢原子被-C(＝O)Rg、-C(＝O)ORg、-C(＝O)NRgRh、-CH2SO2Rg、-CH2SO2NRgRh替换的任何上述基团。在上文中，Rg和Rh相同或不同，并且独立地是氢、烷基、烯基、炔基、烷氧基、烷基氨基、硫代烷基、芳基、芳烷基、环烷基、环烯基、环炔基、环烷基烷基、卤代烷基、卤代烯基、卤代炔基、杂环基、N-杂环基、杂环基烷基、杂芳基、N-杂芳基和/或杂芳基烷基。“取代的”还意指其中一个或多个原子被氨基、氰基、羟基、亚氨基、硝基、氧代、硫代、卤代、烷基、烯基、炔基、烷氧基、烷基氨基、硫代烷基、芳基、芳烷基、环烷基、环烯基、环炔基、环烷基烷基、卤代烷基、卤代烯基、卤代炔基、杂环基、N-杂环基、杂环基烷基、杂芳基、N-杂芳基和/或杂芳基烷基替换的任何上述基团。“取代的”还可意指侧链上的一个或多个原子被烷基、烯基、炔基、酰基、烷基羧酰胺基、烷氧羰基、碳环基、杂环基、芳基或杂芳基替换的氨基酸。另外，前述取代基中的每一者还可任选地被上述取代基中的一者或多者取代。As used herein, the term "substituted" means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamido, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom is replaced by a non-hydrogen atom, such as, but not limited to, halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means any of the above groups in which one or more atoms are replaced by a higher order bond (e.g., a double or triple bond) to a heteroatom (such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imine,oxime , hydrazone, and nitrile). For example, "substituted" includes any of the abovegroups in which oneor more atoms are replaced by-NRgRh ,-NRgC (=O)Rh ,-NRgC (=O)NRgRh , -NRgC(=O) ORh,-NRgSO2Rh ,-OC( =O)NRgRh,-ORg ,-SRg,-SORg ,-SO2Rg ,-OSO2Rg,-SO2ORg , =NSO2Rg,and-SO2NRgRh . "Substituted"also means any of the above groups wherein one or more hydrogen atoms are replaced by-C (=O)Rg , -C(=O)ORg , -C(=O)NRgRh, -CH2SO2Rg,-CH2SO2NRgRh . In the above,Rg andRh are the same or different and are independently hydrogen, alkyl,alkenyl , alkynyl, alkoxy, alkylamino, thioalkyl,aryl , aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. "Substituted" also means any of the above groups in which one or more atoms are replaced by amino, cyano, hydroxy, imino, nitro, oxo, thio, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. "Substituted" may also mean an amino acid in which one or more atoms on the side chain are replaced by alkyl, alkenyl, alkynyl, acyl, alkylcarboxamido, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. In addition, each of the aforementioned substituents may also be optionally substituted by one or more of the above substituents.
如本文所用，“受试者”意指个体。因此，“受试者”可包括驯养动物(例如，猫、狗等)、家畜(例如，牛、马、猪、绵羊、山羊等)、实验动物(例如，小鼠、兔、大鼠、豚鼠等)和鸟。“受试者”还可包括哺乳动物，诸如灵长类动物或人。因此，受试者可以是人或兽医患者。术语“患者”是指在临床医生(例如，医师)的治疗下的受试者。As used herein, "subject" means an individual. Thus, "subject" may include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" may also include mammals, such as primates or humans. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., physician).
术语“抑制”是指活性、反应、疾患、疾病或其他生物参数的减少。这可包括但不限于活性、反应、疾患或疾病的完全消除。这还可包括，例如，与天然或对照水平相比，活性、反应、疾患或疾病减少10％。因此，减少可以是与天然或对照水平相比10％、20％、30％、40％、50％、60％、70％、80％、90％、100％或其间的任何减少量的减少。The term "inhibit" refers to a reduction in an activity, reaction, disorder, disease or other biological parameter. This may include, but is not limited to, complete elimination of an activity, reaction, disorder or disease. This may also include, for example, a 10% reduction in an activity, reaction, disorder or disease compared to a natural or control level. Thus, a reduction may be a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any amount of reduction therebetween compared to a natural or control level.
“减少(reduce)”或所述词的其他形式，诸如“减少(reducing/reduction)”，意指事件或特征(例如，肿瘤生长)的降低。应当理解，这典型地与一些标准或预期值有关，换句话讲，它是相对的，但并不总是需要参考标准或相对值。例如，“减少肿瘤生长”意指相对于标准或对照(例如，未治疗的肿瘤)减少肿瘤的生长速率。"Reduce" or other forms of the word, such as "reducing" or "reduction", means a decrease in an event or characteristic (e.g., tumor growth). It will be understood that this is typically relative to some standard or expected value, in other words, it is relative, but does not always require reference to a standard or relative value. For example, "reduce tumor growth" means reducing the growth rate of a tumor relative to a standard or control (e.g., an untreated tumor).
术语“治疗”是指旨在治愈、改善、稳定或预防疾病、病理状况或病症而对患者进行的医学管理。所述术语包括积极治疗，即专门针对改善疾病、病理状况或病症的治疗，并且还包括病因治疗，即针对消除相关疾病、病理状况或病症的病因的治疗。另外，所述术语包括姑息治疗，即旨在缓解症状而非治愈疾病、病理状况或病症的治疗；预防性治疗，即旨在最小化或部分或完全抑制相关疾病、病理状况或病症的发展的治疗；和支持性治疗，即用于补充另一种旨在改善相关疾病、病理状况或病症的特定疗法的治疗。The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to the amelioration of a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed to the elimination of the cause of the disease, pathological condition or disorder in question. In addition, the term includes palliative treatment, i.e., treatment directed to the relief of symptoms rather than the cure of a disease, pathological condition or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the development of the disease, pathological condition or disorder in question; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to the amelioration of the disease, pathological condition or disorder in question.
术语“治疗有效”是指所用组合物的量足以改善疾病或病症的一种或多种病因或症状。此类改善仅需要减少或改变，而不必消除。The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or disorder. Such improvement only needs to be reduced or altered, not eliminated.
术语“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没有过度的毒性、刺激、过敏反应或其他问题或并发症的与合理的效益/风险比相称的那些化合物、材料、组合物和/或剂型。The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
术语“载剂”意指化合物、组合物、物质或结构，当与化合物或组合物组合时，其有助于或促进化合物或组合物的制备、储存、施用、递送、有效性、选择性或用于其预期用途或目的的任何其他特征。例如，可选择载剂以使活性成分的任何降解最小化并使受试者中的任何不良副作用最小化。The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or promotes the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in a subject.
如本文所用，术语“药学上可接受的载剂”是指无菌水性或非水性溶液、分散体、悬浮液或乳液，以及用于在临近使用前复溶成无菌可注射溶液或分散体的无菌粉末。合适的水性和非水性载剂、稀释剂、溶剂或媒介物的实例包括水、乙醇、多元醇(诸如甘油、丙二醇、聚乙二醇等)、羧甲基纤维素及其合适的混合物、植物油(诸如橄榄油)和可注射有机酯诸如油酸乙酯。适当的流动性可例如通过使用包衣材料诸如卵磷脂、在分散体的情况下通过维持所需的粒度以及通过使用表面活性剂来维持。这些组合物还可含有佐剂，诸如防腐剂、润湿剂、乳化剂和分散剂。可通过包含各种抗细菌剂和抗真菌剂诸如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸等来确保防止微生物的作用。还可期望包括等渗剂，诸如糖、氯化钠等。可注射制剂可例如通过经细菌截留过滤器过滤或通过掺入无菌固体组合物形式的灭菌剂来灭菌，所述无菌固体组合物可在临近使用前溶解或分散在无菌水或其他无菌可注射介质中。合适的惰性载剂可包括糖诸如乳糖。As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. It can be ensured that the action of microorganisms is prevented by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers may include sugars such as lactose.
如本文所用，术语“序列同一性”是指两个多肽序列之间的氨基酸相同并且在相同的相对位置中的百分比。因此，与另一个多肽序列相比，一个多肽序列具有一定百分比的序列同一性。对于序列比较，典型地将一个序列用作参考序列，将其与测试序列进行比较。本领域普通技术人员将理解，如果两个序列在对应位置含有同一的残基，则通常认为它们是“基本上同一的”。在实施方案中，两个氨基酸序列之间的序列同一性使用在EMBOSS包(EMBOSS：The European Molecular Biology Open Software Suite，Rice等人，2000，Trends Genet.16：276-277)的Needle程序中实现的Needleman-Wunsch算法(Needleman和Wunsch，1970，J.Mol.Biol.48：443-453)(截至提交之日存在的版本)来确定。所用参数是空位开放罚分10、空位延伸罚分0.5以及EBLOSUM62(BLOSUM62的EMBOSS版本)替换矩阵。标记为“最长同一性”的Needle输出(使用-nobrief选项获得)用作同一性百分比，并且计算如下：(同一的残基×100)/(比对长度-比对中的空位总数)As used herein, the term "sequence identity" refers to the percentage of amino acids identical and in the same relative position between two polypeptide sequences. Therefore, compared with another polypeptide sequence, a polypeptide sequence has a certain percentage of sequence identity. For sequence comparison, a sequence is typically used as a reference sequence, which is compared with a test sequence. It will be appreciated by those of ordinary skill in the art that if two sequences contain identical residues in corresponding positions, they are generally considered to be "substantially identical". In an embodiment, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) (version as of the date of submission). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the identity percentage and is calculated as follows: (identical residues × 100) / (total number of gaps in the alignment length-alignment)
在实施方案中，序列同一性可使用Smith-Waterman算法以截至提交之日存在的版本来确定。In an embodiment, sequence identity may be determined using the Smith-Waterman algorithm with the version existing as of the date of submission.
如本文所用，“序列同源性”是指两个多肽序列之间的氨基酸同源并且在相同的相对位置中的百分比。因此，与另一个多肽序列相比，一个多肽序列具有一定百分比的序列同源性。如本领域普通技术人员将理解，如果两个序列在对应位置含有同源残基，则通常认为它们是“基本上同源的”。同源残基可以是同一的残基。或者，同源残基可以是具有适当相似的结构和/或功能特征的不同一残基。例如，如本领域普通技术人员所熟知的，某些氨基酸典型地被分类为“疏水性”或“亲水性”氨基酸，和/或被分类为具有“极性”或“非极性”侧链，并且一个氨基酸取代另一个相同类型的氨基酸通常可被认为是“同源”取代。As used herein, "sequence homology" refers to the percentage of amino acids between two polypeptide sequences that are homologous and in the same relative position. Thus, a polypeptide sequence has a certain percentage of sequence homology compared to another polypeptide sequence. As will be appreciated by those of ordinary skill in the art, if two sequences contain homologous residues at corresponding positions, they are generally considered to be "substantially homologous". Homologous residues can be identical residues. Alternatively, homologous residues can be different residues with appropriately similar structural and/or functional characteristics. For example, as is well known to those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or are classified as having "polar" or "non-polar" side chains, and substitution of one amino acid for another of the same type can generally be considered a "homologous" substitution.
如本领域熟知的，可使用多种算法中的任一种来比较氨基酸序列，所述算法包括可在商业计算机程序中获得的那些算法，诸如截至提交之日存在的BLASTP、空位BLAST和PSI-BLAST。在以下中描述了示例性的此类程序：Altschul等人，Basic local alignmentsearch tool，J.Mol.Biol.，215(3)：403-410，1990；Altschul等人，Methods inEnzymology；Altschul等人，“Gapped BLAST and PSI-BLAST：a new generation ofprotein database search programs”，Nucleic Acids Res.25：3389-3402，1997；Baxevanis等人，Bioinformatics A Practical Guide to the Analysis of Genes andProteins，Wiley，1998；和Misener等人，(编)，Bioinformatics Methods and Protocols(Methods in Molecular Biology，第132卷)，Humana Press，1999。除了鉴定同源序列之外，上述程序典型地还提供同源程度的指示。As is well known in the art, amino acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTP, Gapped BLAST, and PSI-BLAST as of the date of submission. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above programs typically also provide an indication of the degree of homology.
如本文所用，术语“反义化合物”和“AC”可互换使用，是指与同其(AC)杂交的靶核酸分子至少部分互补的聚合核酸结构(也可称为寡核苷酸或多核苷酸)。AC可以是短(在实施方案中，小于50个碱基对)多核苷酸或包含与靶前mRNA链中的靶序列互补的序列的多核苷酸同源物。AC可由天然核酸、合成核酸、核酸同源物或它们的任何组合形成。在实施方案中，AC包含寡核苷。在实施方案中，AC包含反义寡核苷酸。在实施方案中，AC包含缀合基团。AC的非限制性实例包括但不限于引物、探针、反义寡核苷酸、外部引导序列(EGS)寡核苷酸、可变剪接子、siRNA、寡核苷酸、寡核苷、寡核苷酸类似物、寡核苷酸模拟物以及这些的嵌合组合。因此，这些化合物可以单链、双链、环状、支链或发夹的形式引入，并且可含有结构元件，诸如内部或末端凸起或环。寡聚双链化合物可以是杂交形成双链化合物的两条链，或者是具有足够的自身互补性以允许杂交并形成完全或部分双链化合物的单链。在实施方案中，AC调节(增加、减少或改变)靶核酸的表达。可对聚合核酸结构进行各种修饰，诸如二氨基磷酸酯吗啉代(PMO)。因此，如本文所用的AC涵盖本文所述的任何修饰，诸如PMO。As used herein, the terms "antisense compound" and "AC" are used interchangeably and refer to a polymeric nucleic acid structure (also referred to as an oligonucleotide or polynucleotide) that is at least partially complementary to a target nucleic acid molecule that hybridizes with it (AC). AC can be a short (in an embodiment, less than 50 base pairs) polynucleotide or a polynucleotide homolog comprising a sequence complementary to a target sequence in a target pre-mRNA chain. AC can be formed by natural nucleic acids, synthetic nucleic acids, nucleic acid homologs, or any combination thereof. In an embodiment, AC comprises an oligonucleoside. In an embodiment, AC comprises an antisense oligonucleotide. In an embodiment, AC comprises a conjugated group. Non-limiting examples of AC include, but are not limited to, primers, probes, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, variable splicing subs, siRNA, oligonucleotides, oligonucleoside, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these. Therefore, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched, or hairpins, and can contain structural elements, such as internal or terminal protrusions or loops. Oligomeric double-stranded compounds can be two chains that hybridize to form double-stranded compounds, or have enough self-complementarity to allow hybridization and form a single strand of a complete or partial double-stranded compound. In an embodiment, AC regulates (increases, decreases or changes) the expression of a target nucleic acid. Various modifications can be made to the polymeric nucleic acid structure, such as diaminophosphoryl morpholino (PMO). Therefore, AC as used herein encompasses any modification described herein, such as PMO.
如本文所用，术语“前mRNA”和“初级转录物”是指DNA转录后直接新合成的真核mRNA分子。前mRNA必须用5’帽进行盖帽，用3’聚A尾修饰，并剪接以产生成熟的mRNA序列。As used herein, the terms "pre-mRNA" and "primary transcript" refer to newly synthesized eukaryotic mRNA molecules directly after DNA transcription. Pre-mRNA must be capped with a 5' cap, modified with a 3' poly A tail, and spliced to produce a mature mRNA sequence.
如本文所使用，术语“靶向”或“靶向于”是指反义化合物(AC)与靶核酸分子或靶核酸分子区的缔合。在实施方案中，AC能够在生理条件下与靶核酸杂交。在实施方案中，AC靶向靶核酸内的特定部分或位点，例如，具有至少一种可识别的结构、功能或特征的靶核酸的一部分，诸如特定外显子或内含子，或外显子或内含子内的选定核碱基或基序。As used herein, the term "targeting" or "targeting to" refers to the association of an antisense compound (AC) with a target nucleic acid molecule or a region of a target nucleic acid molecule. In an embodiment, the AC is capable of hybridizing to a target nucleic acid under physiological conditions. In an embodiment, the AC is targeted to a specific portion or site within a target nucleic acid, for example, a portion of a target nucleic acid having at least one identifiable structure, function or feature, such as a specific exon or intron, or a selected nucleobase or motif within an exon or intron.
如本文所用，术语“靶核酸”和“靶序列”是指具有与反义化合物结合或杂交的核酸序列的核酸分子。靶核酸包括但不限于RNA(包括但不限于前mRNA和mRNA或其部分)、源自此种RNA的cDNA以及非翻译RNA，诸如miRNA。例如，在实施方案中，靶核酸可以是表达与特定病症或疾病病况相关的细胞基因(或由此种基因转录的mRNA)，或感染原的核酸分子。在实施方案中，靶核酸是靶RNA。在实施方案中，靶核酸是靶mRNA。在实施方案中，靶核酸是靶前mRNA。As used herein, the terms "target nucleic acid" and "target sequence" refer to nucleic acid molecules having a nucleic acid sequence that binds or hybridizes to an antisense compound. Target nucleic acids include, but are not limited to, RNA (including, but not limited to, pre-mRNA and mRNA or portions thereof), cDNA derived from such RNA, and non-translated RNA, such as miRNA. For example, in embodiments, the target nucleic acid can be a nucleic acid molecule expressing a cellular gene (or mRNA transcribed from such a gene) associated with a particular disorder or disease condition, or an infectious agent. In embodiments, the target nucleic acid is a target RNA. In embodiments, the target nucleic acid is a target mRNA. In embodiments, the target nucleic acid is a target pre-mRNA.
如本文所用，术语“剪接”和“处理”是指转录后前mRNA的修饰，其中内含子被去除并且外显子被接合。剪接发生在由五个小核核糖核蛋白(snRNP)构成的大RNA-蛋白质复合物(称为剪接体)催化的一系列反应中。在内含子内，剪接需要3’剪接位点、5’剪接位点和分支位点。snRNP的RNA组分与内含子相互作用，并且可能参与催化As used herein, the terms "splicing" and "processing" refer to the modification of pre-mRNA after transcription, in which introns are removed and exons are joined. Splicing occurs in a series of reactions catalyzed by a large RNA-protein complex (called the spliceosome) composed of five small nuclear ribonucleoproteins (snRNPs). Within an intron, splicing requires a 3' splice site, a 5' splice site, and a branch site. The RNA component of the snRNP interacts with the intron and may be involved in catalyzing
如本文所用，术语“外显子”是指前mRNA的一部分，其在剪接后典型地包含在成熟mRNA中。As used herein, the term "exon" refers to a portion of a pre-mRNA that is typically included in the mature mRNA after splicing.
如本文所用，术语“内含子”是指前mRNA的一部分，其在剪接后典型地不包含在成熟mRNA中。As used herein, the term "intron" refers to a portion of a pre-mRNA that is typically not included in the mature mRNA after splicing.
如本文所用，术语“侧翼”是指紧邻所缔合外显子上游(5’)或下游(3’)的内含子。例如，外显子44的5’侧翼内含子是指紧邻外显子44上游(即，直接偶联到外显子44的5’端)的内含子。例如，外显子44的3’侧翼内含子是指紧邻外显子44下游(即，直接偶联到外显子44的5’端)的内含子。As used herein, the term "flanking" refers to an intron that is immediately upstream (5') or downstream (3') of an associated exon. For example, the 5' flanking intron of exon 44 refers to an intron that is immediately upstream of exon 44 (i.e., directly coupled to the 5' end of exon 44). For example, the 3' flanking intron of exon 44 refers to an intron that is immediately downstream of exon 44 (i.e., directly coupled to the 5' end of exon 44).
“靶前mRNA”是包含与AC杂交的靶序列的前mRNA。A "target pre-mRNA" is a pre-mRNA comprising a target sequence that hybridizes to an AC.
“靶mRNA”是由靶前mRNA序列剪接产生的mRNA序列。在实施方案中，靶mRNA不编码功能性蛋白质。在实施方案中，靶mRNA保留一个或多个内含子序列。"Target mRNA" is an mRNA sequence produced by splicing of a target pre-mRNA sequence. In an embodiment, the target mRNA does not encode a functional protein. In an embodiment, the target mRNA retains one or more intron sequences.
如本文所用，术语“基因”是指具有涵盖与基因产物的表达相关的5’启动子区，以及任何内含子和外显子区，以及与基因产物的表达相关的3’非翻译区(″UTR″)的核酸序列的核酸分子。As used herein, the term "gene" refers to a nucleic acid molecule having a nucleic acid sequence that encompasses a 5' promoter region associated with expression of a gene product, as well as any intronic and exonic regions, and a 3' untranslated region ("UTR") associated with expression of a gene product.
本公开的“靶基因”是指编码靶前mRNA的基因。The "target gene" of the present disclosure refers to a gene encoding a target pre-mRNA.
“靶蛋白”是指由靶mRNA编码的氨基酸序列。在实施方案中，靶蛋白可以不是功能性蛋白质。"Target protein" refers to an amino acid sequence encoded by a target mRNA. In an embodiment, the target protein may not be a functional protein.
“野生型靶蛋白”是指由野生型、正常或未突变形式的靶基因产生的天然的功能性蛋白质异构体。野生型靶蛋白还指由已正确剪接的靶前mRNA产生的蛋白质。"Wild-type target protein" refers to the natural functional protein isoform produced by the wild-type, normal or unmutated form of the target gene. The wild-type target protein also refers to the protein produced by the correctly spliced target pre-mRNA.
如本文所用，术语“转录物”是指从DNA转录的RNA分子，并且包括但不限于mRNA、成熟mRNA、前mRNA和部分处理的RNA。As used herein, the term "transcript" refers to an RNA molecule transcribed from DNA, and includes, but is not limited to, mRNA, mature mRNA, pre-mRNA, and partially processed RNA.
如本文所用，“再剪接靶蛋白”是指由与AC杂交的靶前mRNA剪接而产生的mRNA编码的蛋白质。再剪接靶蛋白可与野生型靶蛋白同一，可与野生型靶蛋白同源，可以是野生型靶蛋白的功能变体，或者可以是野生型靶蛋白的活性片段。As used herein, "re-splicing target protein" refers to a protein encoded by mRNA generated by splicing of target pre-mRNA hybridized with AC. The re-splicing target protein may be identical to the wild-type target protein, may be homologous to the wild-type target protein, may be a functional variant of the wild-type target protein, or may be an active fragment of the wild-type target protein.
如本文所用，“功能片段”或“活性片段”是指真核野生型靶蛋白的一部分，其表现出活性，诸如全长野生型靶蛋白的一种或多种活性，或具有另一种活性。在实施方案中，共享野生型靶蛋白的至少一种生物活性的再剪接靶蛋白被认为是野生型靶蛋白的活性片段。活性可以是全长野生型靶蛋白活性的任何百分比(即，或多或少)，包括但不限于与野生型目标蛋白相比的活性的约1％，活性的约2％、约3％、约4％、约5％、约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约95％、约96％、约97％、约98％、约99％、约100％、约200％、约300％、约400％、约500％或更多(包括这些值之间的所有值和范围)。因此，在实施方案中，活性片段可保留野生型靶蛋白的一种或多种生物活性的至少一部分。在实施方案中，活性片段可增强野生型靶蛋白的一种或多种生物活性。As used herein, "functional fragment" or "active fragment" refers to a part of a eukaryotic wild-type target protein that exhibits activity, such as one or more activities of a full-length wild-type target protein, or has another activity. In an embodiment, the re-splicing target protein of at least one biological activity of a shared wild-type target protein is considered to be an active fragment of a wild-type target protein. Activity can be any percentage (i.e., more or less) of the activity of the full-length wild-type target protein, including but not limited to about 1% of the activity compared to the wild-type target protein, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 200%, about 300%, about 400%, about 500% or more (including all values and ranges between these values). Thus, in embodiments, an active fragment may retain at least a portion of one or more biological activities of a wild-type target protein. In embodiments, an active fragment may enhance one or more biological activities of a wild-type target protein.
如本文所用，术语″核苷″意指包含核碱基和糖的糖基胺。核苷包括但不限于天然核苷、无碱基核苷、修饰的核苷和具有模拟碱基和/或糖基团的核苷。“天然核苷”或“未修饰的核苷”是包含天然核碱基和天然糖的核苷。天然核苷包括RNA核苷和DNA核苷。As used herein, the term "nucleoside" means a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides with simulated bases and/or sugar groups. "Natural nucleosides" or "unmodified nucleosides" are nucleosides comprising natural nucleobases and natural sugars. Natural nucleosides include RNA nucleosides and DNA nucleosides.
如本文所用，术语“天然糖”是指未对其在RNA(2’-OH)或DNA(2’-H)中的天然存在形式进行修饰的核苷的糖。As used herein, the term "natural sugar" refers to the sugar of a nucleoside that is unmodified from its naturally occurring form in RNA (2'-OH) or DNA (2'-H).
如本文所用，术语“核苷酸”是指具有与糖共价连接的磷酸基团的核苷。核苷酸可用多种取代基中的任一种进行修饰。As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group covalently linked to a sugar. Nucleotides can be modified with any of a variety of substituents.
如本文所用，术语“核碱基”是指核苷或核苷酸的碱基部分。核碱基可包含能够与另一核酸的碱基氢键合的任何原子或原子团。天然核碱基是未对其在RNA或DNA中天然存在形式进行修饰的核碱基。As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid. A natural nucleobase is a nucleobase that has not been modified from its naturally occurring form in RNA or DNA.
如本文所用，术语“杂环碱基部分”是指包含杂环的核碱基。As used herein, the term "heterocyclic base moiety" refers to a nucleobase comprising a heterocyclic ring.
如本文所用，“寡核苷”是指其中核苷间键联不含有磷原子的寡核苷酸。As used herein, "oligonucleoside" refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
如本文所用，术语“寡核苷酸”是指包含多个连接的核苷酸或核苷的寡聚化合物。在某些实施方案中，寡核苷酸的一个或多个核苷酸被修饰。在实施方案中，寡核苷酸包含核糖核酸(RNA)或脱氧核糖核酸(DNA)。在实施方案中，寡核苷酸由天然和/或修饰的核碱基、糖和共价核苷间键联构成，并且还可包括非核酸缀合物。As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of connected nucleotides or nucleosides. In certain embodiments, one or more nucleotides of an oligonucleotide are modified. In embodiments, an oligonucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In embodiments, an oligonucleotide is composed of natural and/or modified nucleobases, sugars, and covalent internucleoside linkages, and may also include non-nucleic acid conjugates.
如本文所用，″核苷间键联″是指相邻核苷之间的共价键联。As used herein, "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides.
如本文所用，″天然的核苷间键联″是指3’至5’磷酸二酯键联。As used herein, "natural internucleoside linkage" refers to a 3' to 5' phosphodiester linkage.
如本文所用，术语“修饰的核苷间键联”是指除了天然存在的核苷间键联之外的核苷或核苷酸之间的任何键联。As used herein, the term "modified internucleoside linkage" refers to any linkage between nucleosides or nucleotides other than a naturally occurring internucleoside linkage.
如本文所用，术语“嵌合反义化合物”或“嵌合AC”是指具有至少一个糖、核碱基和/或核苷间键联的反义化合物，与同一寡聚化合物内的其他糖、核碱基和核苷间键联相比，所述糖、核碱基和/或核苷间键联被区别地修饰。其余的糖、核碱基和核苷间键联可独立地修饰或未修饰。一般来说，嵌合寡聚化合物将具有修饰的核苷，这些核苷可位于分离的位置或在将限定特定基序的区中组合在一起。修饰和/或模拟基团的任何组合可包含如本文所述的嵌合寡聚化合物。As used herein, the term "chimeric antisense compound" or "chimeric AC" refers to an antisense compound having at least one sugar, nucleobase, and/or internucleoside linkage that is differentially modified compared to other sugars, nucleobases, and internucleoside linkages within the same oligomeric compound. The remaining sugars, nucleobases, and internucleoside linkages may be independently modified or unmodified. In general, a chimeric oligomeric compound will have modified nucleosides that may be located in separate locations or grouped together in a region that will define a specific motif. Any combination of modifications and/or mimetic groups may comprise a chimeric oligomeric compound as described herein.
如本文所用，术语“混合主链反义寡核苷酸”是指其中反义寡核苷酸的至少一个核苷间键联不同于反义寡核苷酸的至少一个其他核苷酸间键联的反义寡核苷酸。As used herein, the term "mixed-backbone antisense oligonucleotide" refers to an antisense oligonucleotide in which at least one internucleoside linkage of the antisense oligonucleotide is different from at least one other internucleoside linkage of the antisense oligonucleotide.
如本文所用，术语″核碱基互补性″是指能够与另一核碱基进行碱基配对的核碱基。例如，在DNA中，腺嘌呤(A)与胸腺嘧啶(T)互补。例如，在RNA中，腺嘌呤(A)与尿嘧啶(U)互补。在实施方案中，互补核碱基是指反义化合物中能够与其靶核酸的核碱基进行碱基配对的核碱基。例如，如果反义化合物的某个位置处的核碱基能够与靶核酸的某个位置处的核碱基氢键合，那么寡核苷酸与靶核酸之间的氢键合的位置被认为在所述核碱基对处是互补的。As used herein, the term "nucleobase complementarity" refers to a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In an embodiment, a complementary nucleobase refers to a nucleobase in an antisense compound that can base pair with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound can hydrogen bond with a nucleobase at a certain position of a target nucleic acid, the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at the nucleobase pair.
如本文所用，术语“非互补核碱基”是指一对彼此不形成氢键或以其他方式支持杂交的核碱基。As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not hydrogen bond with each other or otherwise support hybridization.
如本文所用，术语“互补”是指寡聚化合物通过核碱基互补性与另一种寡聚化合物或核酸杂交的能力。在实施方案中，当每个分子中足够数量的对应位置被可彼此键合以允许反义化合物与靶标之间的稳定缔合的核碱基占据时，反义化合物和其靶标彼此互补。本领域技术人员认识到，在不消除寡聚化合物保持缔合的能力的情况下包含错配是可能的。因此，本文描述了反义化合物，其可包含至多约20％错配的核苷酸(即，与靶标的对应核苷酸不核碱基互补)。优选地，反义化合物含有不超过约15％、更优选不超过约10％、最优选不超过5％错配或没有错配。其余核苷酸是核碱基互补的或不会破坏杂交(例如，通用碱基)。本领域普通技术人员将认识到，本文提供的化合物与靶核酸至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％核碱基互补。As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize with another oligomeric compound or nucleic acid by core base complementarity. In an embodiment, when a sufficient number of corresponding positions in each molecule are occupied by core bases that can be bonded to each other to allow the stable association between antisense compounds and targets, antisense compounds and their targets complement each other. It is recognized by those skilled in the art that it is possible to include mismatches without eliminating the ability of oligomeric compounds to maintain association. Therefore, antisense compounds are described herein, which may include up to about 20% mismatched nucleotides (i.e., not core base complementary to the corresponding nucleotides of the target). Preferably, antisense compounds contain no more than about 15%, more preferably no more than about 10%, most preferably no more than 5% mismatches or no mismatches. The remaining nucleotides are core base complementary or will not destroy hybridization (e.g., universal bases). It will be recognized by those of ordinary skill in the art that compounds provided herein are complementary to target nucleic acids at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% core bases.
如本文所使用，“杂交”意指互补寡聚化合物(例如，反义化合物和其靶核酸)的配对。虽然不限于具体机制，但配对的最常见机制涉及氢键合，所述氢键合可为互补核苷或核苷酸碱基(核碱基)之间的沃森-克里克、霍斯汀或反向霍斯汀氢键合。例如，天然碱基腺嘌呤是与天然核碱基胸苷和尿嘧啶互补的核碱基，其通过形成氢键来配对。天然碱基鸟嘌呤是与天然碱基胞嘧啶和5-甲基胞嘧啶互补的核碱基。杂交可在不同情形下发生。As used herein, "hybridization" means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). Although not limited to a specific mechanism, the most common mechanism of pairing involves hydrogen bonding, which can be Watson-Crick, Hostin, or reverse Hostin hydrogen bonding between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is a nucleobase complementary to the natural nucleobases thymidine and uracil, which pair by forming hydrogen bonds. The natural base guanine is a nucleobase complementary to the natural bases cytosine and 5-methylcytosine. Hybridization can occur in different situations.
如本文所用，术语“特异性杂交”是指寡聚化合物与一个核酸位点杂交比它与另一个核酸位点杂交具有更大亲和力的能力。在实施方案中，反义寡核苷酸与多于一个靶位点特异性杂交。在实施方案中，寡聚化合物在严格杂交条件下与其靶标特异性杂交。As used herein, the term "specific hybridization" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site. In embodiments, an oligomeric compound specifically hybridizes to its target under stringent hybridization conditions.
术语“调节(modulate/modulating/modulation)”是指与调节前的表达、功能或活性水平相比时对表达、功能或活性的扰动。调节可包括表达、功能或活性的增加(刺激或诱导)或减少(抑制或降低)。在一个实施方案中，调节可包括扰动前mRNA处理期间的剪接位点选择。The term "modulate/modulating/modulation" refers to a disturbance in expression, function or activity when compared to the level of expression, function or activity before modulation. Modulation may include an increase (stimulation or induction) or a decrease (inhibition or reduction) in expression, function or activity. In one embodiment, modulation may include splice site selection during pre-mRNA processing of the perturbation.
术语“抑制(inhibit/inhibiting/inhibition)”是指活性、表达、功能或其他生物学参数的降低，并且可包括但不要求活性、表达、功能或其他生物学参数的完全消除。抑制可包括，例如，与对照相比，活性、反应、疾患或疾病至少约10％减少。在实施方案中，基因或蛋白质的表达、活性或功能降低了统计学上显著的量。The term "inhibit/inhibiting/inhibition" refers to a decrease in activity, expression, function, or other biological parameter, and may include but not require complete elimination of activity, expression, function, or other biological parameter. Inhibition may include, for example, at least about a 10% decrease in activity, response, disorder, or disease compared to a control. In embodiments, the expression, activity, or function of a gene or protein is reduced by a statistically significant amount.
如本文所用，术语“表达”是指将基因的编码信息转化为细胞中存在和操作的结构的所有功能和步骤。此类结构包括但不限于转录和翻译的产物。As used herein, the term "expression" refers to all functions and steps that convert the coded information of a gene into structures that exist and operate in a cell. Such structures include, but are not limited to, products of transcription and translation.
如本文所用，术语“2’-修饰的”或“2’-取代的”意指在2’位包含除H或OH之外的取代基的糖。2’-修饰的单体包括但不限于BNA和具有2’-取代基的单体(例如，核苷和核苷酸)，所述取代基诸如烯丙基、氨基、叠氮基、硫代、O-烯丙基、O-C1-C10烷基、-OCF3、O-(CH2)2-O-CH3、2’-O(CH2)2SCH3、O-(CH2)2-O-N(Rm)(Rn)或O-CH2-C(＝O)-N(Rm)(Rn)，其中每个Rm和Rn独立地是H或取代或未取代的C1-C10烷基。As used herein, the term "2'-modified" or "2'-substituted" means a sugar comprising a substituent other than H or OH at the 2' position. 2'-modified monomers include, but are not limited to, BNA and monomers (e.g., nucleosides and nucleotides) having 2'-substituents such as allyl, amino, azido, thio, O-allyl, OC1 -C10 alkyl, -OCF3 , O-(CH2 )2 -O-CH3 , 2'-O(CH2 )2 SCH3 , O-(CH2 )2 -ON(Rm )(Rn ) or O-CH2 -C(＝O)-N(Rm )(Rn ), wherein each R m and R n is independently H or substituted or unsubstituted C1 -C10 alkyl.
如本文所用，术语″MOE″是指2’-O-甲氧基乙基取代基。As used herein, the term "MOE" refers to a 2'-O-methoxyethyl substituent.
如本文所用，术语“高亲和力修饰的核苷酸”是指具有至少一个修饰的核碱基、核苷间键联或糖部分的核苷酸，使得所述修饰增加包含修饰的核苷酸的反义化合物与靶核酸的亲和力。高亲和力修饰包括但不限于BNA、LNA和2’-MOE。As used herein, the term "high affinity modified nucleotide" refers to a nucleotide having at least one modified nucleobase, internucleoside linkage, or sugar moiety, such that the modification increases the affinity of the antisense compound comprising the modified nucleotide to the target nucleic acid. High affinity modifications include, but are not limited to, BNA, LNA, and 2'-MOE.
如本文所用，术语“模拟物”是指取代AC中糖、核碱基和/或核苷间键联的基团。一般来说，使用模拟物替代糖或糖-核苷间键联组合，并且维持核碱基以便与所选靶标杂交。糖模拟物的代表性实例包括但不限于环己烯基或吗啉代。糖-核苷间键联组合的模拟物的代表性实例包括但不限于通过不带电荷的非手性键联来连接的肽核酸(PNA)和吗啉代基团。在一些情况下，使用模拟物替代核碱基。代表性核碱基模拟物是本领域熟知的，并且包括但不限于三环吩噁嗪类似物和通用碱基(Berger等人，Nuc Acid Res.2000，28：2911-14，以引用方式并入本文)。糖、核苷和核碱基模拟物的合成方法是本领域技术人员熟知的。As used herein, the term "mimetic" refers to a group that replaces a sugar, a nucleobase, and/or an internucleoside linkage in AC. In general, a mimetic is used to replace a sugar or a sugar-nucleoside linkage combination, and the nucleobase is maintained to hybridize with a selected target. Representative examples of sugar mimics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimics of a sugar-nucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups connected by uncharged non-chiral linkages. In some cases, mimics are used to replace nucleobases. Representative nucleobase mimics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28: 2911-14, incorporated herein by reference). The synthesis methods of sugar, nucleoside and nucleobase mimics are well known to those skilled in the art.
如本文所用，术语“双环核苷”或“BNA”是指其中核苷的呋喃糖部分包括连接呋喃糖环上的两个原子的桥，从而形成双环环体系的核苷。BNA包括但不限于α-L-LNA、β-D-LNA、ENA、氧氨基BNA(2’-O-N(CH3)-CH2-4’)和氨氧基BNA(2’-N(CH3)-O-CH2-4’)。As used herein, the term "bicyclic nucleoside" or "BNA" refers to a nucleoside in which the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring, thereby forming a bicyclic ring system. BNAs include, but are not limited to, α-L-LNA, β-D-LNA, ENA, oxyamino BNA (2'-ON(CH3 )-CH2 -4') and aminooxy BNA (2'-N(CH3 )-O-CH2 -4').
如本文所用，术语“4’至2’双环核苷”是指其中连接呋喃糖环的两个原子的桥桥接呋喃糖环的4’碳原子和2’碳原子，从而形成双环环体系的BNA。As used herein, the term "4' to 2' bicyclic nucleoside" refers to a BNA in which the bridge connecting the two atoms of the furanose ring bridges the 4' carbon atom and the 2' carbon atom of the furanose ring, thereby forming a bicyclic ring system.
如本文所用，“锁核酸”或“LNA”是指修饰的核苷酸，使得核糖基糖环的2’-羟基基团经由亚甲基基团连接到糖环的4’碳原子，从而形成2’-C，4’-C-氧基亚甲基键联。LNA包括但不限于α-L-LNA和β-D-LNA。As used herein, "locked nucleic acid" or "LNA" refers to a modified nucleotide such that the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring via a methylene group, thereby forming a 2'-C, 4'-C-oxymethylene linkage. LNA includes, but is not limited to, α-L-LNA and β-D-LNA.
如本文所用，术语“帽结构”或“末端帽部分”是指已并入AC任一端的化学修饰。As used herein, the term "cap structure" or "terminal cap moiety" refers to a chemical modification that has been incorporated into either end of an AC.
如本文所用，术语“剂量单位”是指提供药剂的形式。在实施方案中，剂量单位是包含冻干反义寡核苷酸的小瓶。在实施方案中，剂量单位是包含复溶反义寡核苷酸的小瓶。As used herein, the term "dosage unit" refers to the form in which a medicament is provided. In an embodiment, the dosage unit is a vial containing a lyophilized antisense oligonucleotide. In an embodiment, the dosage unit is a vial containing a reconstituted antisense oligonucleotide.
已描述了本发明的多个实施方案。然而，应当理解，在不背离本发明的精神和范围的情况下，可进行各种修改。其他实施方案在以下权利要求书的范围内。Several embodiments of the invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Other embodiments are within the scope of the following claims.
本说明书中提到的所有出版物、专利和专利申请都指示本发明所属领域技术人员的技术水平。所有出版物、专利和专利申请都以引用方式并入本文，引用的程度就如同已特定地和个别地指示将各个别出版物或专利申请以引用方式并入一般。All publications, patents and patent applications mentioned in this specification are indicative of the technical level of those skilled in the art to which the invention belongs. All publications, patents and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application had been specifically and individually indicated to be incorporated by reference.
实施例Example
实施例1.寡核苷酸与细胞穿透肽的缀合.Example 1. Conjugation of oligonucleotides to cell-penetrating peptides.
寡核苷酸与细胞穿透肽的缀合.如图1A中所示，5’硫代磷酸酯末端具有(NH2-(CH2)5-CH2-)接头的寡核苷酸经由肽上的羧酸酯或N-羟基琥珀酰亚胺酯(NHS酯)官能团与CPP缀合。如图1B中所示，寡核苷酸经由酰胺键形成(左)或点击化学与细胞穿透肽(CPP)缀合。接头/CPP安装在寡核苷酸的5’端或3’端上。Conjugation of oligonucleotides to cell penetrating peptides. As shown in FIG1A , an oligonucleotide with a (NH2-(CH2)5 -CH2-) linker at the 5' phosphorothioate terminus is conjugated to a CPP via a carboxylate or N-hydroxysuccinimide ester (NHS ester) functional group on the peptide. As shown in FIG1B , an oligonucleotide is conjugated to a cell penetrating peptide (CPP) via amide bond formation (left) or click chemistry. The linker/CPP is installed on the 5' or 3' end of the oligonucleotide.
具有PEG间隔区的寡核苷酸-肽缀合物的合成.如图2A和图2B中所示，合成了寡核苷酸-肽缀合物，其不具有(图2A)和具有(图2B)插入寡核苷酸部分与肽之间的PEG(聚乙二醇)接头。图中的“R”代表棕榈酰基。Synthesis of oligonucleotide-peptide conjugates with PEG spacers. As shown in Figures 2A and 2B, oligonucleotide-peptide conjugates were synthesized without (Figure 2A) and with (Figure 2B) a PEG (polyethylene glycol) linker inserted between the oligonucleotide portion and the peptide. "R" in the figure represents a palmitoyl group.
各种基因靶标的寡核苷酸-肽缀合物的合成.靶向表7中所示的DMD或AC的外显子44的示例性反义化合物(AC)，例如，表6A-6M中的那些或其反向互补序列，与本文公开的CPP或EEV缀合。在实施方案中，AC与具有氨基酸序列FfΦRrRrQ(EEV-1)的环肽缀合。在实施方案中，AC与具有氨基酸序列FfΦRrRrQ的环肽缀合，所述氨基酸序列与脂质基团(R1)(EEV-1(R1))缀合。在实施方案中，AC与具有序列环(FfΦRrRrQ)-PEG12-OH的内体逃逸载体(EEV)缀合。在实施方案中，AC与具有氨基酸序列FGFGRGRQ的环肽缀合。在实施方案中，AC与具有序列Ac-PKKKRKV-AEEA-Lys-(环[FGFGRGRQ])-PEG12-OH的EEV缀合。Synthesis of oligonucleotide-peptide conjugates for various gene targets. Exemplary antisense compounds (AC) targeting exon 44 of DMD or AC shown in Table 7, e.g., those in Tables 6A-6M or their reverse complement sequences, are conjugated to CPPs or EEVs disclosed herein. In an embodiment, AC is conjugated to a cyclic peptide having the amino acid sequence FfΦRrRrQ (EEV-1). In an embodiment, AC is conjugated to a cyclic peptide having the amino acid sequence FfΦRrRrQ, which is conjugated to a lipid group (R1) (EEV-1 (R1)). In an embodiment, AC is conjugated to an endosomal escape vector (EEV) having the sequence cyclic (FfΦRrRrQ)-PEG12-OH. In an embodiment, AC is conjugated to a cyclic peptide having the amino acid sequence FGFGRGRQ. In an embodiment, AC is conjugated to an EEV having the sequence Ac-PKKKRKV-AEEA-Lys-(cyclic [FGFGRGRQ])-PEG12-OH.
表7：靶向DMD的外显子44的ACTable 7: ACs targeting exon 44 of DMD
实施例2.使用与寡核苷酸缀合的细胞穿透肽来校正DMD小鼠模型中抗肌萎缩蛋白的外显子23剪接.Example 2. Use of cell-penetrating peptides conjugated to oligonucleotides to correct exon 23 splicing of dystrophin in a DMD mouse model.
小鼠.本研究使用MDX小鼠(Sicinski等人(1989)“The molecular basis ofmuscular dystrophy in the mdx mouse：a point mutation.Science.244(4912)：1578-80)，其含有C至T突变，导致在X染色体上的抗肌萎缩蛋白肌营养不良基因(Dmd)的外显子23内2983位的终止密码子。表达这种突变等位基因的小鼠产生截短的抗肌萎缩蛋白，因此是杜兴氏肌营养不良(“DMD”)的模型。Mice. This study used the mdx mouse (Sicinski et al. (1989) "The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 244(4912): 1578-80), which contains a C to T mutation resulting in a stop codon at position 2983 within exon 23 of the dystrophin gene (Dmd) on chromosome X. Mice expressing this mutant allele produce truncated dystrophin and are therefore a model for Duchenne muscular dystrophy ("DMD").
研究设计.使用MDX小鼠来评价组合物跳跃外显子23从而治疗DMD的能力。本研究中使用的AC是具有以下序列的二氨基磷酸酯吗啉代寡聚物(PMO)：5’-GCTATTACCTTAACCCA-3’(PMO-MDX-23)，其靶向外显子23。PMO-MDX-23与具有以下序列的内体逃逸载体(EEV)缀合：c环(FfΦRrRrQ)-PEG12-OH(EEV-1)，形成EEV-PMO缀合物(EEV-PMO-MDX-23)。不具有EEV的PMO称为PMO-MDX-23。EEV-PMO-MDX-23的制备示意图如图4A中所示。Study design. MDX mice were used to evaluate the ability of the composition to skip exon 23 and thus treat DMD. The AC used in this study was a phosphorodiamidate morpholino oligomer (PMO) with the following sequence: 5’-GCTATTACCTTAACCCA-3’ (PMO-MDX-23), which targets exon 23. PMO-MDX-23 was conjugated to an endosomal escape vector (EEV) with the following sequence: c-loop (FfΦRrRrQ)-PEG12-OH (EEV-1) to form an EEV-PMO conjugate (EEV-PMO-MDX-23). PMO without EEV is called PMO-MDX-23. A schematic diagram of the preparation of EEV-PMO-MDX-23 is shown in Figure 4A.
EEV-PMO-MDX-23和PMO-MDX-23以以下剂量肌内(IM)或静脉内(IV)施用于MDX小鼠：1mpk、3mpk、10mpk、30mpk；(mpk：化合物毫克数/体重千克数)。从组织样品中提取总RNA，并通过RT-PCR进行分析，以可视化剪接校正的效率。EEV-PMO-MDX-23 and PMO-MDX-23 were administered intramuscularly (IM) or intravenously (IV) to MDX mice at the following doses: 1 mpk, 3 mpk, 10 mpk, 30 mpk; (mpk: mg of compound/kg of body weight). Total RNA was extracted from tissue samples and analyzed by RT-PCR to visualize the efficiency of splicing correction.
通过RT-PCR和蛋白印迹检测剪接校正.如图4B中所示，用EEV-PMO-MDX-23(1mpk，IM或3mpk，IM)治疗的MDX小鼠产生缺乏外显子23的抗肌萎缩蛋白，而单独用PMO-MDX-23治疗的MDX小鼠仅产生含有外显子23的抗肌萎缩蛋白(与未治疗对照相似)。Splicing correction was detected by RT-PCR and Western blotting. As shown in Figure 4B, MDX mice treated with EEV-PMO-MDX-23 (1 mpk, IM or 3 mpk, IM) produced anti-dystrophin lacking exon 23, while MDX mice treated with PMO-MDX-23 alone produced only anti-dystrophin containing exon 23 (similar to untreated controls).
向MDX小鼠静脉内(IV)施用10mpk PMO-MDX-23和EEV-PMO-MDX-23。各个肌肉组(例如四头肌、胫骨前肌、横膈膜和心脏)中的抗肌萎缩蛋白产物如图4C中所示，证明与单独递送PMO-MDX-23相比，向MDX小鼠递送EEV-PMO-MDX-23导致四头肌、胫骨前肌、横膈膜和心脏中抗肌萎缩蛋白剪接的校正(例如，切除外显子23的抗肌萎缩蛋白)。10 mpk of PMO-MDX-23 and EEV-PMO-MDX-23 were administered intravenously (IV) to MDX mice. The dystrophin production in various muscle groups (e.g., quadriceps, tibialis anterior, diaphragm, and heart) is shown in FIG4C , demonstrating that delivery of EEV-PMO-MDX-23 to MDX mice resulted in correction of dystrophin splicing (e.g., excision of exon 23 dystrophin) in quadriceps, tibialis anterior, diaphragm, and heart compared to delivery of PMO-MDX-23 alone.
图5A至图5D示出了以下IV剂量方案对外显子跳跃功效的影响：10mpk或30mpk，1周给药一次。值得注意的是，30mpk EEV-PMO-MDX-231周给药一次导致所有四种组织中外显子跳跃的百分比最高：四头肌(图5A)、胫骨前肌(图5B)、横膈膜(图5C)和心脏(图5D)。Figures 5A to 5D show the effect of the following IV dosing regimens on exon skipping efficacy: 10 mpk or 30 mpk, administered once a week. Notably, 30 mpk EEV-PMO-MDX-23 administered once a week resulted in the highest percentage of exon skipping in all four tissues: quadriceps (Figure 5A), tibialis anterior (Figure 5B), diaphragm (Figure 5C), and heart (Figure 5D).
图6A至图6D示出了以每周两次10mpk、每周一次10mpk、每两周一次10mpk和每周一次30mpk给药EEV-PMO-MDX-23后，胫骨前肌(图6A)、四头肌(图6B)、横膈膜(图6C)和心脏(图6D)中外显子23剪接校正的百分比(如通过RT-PCR测定)。Figures 6A to 6D show the percentage of exon 23 splicing correction (as determined by RT-PCR) in the tibialis anterior muscle (Figure 6A), quadriceps (Figure 6B), diaphragm (Figure 6C), and heart (Figure 6D) following administration of EEV-PMO-MDX-23 at 10 mpk twice per week, 10 mpk once per week, 10 mpk once every two weeks, and 30 mpk once per week.
图7A至图7D示出了递送PMO-MDX-23或EEV-PMO-MDX-23后通过蛋白印迹法检测的四头肌(图7A)、胫骨前肌(TA)(图7B)、横膈膜(图7C)和心脏(图7D)中外显子-23校正抗肌萎缩蛋白的量。Figures 7A to 7D show the amount of exon-23 corrected dystrophin detected by Western blotting in quadriceps (Figure 7A), tibialis anterior (TA) (Figure 7B), diaphragm (Figure 7C), and heart (Figure 7D) after delivery of PMO-MDX-23 or EEV-PMO-MDX-23.
图8A至图8D示出了静脉内递送10mpk或30mpk EEV-PMO-MDX-23-1后横膈膜(图8A)、心脏(图8B)、四头肌(图8C)和胫骨前肌(图8D)中外显子23校正抗肌萎缩蛋白和α-肌动蛋白的蛋白印迹。8A-8D show Western blots of exon 23 corrected dystrophin and α-actin in diaphragm ( FIG. 8A ), heart ( FIG. 8B ), quadriceps ( FIG. 8C ), and tibialis anterior ( FIG. 8D ) muscles following intravenous delivery of 10 mpk or 30 mpk of EEV-PMO-MDX-23-1.
图9A至图9B示出了用30mpk EEV-PMO-MDX-23或30mpk PMO-MDX-23治疗后两周(图9A)和四周(图9B)MDX小鼠中的抗肌萎缩蛋白水平。9A-9B show dystrophin levels in MDX mice two weeks ( FIG. 9A ) and four weeks ( FIG. 9B ) after treatment with 30 mpk EEV-PMO-MDX-23 or 30 mpk PMO-MDX-23.
图10A至图10D示出了施用30mpk PMO-MDX-23或30mpk EEV-PMO-MDX-23的MDX小鼠胫骨前肌(图10A)、四头肌(图10B)、横膈膜(图10C)和心脏(图10D)中外显子23校正的百分比。与单独施用PMO-MDX-23的小鼠相比，施用EEV-PMO-MDX-23的小鼠表现出增强的剪接校正。Figures 10A to 10D show the percentage of exon 23 correction in the tibialis anterior muscle (Figure 10A), quadriceps muscle (Figure 10B), diaphragm (Figure 10C), and heart (Figure 10D) of MDX mice administered 30 mpk PMO-MDX-23 or 30 mpk EEV-PMO-MDX-23. Mice administered EEV-PMO-MDX-23 showed enhanced splicing correction compared to mice administered PMO-MDX-23 alone.
图11A至图11C示出了单一IV剂量(40mg/kg)的EEV-PMO-MDX-23后至多8周在mdx小鼠中观测到的心脏(图11A)、胫骨前肌(图11B)和横膈膜(图11C)中的外显子23跳跃和抗肌萎缩蛋白校正的高且持续的水平。Figures 11A-11C show high and sustained levels of exon 23 skipping and dystrophin correction observed in the heart (Figure 11A), tibialis anterior muscle (Figure 11B), and diaphragm (Figure 11C) in mdx mice up to 8 weeks after a single IV dose (40 mg/kg) of EEV-PMO-MDX-23.
实施例3.使用与寡核苷酸缀合的细胞穿透肽来校正DMD的D2-mdx小鼠模型中抗肌萎缩蛋白的外显子23剪接.Example 3. Use of cell-penetrating peptides conjugated to oligonucleotides to correct exon 23 splicing of dystrophin in the D2-mdx mouse model of DMD.
小鼠.本研究使用D2-mdx小鼠模型。(Fukada等人(2010)“Genetic backgroundaffects properties of satellite cells and mdx phenotypes.Am.J.Path.176(5)：2414-24)。Mice. The D2-mdx mouse model was used in this study. (Fukada et al. (2010) "Genetic background affects properties of satellite cells and mdx phenotypes. Am. J. Path. 176(5): 2414-24).
每月重复20mg/kg剂量的EEV-PMO-MDX-23-2(EEV＝Ac-PKKKRKV-miniPEG-K(环(GfFGrGrQ))-PEG12-OH；PMO＝5’-GGCCAAACCTCGGCTTACCTGAAAT-3’)或PMO-MDX-23-2(5’-GGCCAAACCTCGGCTTACCTGAAAT-3’)后在不同肌肉组(n＝6)中观察到稳健的外显子23跳跃。图12A至图12D示出了D2-mdx小鼠在心脏(图12A)、横膈膜(图12B)、胫骨前肌(图12C)和三头肌(图12D)中表现出广泛的抗肌萎缩蛋白表达和肌肉完整性的恢复。Robust exon 23 skipping was observed in different muscle groups (n=6) following repeated monthly 20 mg/kg doses of EEV-PMO-MDX-23-2 (EEV=Ac-PKKKRKV-miniPEG-K(cyclo(GfFGrGrQ))-PEG12 -OH; PMO=5'-GGCCAAACCTCGGCTTACCTGAAAT-3') or PMO-MDX-23-2 (5'-GGCCAAACCTCGGCTTACCTGAAAT-3'). Figures 12A to 12D show that D2-mdx mice exhibit widespread dystrophin expression and restoration of muscle integrity in the heart (Figure 12A), diaphragm (Figure 12B), tibialis anterior (Figure 12C), and triceps (Figure 12D).
肌聚糖与抗肌萎缩蛋白和其他抗肌萎缩蛋白相关蛋白相互作用，形成抗肌萎缩蛋白相关糖蛋白复合物(DAGC)。DAGC保护肌膜免受收缩引起的损伤。在D2-mdx小鼠模型中，抗肌萎缩蛋白的缺失导致α-肌聚糖的缺失。D2-mdx小鼠接受4xQ4W(20mg/kg)IV注射PMO-MDX-23-2或EEV-PMO-MDX-23-2治疗。EEV-PMO-MDX-23-2处理的肌肉表现出抗肌萎缩蛋白和α-肌聚糖几乎完全恢复，而PMO-MDX-23-2治疗的小鼠则显示抗肌萎缩蛋白或α-肌聚糖的恢复有限(数据未示出)。Sarcoglycan interacts with dystrophin and other dystrophin-associated proteins to form the dystrophin-associated glycoprotein complex (DAGC). DAGC protects the sarcolemma from contraction-induced damage. In the D2-mdx mouse model, the loss of dystrophin leads to the loss of α-sarcoglycan. D2-mdx mice were treated with 4xQ4W (20 mg/kg) IV injections of PMO-MDX-23-2 or EEV-PMO-MDX-23-2. Muscles treated with EEV-PMO-MDX-23-2 showed almost complete restoration of dystrophin and α-sarcoglycan, while mice treated with PMO-MDX-23-2 showed limited restoration of dystrophin or α-sarcoglycan (data not shown).
向D2-mdx小鼠施用20mg/kg的PMO-MDX-23-2或EEV-PMO-MDX-23-2(每月)。将肌酸激酶(CK)水平(图13A)和功能读数(线挂时间(图13B)和正常化握力(图13C))与野生型(DBA/2J)和媒介物(盐水)处理的小鼠进行比较。与单独使用PMO-MDX-23-2相比时，用EEV-PMO-MDX-23治疗使血清CK水平正常化并显著改善肌肉功能(图13A至图13C)。D2-mdx mice were administered 20 mg/kg of PMO-MDX-23-2 or EEV-PMO-MDX-23-2 (monthly). Creatine kinase (CK) levels (FIG. 13A) and functional readouts (wire hanging time (FIG. 13B) and normalized grip strength (FIG. 13C)) were compared to wild-type (DBA/2J) and vehicle (saline) treated mice. Treatment with EEV-PMO-MDX-23 normalized serum CK levels and significantly improved muscle function when compared to PMO-MDX-23-2 alone (FIG. 13A to FIG. 13C).
实施例4：施用EEV-PMO-MDX-23-2后对D2MDX小鼠的持续时间和重复剂量效应Example 4: Duration and Repeated Dose Effects in D2MDX Mice Following Administration of EEV-PMO-MDX-23-2
方法：向D2/MDX小鼠给药20、40或80mpk的EEV-PMO-M DX-23-2(EEV＝Ac-PKKKRKV-miniPEG-K(环(GfFGrGrQ))-PEG12-OH；PMO＝5’-GGCCAAACCTCGGCTTACCTGAAAT-3’)或PMO-MDX-23-2Methods: D2/MDX mice were administered 20, 40, or 80 mpk of EEV-PMO-MDX-23-2 (EEV = Ac-PKKKRKV-miniPEG-K(cyclo(GfFGrGrQ))-PEG12 -OH; PMO = 5'-GGCCAAACCTCGGCTTACCTGAAAT-3') or PMO-MDX-23-2
(5’-GGCCAAACCTCGGCTTACCTGAAAT-3’)，在D2/MDX小鼠模型中诱导外显子23跳跃。在1、2、4和8周时收获组织以测试单剂量持续时间效应和单剂量范围发现。向D2/MDX小鼠每周给药40mpk，持续4周，并在最终剂量后1周处死小鼠以测试重复剂量效应。(5'-GGCCAAACCTCGGCTTACCTGAAAT-3'), induced exon 23 skipping in the D2/MDX mouse model. Tissues were harvested at 1, 2, 4, and 8 weeks to test single-dose duration effects and single-dose range finding. D2/MDX mice were dosed with 40 mpk weekly for 4 weeks and mice were sacrificed 1 week after the final dose to test repeated dose effects.
结果：单剂量后，在所有4个组织中均观察到外显子跳跃(图14A至图14D)，以及抗肌萎缩蛋白的产生。外显子跳跃在注射后2周达到峰值，并在骨骼肌中维持至少8周：图15A(三头肌)和图15B(胫骨前肌)。4周和8周后，在横膈膜(图15C)和心脏(图15D)中观察到外显子跳跃下降。每周给药4次后，在所有4个组织中，特别是在心肌中(图16)观察到累积的外显子跳跃(最后一剂后1周收集组织)。Results: After a single dose, exon skipping was observed in all 4 tissues (Figures 14A to 14D), as well as dystrophin production. Exon skipping peaked 2 weeks after injection and was maintained in skeletal muscle for at least 8 weeks: Figure 15A (triceps) and Figure 15B (tibialis anterior). After 4 and 8 weeks, a decrease in exon skipping was observed in the diaphragm (Figure 15C) and heart (Figure 15D). After 4 weekly doses, cumulative exon skipping was observed in all 4 tissues, especially in the myocardium (Figure 16) (tissues were collected 1 week after the last dose).
实施例5：D2MDX中的功能测定Example 5: Functional assay in D2MDX
方法：每2周对6组雄性D2/MDX和DBA/2J(野生型)小鼠(每组n＝8)进行静脉内给药，总共6剂，包括媒介物(盐水)、PMO-MDX-2(5’-GGCCAAACCTCGGCTTACCTGAAAT-3’)或靶向外显子23的两种EEV-PMO构建体之一(EEV-PMO-MDX23-2：EEV＝Ac-PKKK RKV-miniPEG-K(环(GfFGrGrQ))-PEG12-OH；PMO＝5’-GGCCAAA CCTCGGCTTACCTGAAAT-3’；和EEV-PMO-MDX-23-3：EEV＝A c-PKKKRKV-Lys(FfΦGrGrQ)-PEG12-K(N3)-NH2；PMO＝Methods: Six groups of male D2/MDX and DBA/2J (wild-type) mice (n=8 per group) were dosed intravenously every 2 weeks for a total of 6 doses with vehicle (saline), PMO-MDX-2 (5'-GGCCAAACCTCGGCTTACCTGAAAT-3'), or one of two EEV-PMO constructs targeting exon 23 (EEV-PMO-MDX23-2: EEV = Ac-PKKK RKV-miniPEG-K(cyclo(GfFGrGrQ))-PEG12 -OH; PMO = 5'-GGCCAAA CCTCGGCTTACCTGAAAT-3'; and EEV-PMO-MDX-23-3: EEV = Ac-PKKKRKV-Lys(FfΦGrGrQ)-PEG12 -K(N3 )-NH2 ; PMO = 5'-GGCCAAA CCTCGGCTTACCTGAAAT-3';).
5’-GGCCAAACCTCGGCTTACCTGAAAT-3’-C4COT)。C4COT＝环辛-2-炔-1-O-(CH2)4-O-C(O)。剂量列于图中。每4周测定一次肌酸激酶水平、握力和线挂时间，共测定4次。5'-GGCCAAACCTCGGCTTACCTGAAAT-3'-C4COT). C4COT = cyclooct-2-yne-1-O-(CH2 )4 -OC(O). Doses are listed in the figure. Creatine kinase levels, grip strength and thread-hanging time were measured once every 4 weeks, for a total of 4 times.
结果：首次注射后2周，EEV-PMO-MDX-23-2 80mpk Q2W治疗的线挂时间略高于其他组，并且继续显示出统计学上显著的改善，并在首次注射后4周和8周与媒介物D2.mdx组相比均有所增加(图17)。治疗12周后，EEV-PMO-MDX-23-2 80mpk Q2W在统计学上与WT动物没有区别(图17)。与媒介物D2.mdx组相比，使用负载剂量(分别为80mpk和30mpk)的EEV-PMO-MDX-23-2 40pmk Q2W和EEV-PMO-MDX-23-3 15mpk Q2W治疗从第一次治疗后8周开始显示出显著更高的线挂时间，一直稳定到治疗12周，此时表型改善的迹象首次变得明显(图17)。单独的PMO-MDX-23-2治疗似乎遵循与媒介物D2.mdx组和媒介物对照组相同的趋势。Results: 2 weeks after the first injection, EEV-PMO-MDX-23-2 80 mpk Q2W treatment had slightly higher thread hanging time than other groups and continued to show statistically significant improvement and increase compared to the vehicle D2.mdx group at both 4 and 8 weeks after the first injection (Figure 17). After 12 weeks of treatment, EEV-PMO-MDX-23-2 80 mpk Q2W was statistically indistinguishable from WT animals (Figure 17). EEV-PMO-MDX-23-2 40 pmk Q2W and EEV-PMO-MDX-23-3 15 mpk Q2W treatments with loading doses (80 mpk and 30 mpk, respectively) showed significantly higher thread hanging time starting 8 weeks after the first treatment and remained stable until 12 weeks of treatment, when signs of phenotypic improvement first became apparent compared to the vehicle D2.mdx group (Figure 17). PMO-MDX-23-2 treatment alone appeared to follow the same trend as the vehicle D2.mdx group and the vehicle control group.
在4个时间点测定血清肌酸激酶(CK)水平：给药前，以及4、8和12周时。用EEV-PMO-MDX-23-2或EEV-PMO-MDX-23-3治疗的小鼠显示出血清CK显著降低，接近野生型。用PMO-MDX-23-2治疗的小鼠在治疗后的所有时间点均未显示出显著下降(图18A至图18C)。Serum creatine kinase (CK) levels were measured at 4 time points: before dosing, and at 4, 8, and 12 weeks. Mice treated with EEV-PMO-MDX-23-2 or EEV-PMO-MDX-23-3 showed a significant decrease in serum CK, close to wild type. Mice treated with PMO-MDX-23-2 did not show a significant decrease at all time points after treatment (Figures 18A to 18C).
在给药前(图19A)和12周时(图19B)测量握力。对于接受治疗的小鼠，观察到握力呈剂量依赖性增加。媒介物和PMO治疗的小鼠没有显示出显著的改善。Grip strength was measured before dosing (Figure 19A) and at 12 weeks (Figure 19B). A dose-dependent increase in grip strength was observed for treated mice. Vehicle and PMO treated mice did not show significant improvement.
实施例6.使用与寡核苷酸缀合的细胞穿透肽来校正hDMD的外显子44剪接Example 6. Correction of exon 44 splicing in hDMD using cell-penetrating peptides conjugated to oligonucleotides
目的：本研究采用hDMD和CD1小鼠模型以及NHP模型来研究包含反义化合物和细胞穿透肽的化合物的效应。每种化合物均含有环外序列PKKKRKV。Objective: This study used hDMD and CD1 mouse models and NHP models to investigate the effects of a compound containing an antisense compound and a cell-penetrating peptide. Each compound contains the exocyclic sequence PKKKRKV.
评价的化合物：本研究评价了EEV-PMO-DMD44-1、EEV-PMO-DMD44-2和EEV-PMO-DMD44-3。这些化合物的序列信息如表8中所示。Compounds evaluated: EEV-PMO-DMD44-1, EEV-PMO-DMD44-2 and EEV-PMO-DMD44-3 were evaluated in this study. The sequence information of these compounds is shown in Table 8.
化合物合成和纯化：根据以下程序合成化合物。使TFA-赖氨酸保护的cCPP与表8的AC反应，随后脱保护以提供cCPP-AC缀合物。简而言之，通过使cCPP与HATU(2.0当量)和DIPEA(2.0当量)在DMSO(10mM，1.8mL)中反应来将其预激活。在室温下10min后，将预激活的溶液与AC在DMSO中的溶液(10mM，1.8mL)组合并充分混合。将反应在室温下孵育2小时。通过LCMS(Q-TOF)监测反应，使用BEH C18柱(1.7μm，2.1mm×50mm)，缓冲液A：水(0.1％FA)，缓冲液B：乙腈(0.1％FA)，流速：0.4mL/min，从2％缓冲液B开始并在3.4min内逐渐增加至98％。完成后，通过用0.2M KCl(水溶液)pH 12(36mL)稀释反应混合物来引发TFA保护的赖氨酸的原位脱保护。使用上述分析方法，通过LCMS(Q-TOF)监测反应。将粗混合物直接上样到C18反相柱(Oligo clarity柱，150mm*21.2mm)上。然后使用含0.1％FA和乙腈的水作为溶剂并以20mL/min的流速，在60min内使用5％-20％的梯度纯化粗产物。合并含有所需产物的级分，并使用0.5M NaOH将溶液的pH值调节至7。将溶液冷冻并冻干，得到白色粉末。通过在1M NaCl水溶液中复溶cCPP-AC缀合物并通过3-kD MW-截止amicon管重复洗涤(以3500rpm离心20-40min)，用氯化物交换甲酸盐。所述过程用1M NaCl进行三次，并用盐水(0.9％NaCl，无菌，无内毒素)进行三次。评估最终滤液的电导率以确认适当的盐浓度。将溶液进一步用盐水稀释至期望的制剂浓度，并在生物安全柜中无菌过滤。过滤后重新测量每种制剂的浓度。Compound synthesis and purification: Compounds were synthesized according to the following procedure. TFA-lysine protected cCPP was reacted with AC of Table 8, followed by deprotection to provide cCPP-AC conjugates. Briefly, cCPP was preactivated by reacting it with HATU (2.0 equiv.) and DIPEA (2.0 equiv.) in DMSO (10 mM, 1.8 mL). After 10 min at room temperature, the preactivated solution was combined with a solution of AC in DMSO (10 mM, 1.8 mL) and mixed thoroughly. The reaction was incubated at room temperature for 2 hours. The reaction was monitored by LCMS (Q-TOF) using a BEH C18 column ( 1.7μm, 2.1mm×50mm), buffer A: water (0.1% FA), buffer B: acetonitrile (0.1% FA), flow rate: 0.4mL/min, starting from 2% buffer B and gradually increasing to 98% in 3.4min. After completion, the in situ deprotection of TFA-protected lysine was initiated by diluting the reaction mixture with 0.2M KCl (aqueous solution) pH 12 (36mL). The reaction was monitored by LCMS (Q-TOF) using the above analytical method. The crude mixture was directly loaded onto a C18 reverse phase column (Oligo clarity column, 150mm*21.2mm). The crude product was then purified using a gradient of 5%-20% in 60min using water containing 0.1% FA and acetonitrile as solvent and at a flow rate of 20mL/min. The fractions containing the desired product were combined and the pH of the solution was adjusted to 7 using 0.5M NaOH. The solution was frozen and lyophilized to obtain a white powder. The cCPP-AC conjugate was redissolved in a 1M NaCl aqueous solution and washed repeatedly through a 3-kD MW-cutoff amicon tube (centrifuged at 3500rpm for 20-40min), exchanging formate with chloride. The process was performed three times with 1M NaCl and three times with saline (0.9% NaCl, sterile, endotoxin-free). The conductivity of the final filtrate was assessed to confirm the appropriate salt concentration. The solution was further diluted with saline to the desired formulation concentration and sterile filtered in a biosafety cabinet. The concentration of each formulation was remeasured after filtration.
获得EEV-PMO-DMD44-1，产率为74％。通过液相色谱-质谱四极杆飞行时间质谱(QTOF-LCMS)评估每种制剂的纯度和身份。EEV-PMO-DMD44-1通过RP-FA确定为99％纯，并且通过CEX确定为78％纯。C411H661N173O130P24的MW计算值为10849.26。通过QTOF-LCMS鉴定的MW为10850.95。进一步测定制剂的内毒素量、残留游离肽、FA含量和pH值。EEV-PMO-DMD44-1 was obtained with a yield of 74%. The purity and identity of each preparation were assessed by liquid chromatography-mass spectrometry quadrupole time-of-flight mass spectrometry (QTOF-LCMS). EEV-PMO-DMD44-1 was determined to be 99% pure by RP-FA and 78% pure by CEX. The MW calculated for C411 H661 N173 O130 P24 was 10849.26. The MW identified by QTOF-LCMS was 10850.95. The endotoxin content, residual free peptide, FA content and pH value of the preparation were further determined.
获得EEV-PMO-DMD44-2，产率为70％。通过QTOF-LCMS评估每种制剂的纯度和身份。EEV-PMO-DMD44-2通过RP-FA确定为99％纯，并且通过CEX确定为78％纯。C411H661N173O130P24的MW计算值为10849.26。通过QTOF-LCMS鉴定的MW为10850.88。EEV-PMO-DMD44-2 was obtained in 70% yield. The purity and identity of each preparation was assessed by QTOF-LCMS. EEV-PMO-DMD44-2 was determined to be 99% pure by RP-FA and 78% pure by CEX. The calculated MW forC 411 H661 N173 O130 P24 was 10849.26. The identified MW by QTOF-LCMS was 10850.88.
获得EEV-PMO-DMD44-3，产率为68％。通过QTOF-LCMS评估每种制剂的纯度和身份。EEV-PMO-DMD44-3通过RP-FA确定为86.3％纯(杂质为未反应的AC)。C422H669N173O130P24的MW计算值为10989.45。通过QTOF-LCMS鉴定的MW为10990.07。EEV-PMO-DMD44-3 was obtained in 68% yield. The purity and identity of each preparation was assessed by QTOF-LCMS. EEV-PMO-DMD44-3 was determined to be 86.3% pure by RP-FA (the impurity was unreacted AC). The calculated MW ofC 422 H669 N173 O130 P24 was 10989.45. The identified MW by QTOF-LCMS was 10990.07.
hDMD小鼠模型：hDMD小鼠订购自Jackson Lab(STOCK Tg(DMD)72Thoen/J；保藏号：018900)并在室内繁殖。在Transnetyx处对半合子小鼠进行进一步基因分型。所有组通过静脉内(iv)注射以每只动物5mL/kg给药，并在注射后5天后处死。通过CO2窒息对所有动物实施安乐死，随后经由心脏穿刺进行末端采血。将最大可获得体积的全血收集到肝素锂管中并处理成血浆。通过Testing Facility(IDEXX)分析一部分血浆的临床化学特性，并将其余部分在标称-70℃下冷冻储存。收获组织(三头肌、TA、横膈膜、心脏、肾、肝、脑)，并在液氮中快速冷冻，并储存在-80℃下用于进一步评价外显子跳跃和药物浓度测量。将动物进行年龄匹配并根据表9分配到八(8)个治疗组中。组1-1(3只纯合子hDMD小鼠，6周龄)、组1-2(3只纯合子hDMD小鼠，6周龄)、组1-3(1只雄性、1只雌性，半合子hDMD，11周龄)、组1-4(1只雄性、1只雌性，半合子DMD，11周龄)分别接受10、20、40和80毫克/千克体重(mpk)的EEV-PMO-DMD44-1。组2-1(3只纯合子hDMD小鼠，6周龄)、组2-2(3只纯合子hDMD小鼠，6周龄)、组2-3(1只雄性、1只雌性，半合子hDMD，11周龄)、组2-4(1只雄性、1只雌性，半合子DMD，11周龄)分别接受10、20、40和80mpk的EEV-PMO-DMD44-2。所有动物存活直至其预定的安乐死时间。按照方案收集组织。通过LC-MS量化各种组织样品中AC和cCPP-AC的量。通过RT-PCR分析不同组织中的外显子跳跃并量化外显子44校正。hDMD mouse model: hDMD mice were ordered from Jackson Lab (STOCK Tg (DMD) 72 Thoen / J; deposit number: 018900) and bred in house. Hemizygous mice were further genotyped at Transnetyx. All groups were administered with 5 mL / kg per animal by intravenous (iv) injection and sacrificed 5 days after injection. All animals were euthanized byCO2 asphyxiation, followed by terminal blood sampling via cardiac puncture. The maximum available volume of whole blood was collected into heparin lithium tubes and processed into plasma. The clinical chemistry properties of a portion of the plasma were analyzed by Testing Facility (IDEXX), and the rest was stored frozen at nominal -70 ° C. Tissues (triceps, TA, diaphragm, heart, kidney, liver, brain) were harvested and quickly frozen in liquid nitrogen and stored at -80 ° C for further evaluation of exon skipping and drug concentration measurements. Animals were age matched and assigned to eight (8) treatment groups according to Table 9. Group 1-1 (3 homozygous hDMD mice, 6 weeks old), Group 1-2 (3 homozygous hDMD mice, 6 weeks old), Group 1-3 (1 male, 1 female, hemizygous hDMD, 11 weeks old), and Group 1-4 (1 male, 1 female, hemizygous DMD, 11 weeks old) received 10, 20, 40, and 80 mg/kg body weight (mpk) of EEV-PMO-DMD44-1, respectively. Group 2-1 (3 homozygous hDMD mice, 6 weeks old), Group 2-2 (3 homozygous hDMD mice, 6 weeks old), Group 2-3 (1 male, 1 female, hemizygous hDMD, 11 weeks old), Group 2-4 (1 male, 1 female, hemizygous DMD, 11 weeks old) received 10, 20, 40 and 80 mpk of EEV-PMO-DMD44-2, respectively. All animals survived until their scheduled euthanasia time. Tissues were collected according to the protocol. The amount of AC and cCPP-AC in various tissue samples was quantified by LC-MS. Exon skipping in different tissues was analyzed by RT-PCR and exon 44 correction was quantified.
表9：hDMD实验的实验设计Table 9: Experimental design of hDMD experiments
CD1小鼠模型：使用7周龄的CD1雄性小鼠评价EEV-PMO-DMD44-1和EEV-PMO-DMD44-2的耐受性。它们从Charles River Lab订购，并且在接受后在注射前使它们适应5天。将动物进行年龄匹配并根据表10分配到九(9)个治疗组中。组1(3只小鼠，盐水)；组2-1(3只小鼠)、组2-2(2只小鼠)、组2-3(2只小鼠)、组2-4(2只小鼠)、组2-5(3只小鼠)、组2-6(3只小鼠)分别接受80、100、120、160、200和300mpk的EEV-PMO-DMD44-1。组3-1(3只小鼠)、组3-2(2只小鼠)、组3-3(2只小鼠)、组3-4(2只小鼠)、组3-5(3只小鼠)、组3-6(3只小鼠)分别接受80、100、120、160、200和300mpk的EEV-PMO-DMD44-2。CD1 mouse model: 7-week-old CD1 male mice were used to evaluate the tolerance of EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2. They were ordered from Charles River Lab and were acclimated for 5 days before injection. The animals were age-matched and assigned to nine (9) treatment groups according to Table 10. Group 1 (3 mice, saline); Group 2-1 (3 mice), Group 2-2 (2 mice), Group 2-3 (2 mice), Group 2-4 (2 mice), Group 2-5 (3 mice), Group 2-6 (3 mice) received 80, 100, 120, 160, 200 and 300 mpk of EEV-PMO-DMD44-1, respectively. Group 3-1 (3 mice), group 3-2 (2 mice), group 3-3 (2 mice), group 3-4 (2 mice), group 3-5 (3 mice), and group 3-6 (3 mice) received 80, 100, 120, 160, 200, and 300 mpk of EEV-PMO-DMD44-2, respectively.
表10：CD1小鼠中的耐受性研究的实验设计Table 10: Experimental design of tolerance studies in CD1 mice
NHP模型：根据表11以10mL/kg的剂量体积按照每种化合物(EEV-PMO-DMD44-1和EEV-PMO-DMD44-2)一只雌性动物施用60分钟IV输注。将每种测试制品在盐水中以4mg/mL配制。在表12所示的时间采集血液和尿液用于进一步PK分析。注射后2天对二头肌进行活检。在注射后第7天处死动物，并且取骨骼肌(四头肌、横膈膜、二头肌、三角肌、胫骨前肌(TiA)、平滑肌(食道、主动脉、结肠)和心肌(心室、心房)，粉碎，并储存在-80℃下用于评价外显子跳跃和组织中的生物分布。NHP model: According to Table 11, one female animal was administered a 60-minute IV infusion at a dose volume of 10 mL/kg per compound (EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2). Each test article was prepared in saline at 4 mg/mL. Blood and urine were collected at the times shown in Table 12 for further PK analysis. The biceps were biopsied 2 days after injection. The animals were sacrificed on the 7th day after injection, and skeletal muscle (quadriceps, diaphragm, biceps, deltoid, tibialis anterior (TiA), smooth muscle (esophagus, aorta, colon) and myocardium (ventricle, atrium) were taken, crushed, and stored at -80°C for evaluation of exon skipping and biodistribution in tissues.
表11：NHP研究的实验设计Table 11: Experimental design of NHP study
表12：NHP研究时间点Table 12: NHP study time points
生物分析样品分析：将组织解冻、称重并用掺有1x蛋白酶抑制剂混合物(ThermoFisher Scientific，参考号1860932)的RIPA缓冲液匀浆(w/v，1/5)。将匀浆物在4℃下以5000rpm离心5分钟。将上清液用H2O、乙腈和MeOH的混合物沉淀，并在4℃下以15000rpm离心15分钟。将上清液转移到注射板上，使用与Triple Quad Sciex 4500仪器集成的Shimadzu UPLC进行LC-MS/MS分析。LC-MS/MS测定的动态范围为25ng/g组织至50,000ng/g组织。LC-MS/MS方法的细节在此处和表13中概述。简而言之，使用以下条件操作UPLC：Waters Acquity UPLC BEH C4，300A，1.7um，2.1x150mm；缓冲液A：H2O，0.2％FA；缓冲液B：95％乙腈的H2O溶液，0.2％FA；流速(0.3mL/min)和50℃的柱温。10min运行开始于2％缓冲液B，并且逐渐增加至35％持续3.5min，随后90％持续1min，在90％梯度下停留2.5min并且最后在2％梯度下运行2min。用以下建立7.5min持续时间的MRM方法：正极性；涡轮喷雾离子源；帘幕气体：25；碰撞气体：6；离子喷雾电压：5500；温度：500；离子源气体1∶60；离子源气体2：60。表13的下划线行用于量化完整且对应的代谢物(235-PEG12)。Bioanalytical Sample Analysis: Tissues were thawed, weighed and homogenized (w/v, 1/5) with RIPA buffer spiked with 1x protease inhibitor cocktail (ThermoFisher Scientific, Ref. 1860932). The homogenate was centrifuged at 5000 rpm for 5 minutes at 4°C. The supernatant was precipitated with a mixture ofH2O , acetonitrile and MeOH and centrifuged at 15000 rpm for 15 minutes at 4°C. The supernatant was transferred to an injection plate and LC-MS/MS analysis was performed using a Shimadzu UPLC integrated with a Triple Quad Sciex 4500 instrument. The dynamic range of the LC-MS/MS assay was 25 ng/g tissue to 50,000 ng/g tissue. The details of the LC-MS/MS method are summarized here and in Table 13. Briefly, the UPLC was operated using the following conditions: Waters Acquity UPLC BEH C4, 300A, 1.7um, 2.1x150mm; Buffer A:H2O , 0.2% FA; Buffer B: 95% acetonitrile inH2O , 0.2% FA; flow rate (0.3mL/min) and column temperature of 50°C. A 10 min run started at 2% buffer B and was gradually increased to 35% for 3.5 min, followed by 90% for 1 min, a dwell at 90% gradient for 2.5 min and a final run at 2% gradient for 2 min. An MRM method of 7.5 min duration was established using the following: positive polarity; turbo spray ion source; curtain gas: 25; collision gas: 6; ion spray voltage: 5500; temperature: 500; ion source gas 1:60; ion source gas 2:60. The underlined rows of Table 13 were used to quantify the intact and corresponding metabolites (235-PEG12).
表13：LC-MS/MS测定Table 13: LC-MS/MS determination
通过RT-PCR进行外显子跳跃分析：hDMD小鼠和NHP表达全长人抗肌萎缩蛋白mRNA。AC的递送可改变剪接，并在外显子44跳跃后产生缩短的抗肌萎缩蛋白mRNA。使用1mL RLT裂解缓冲液(Qiagen，目录号79216)将组织匀浆。通过RT-PCR测量剪接校正过程的检测，其中首先将从组织提取的RNA逆转录成cDNA，并使用以下引物组通过一步RT-PCR进一步分析：正向引物5′-GCTCAGGTCGGATTGACATT-3′和反向引物5′-GGGCAACTCTTCCACCAGTA-3′。没有剪接校正的组织的RT-PCR读出产生641bp基因片段，并且在剪接校正之后出现新的493bp基因片段。进行对应于跳跃和未跳跃转录物的条带的相对强度的定量，以评估AC诱导的外显子-44跳跃功效。使用以下等式计算通过RT-PCR检测的剪接校正的程度(百分比)：％校正＝(493bp片段条带的强度)/(493bp片段条带的强度+641bp片段条带的强度)。Exon skipping analysis by RT-PCR: hDMD mice and NHP express full-length human dystrophin mRNA. The delivery of AC can change splicing and produce shortened dystrophin mRNA after exon 44 skipping. Tissues were homogenized using 1mL RLT lysis buffer (Qiagen, catalog number 79216). The detection of the splicing correction process was measured by RT-PCR, in which RNA extracted from the tissue was first reverse transcribed into cDNA and further analyzed by one-step RT-PCR using the following primer set: forward primer 5′-GCTCAGGTCGGATTGACATT-3′ and reverse primer 5′-GGGCAACTCTTCCACCAGTA-3′. The RT-PCR readout of tissues without splicing correction produced a 641bp gene fragment, and a new 493bp gene fragment appeared after splicing correction. The relative intensity of the bands corresponding to the skipped and unskipped transcripts was quantified to evaluate the exon-44 skipping efficacy induced by AC. The degree (percentage) of splicing correction detected by RT-PCR was calculated using the following equation: % correction = (intensity of the 493 bp fragment band)/(intensity of the 493 bp fragment band + intensity of the 641 bp fragment band).
结果：EEV-PMO-DMD44-1、EEV-PMO-DMD44-2和EEV-PMO-DMD44-3在患者肌管中的功效(图21)：评估了各自靶向人抗肌萎缩蛋白(DMD)外显子44的EEV-PMO-DMD44-1、EEV-PMO-DMD44-2和EEV-PMO-DMD44-3在DMD患者来源的肌细胞中的DMD外显子44跳跃。简而言之，在补充有2％马血清和1％鸡胚提取物的PromoCell骨骼肌细胞生长培养基中，用1μM、3μM和10μM的EEV-PMO-DMD44-1、EEV-PMO-DMD44-2和EEV-PMO-DMD44-3处理具有外显子45缺失(DMDΔ45)的患者来源的成肌细胞24小时。24小时后，用DMEM/2％马血清替换含化合物的生长培养基并孵育5天以促进成肌细胞融合以及分化成肌管。洗涤细胞并收获用于RNA提取以评估外显子44跳跃，或在含有蛋白酶抑制剂的RIPA缓冲液中用于蛋白质提取和抗肌萎缩蛋白恢复的Simple Western分析。将抗肌萎缩蛋白水平归一化至HSP90，并相对于未处理的健康样品表示。数据表示为平均值±SD，n＝3-4。未处理的DMDΔ45患者来源的细胞在基线时表达约10％自发DMD外显子44跳跃和约4％抗肌萎缩蛋白。所有三种化合物以剂量依赖性方式导致稳健的外显子跳跃和抗肌萎缩蛋白恢复。Results: EEV-PMO-DMD44-1, EEV-PMO-DMD44-2 and EEV-PMO-DMD44-3 efficacy in patient myotubes (Figure 21): EEV-PMO-DMD44-1, EEV-PMO-DMD44-2 and EEV-PMO-DMD44-3, each targeting human dystrophin (DMD) exon 44, were evaluated for DMD exon 44 skipping in DMD patient-derived myoblasts. Briefly, patient-derived myoblasts with exon 45 deletion (DMDΔ45) were treated with 1 μM, 3 μM and 10 μM of EEV-PMO-DMD44-1, EEV-PMO-DMD44-2 and EEV-PMO-DMD44-3 in PromoCell skeletal muscle cell growth medium supplemented with 2% horse serum and 1% chicken embryo extract for 24 hours. After 24 hours, the compound-containing growth medium was replaced with DMEM/2% horse serum and incubated for 5 days to promote myoblast fusion and differentiation into myotubes. The cells were washed and harvested for RNA extraction to evaluate exon 44 skipping, or for Simple Western analysis of protein extraction and dystrophin recovery in RIPA buffer containing protease inhibitors. Dystrophin levels were normalized to HSP90 and expressed relative to untreated healthy samples. Data are expressed as mean ± SD, n = 3-4. Untreated DMDΔ45 patient-derived cells expressed approximately 10% spontaneous DMD exon 44 skipping and approximately 4% dystrophin at baseline. All three compounds resulted in robust exon skipping and dystrophin recovery in a dose-dependent manner.
图22A和图22B示出了经由IV注射施用EEV-PMO-DMD44-1(图22A)和EEV-PMO-DMD44-2(图22B)的hDMD小鼠中的外显子跳跃。没有观察到严重的副作用。小鼠在注射后、注射后24小时和处死日期之前都是正常的。在IV注射后5天评价测量肝和肾毒性(碱性磷酸酶(ALP)、天冬氨酸转氨酶(AST)、丙氨酸氨基转移酶(ALT)、白蛋白、血尿素氮(BUN)、肌酸酐、钙、磷、氯、钾、钠、BUN/肌酸酐、镁)以及溶血和脂血指数的临床化学。通过临床化学评价，在EEV-PMO-DMD44-1和EEV-PMO-DMD44-2治疗的小鼠中没有检测到显著的毒性。在10、20、40和80mpk IV剂量后5天，评估了各种肌肉组中的组织浓度和外显子跳跃。对于每种剂量的EEV-PMO-DMD44-1，在心脏/三头肌/TiA/横膈膜组织中分别实现了以下外显子跳跃：10mpk(0％、6％、12％、6％)；20mpk(0％、22％、36％、33％)；40mpk(20％、94％、99％、82％)；80mpk(79％、97％、99％、98％)。对于每种剂量的EEV-PMO-DMD44-2，在心脏/三头肌/TiA/横膈膜组织中分别实现了以下外显子跳跃：10mpk(0％、17％、22％、14％)；20mpk(2％、44％、58％、35％)；40mpk(17％、92％、95％、83％)；80mpk(79％、98％、99％、99％)。在携带全尺寸人DMD基因的转基因鼠模型中，在心肌和骨骼肌中观察到EEV-PMO-DMD44-1和EEV-PMO-DMD44-2两者的强剂量依赖性累积和有效外显子跳跃。在较低剂量10mpk和20mpk下，EEV-PMO-DMD44-2药物暴露和功效略高于EEV-PMO-DMD44-1。然而，所述效应在40mpk剂量下开始减弱，其中两种化合物在所有骨骼肌中均导致相同高水平的外显子跳跃(高于80％)。EEV-PMO-DMD44-1的对应组织浓度在100-300ng/g组织范围内，而对于EEV-PMO-DMD44-2，所述范围移至略高的数值，为300-500ng/g组织浓度。有趣的是，EEV-PMO-DMD44-1和EEV-PMO-DMD44-2两者在心脏中的最小有效剂量是用40mpk实现的，分别对应于每克组织浓度170ng和350ng。Figure 22 A and Figure 22 B show exon skipping in hDMD mice of EEV-PMO-DMD44-1 (Figure 22 A) and EEV-PMO-DMD44-2 (Figure 22 B) administered via IV injection.No serious side effects were observed.Mice were normal after injection, 24 hours after injection and before the date of execution.The clinical chemistry of liver and kidney toxicity (alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, blood urea nitrogen (BUN), creatinine, calcium, phosphorus, chlorine, potassium, sodium, BUN/creatinine, magnesium) and hemolysis and lipemia index were evaluated and measured 5 days after IV injection.By clinical chemistry evaluation, significant toxicity was not detected in mice treated with EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2.5 days after 10,20,40 and 80mpk IV dosage, tissue concentration and exon skipping in various muscle groups were assessed. For each dose of EEV-PMO-DMD44-1, the following exon skipping was achieved in heart/triceps/TiA/diaphragm tissues, respectively: 10 mpk (0%, 6%, 12%, 6%); 20 mpk (0%, 22%, 36%, 33%); 40 mpk (20%, 94%, 99%, 82%); 80 mpk (79%, 97%, 99%, 98%). For each dose of EEV-PMO-DMD44-2, the following exon skipping was achieved in heart/triceps/TiA/diaphragm tissues, respectively: 10mpk (0%, 17%, 22%, 14%); 20mpk (2%, 44%, 58%, 35%); 40mpk (17%, 92%, 95%, 83%); 80mpk (79%, 98%, 99%, 99%). In a transgenic mouse model carrying a full-size human DMD gene, strong dose-dependent accumulation and effective exon skipping of both EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 were observed in cardiac and skeletal muscles. At lower doses of 10mpk and 20mpk, EEV-PMO-DMD44-2 drug exposure and efficacy were slightly higher than EEV-PMO-DMD44-1. However, the effect began to wane at the 40 mpk dose, where both compounds caused the same high levels of exon skipping (greater than 80%) in all skeletal muscles. The corresponding tissue concentrations for EEV-PMO-DMD44-1 were in the range of 100-300 ng/g tissue, while for EEV-PMO-DMD44-2, the range shifted to slightly higher values, 300-500 ng/g tissue concentrations. Interestingly, the minimum effective dose in the heart for both EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 was achieved with 40 mpk, corresponding to concentrations of 170 ng and 350 ng per gram of tissue, respectively.
EEV-PMO-DMD44-1在以80、100、160、200和300mpk剂量施用于CD1小鼠的所有剂量中是非常良好耐受的。仅观察到短暂的症状，其在注射后1小时完全消退。在注射后1天和7天没有观察到生物标志物异常。EEV-PMO-DMD44-2是较不耐受的。在EEV-PMO-DMD44-2的最高剂量300mpk下，三只小鼠中的一只在注射后1-3h内死亡。在EEV-PMO-DMD44-2的较低剂量200mpk下，三只小鼠中的一只具有严重的症状(对刺激无反应、耳朵向后拉、呼吸缓慢、难以恢复正常)。这些症状逐渐恶化，并且它们与肌肉抽搐结合。对于160mpk和80mpk的较低剂量没有观察到症状。令人惊讶地，在100mpk下，三只小鼠中的一只在注射后2小时显示出延迟的症状；但它们在注射后1天和7天完全正常。EEV-PMO-DMD44-1 is very well tolerated in all doses of CD1 mice at 80, 100, 160, 200 and 300mpk dosages. Only transient symptoms were observed, which completely disappeared 1 hour after injection. No abnormal biomarkers were observed 1 day and 7 days after injection. EEV-PMO-DMD44-2 is less tolerated. Under the highest dose of EEV-PMO-DMD44-2, 300mpk, one of three mice died within 1-3h after injection. Under the lower dose of EEV-PMO-DMD44-2, 200mpk, one of three mice had serious symptoms (no response to stimulation, ears pulled back, slow breathing, difficult to return to normal). These symptoms gradually worsen, and they are combined with muscle twitching. No symptoms were observed for the lower doses of 160mpk and 80mpk. Surprisingly, at 100 mpk, one of three mice showed delayed symptoms at 2 hours post-injection; but they were completely normal at 1 and 7 days post-injection.
为了进一步证明cCPP-AC缀合物的外显子跳跃的功效，利用NHP。具体地，对具有完整肌肉组织的食蟹猴以40mg/kg施用EEV-PMO-DMD44-1或EEV-PMO-DMD44-2的60分钟IV输注，它们是良好耐受的。更具体地，动物在治疗开始45分钟时经历恶心，这在治疗后大约3小时显著消退，并且动物更警觉，不再弯腰驼背并食用提供的产品。在给药后大约20小时，动物变得(机灵、警觉并且反应灵敏，使得动物在表型上是“正常的”)(BAR)，在笼中没有留下饼干，并观察到食用产品。To further demonstrate the efficacy of exon skipping of cCPP-AC conjugates, NHP was used. Specifically, 60-minute IV infusions of EEV-PMO-DMD44-1 or EEV-PMO-DMD44-2 were administered to cynomolgus monkeys with intact muscle tissue at 40 mg/kg, and they were well tolerated. More specifically, animals experienced nausea 45 minutes after the start of treatment, which significantly subsided about 3 hours after treatment, and the animals were more alert, no longer hunched over and ate the products provided. About 20 hours after administration, the animals became (smart, alert and responsive, making the animals phenotypically "normal") (BAR), no biscuits were left in the cage, and edible products were observed.
在注射后2天和7天没有观察到临床化学组的异常。按照标准方案分析不同组织中的外显子44跳跃百分比。图23A至图23B描绘了EEV-PMO-DMD44-1的外显子跳跃(图23A)和药物暴露(图23B)。图24A至图24B描绘了EEV-PMO-DMD44-2的外显子跳跃(图24A)和药物暴露(图24B)。两种化合物在注射后7天在通过IV给予40mpk的不同肌肉组中均表现出优异的外显子跳跃水平。从功效的观点来看，EEV-PMO-DMD44-1在TiA、横膈膜中表现得更好，并且在心室和心房中表现得不那么显著。在所有骨骼肌中，实现了超过78％的外显子跳跃，最大98.4％在横膈膜中实现。在心脏组织中，40mpk下的EEV-PMO-DMD44-1在心室和心房中分别产生31.9％和23.4％。在平滑肌中，食管显示57.1％的最高功效。EEV-PMO-DMD44-1和EEV-PMO-DMD44-2两者以药理学相关浓度分布于各种组织中。在一些情况下，诸如在心脏组织中的心室和心房以及更显著地在食管和结肠中，相同的组织浓度不转变成相同的功能递送。这可能表明不同组织中的内体逃逸水平可能不同。然而，在骨骼肌中，大约200ng/克组织浓度与超过80％的稳健外显子跳跃相关，而在心脏组织中，800-1000ng/克组织浓度与心房和心室中粗略组合的50％外显子跳跃相关。仅单一剂量40mpk的cCPP-AC缀合物的50％外显子跳跃是非常鼓舞人心的，因为心脏组织对于递送而言是更具挑战性的组织，并且是对于神经肌肉病症(诸如DMD)的治疗而言至关重要的组织。No abnormality of clinical chemistry group was observed 2 days and 7 days after injection. The percentage of exon 44 skipping in different tissues was analyzed according to standard protocols. Figure 23A to Figure 23B depicts exon skipping (Figure 23A) and drug exposure (Figure 23B) of EEV-PMO-DMD44-1. Figure 24A to Figure 24B depicts exon skipping (Figure 24A) and drug exposure (Figure 24B) of EEV-PMO-DMD44-2. Both compounds showed excellent exon skipping levels in different muscle groups given 40mpk by IV 7 days after injection. From the point of view of efficacy, EEV-PMO-DMD44-1 performed better in TiA, diaphragm, and was less significant in ventricle and atrium. In all skeletal muscles, more than 78% of exon skipping was achieved, and the maximum 98.4% was achieved in the diaphragm. In cardiac tissue, EEV-PMO-DMD44-1 at 40mpk produced 31.9% and 23.4% in the ventricle and atrium, respectively. In smooth muscle, the esophagus showed the highest efficacy of 57.1%. Both EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 are distributed in various tissues at pharmacologically relevant concentrations. In some cases, such as in the ventricles and atria in cardiac tissue and more significantly in the esophagus and colon, the same tissue concentration does not translate into the same functional delivery. This may indicate that the level of endosomal escape in different tissues may be different. However, in skeletal muscle, approximately 200ng/gram tissue concentrations are associated with more than 80% robust exon skipping, while in cardiac tissue, 800-1000ng/gram tissue concentrations are associated with roughly combined 50% exon skipping in the atria and ventricles. 50% exon skipping with only a single dose of 40 mpk of the cCPP-AC conjugate is very encouraging as cardiac tissue is a more challenging tissue for delivery and is a tissue of critical importance for the treatment of neuromuscular disorders such as DMD.
患者来源的肌细胞：将用于外显子44跳跃(EEV-PMO-DMD44-1)的化合物添加到DMD患者来源的肌细胞中，并施用于表达全长人抗肌萎缩蛋白基因的hDMD转基因小鼠，以测试人序列特异性PMO的DMD转录校正。图25A示出了在用EEV-PMO-DMD44-1处理的DMD患者来源的肌细胞中稳健的剂量依赖性外显子跳跃和抗肌萎缩蛋白的恢复。与未处理的患者来源的细胞和健康细胞相比，在用EEV-PMO-DMD44-1处理的DMD患者来源的肌细胞中观察到剂量依赖性外显子44跳跃和抗肌萎缩蛋白恢复(分别高达100％和43.7％)。图25B示出了在以10mg/kg至80mg/kg范围内的各种水平施用递增IV剂量的EEV-PMO-DMD44-1后携带全长人DMD基因的整合拷贝的转基因小鼠的心肌和骨骼肌中的剂量依赖性组织暴露和外显子跳跃。给药五天后分别评估外显子跳跃和组织暴露。观察到在翻译相关剂量下高达80％的组织暴露和高达100％的外显子跳跃的剂量依赖性水平。Patient-derived muscle cells: Compounds for exon 44 skipping (EEV-PMO-DMD44-1) were added to DMD patient-derived muscle cells and administered to hDMD transgenic mice expressing the full-length human dystrophin gene to test DMD transcriptional correction of human sequence-specific PMOs. Figure 25A shows robust dose-dependent exon skipping and restoration of dystrophin in DMD patient-derived muscle cells treated with EEV-PMO-DMD44-1. Dose-dependent exon 44 skipping and dystrophin restoration (up to 100% and 43.7%, respectively) were observed in DMD patient-derived muscle cells treated with EEV-PMO-DMD44-1 compared to untreated patient-derived cells and healthy cells. Figure 25B shows dose-dependent tissue exposure and exon skipping in cardiac muscle and skeletal muscle of transgenic mice carrying an integrated copy of the full-length human DMD gene after administration of increasing IV doses of EEV-PMO-DMD44-1 at various levels within the range of 10 mg/kg to 80 mg/kg. Exon skipping and tissue exposure were assessed after administration for five days. Dose-dependent levels of up to 80% tissue exposure and up to 100% exon skipping were observed at translation-related doses.
以10、20、40和80mg/kg静脉内(IV)施用EEV-PMO-DMD44-1后，在hDMD转基因小鼠的心脏(图26A)、胫骨前肌(图26B)和横膈膜(图26C)中观察到剂量依赖性组织暴露和外显子跳跃。Following intravenous (IV) administration of EEV-PMO-DMD44-1 at 10, 20, 40, and 80 mg/kg, dose-dependent tissue exposure and exon skipping were observed in the heart ( FIG. 26A ), tibialis anterior muscle ( FIG. 26B ), and diaphragm ( FIG. 26C ) of hDMD transgenic mice.
将单一30mg/kg IV剂量(1小时输注)的EEV-PMO-DMD44-1施用于NHP，在NHP的血浆中观察到长达50小时的EEV-PMO-DMD44-1的延长的循环半衰期(数据未显示)。这种药代动力学分布提示了预期的组织暴露、靶结合和药效学效应的机会。IV输注后7天，在从EEV-PMO-DMD44-1处理的NHP中分离出的不同肌肉组中观察到稳健的外显子44跳跃(图11B)。因此，在施用EEV-PMO-DMD44-1的NHP中观察到延长的半衰期和高水平(二头肌中几乎90％)的外显子跳跃。A single 30 mg/kg IV dose (1 hour infusion) of EEV-PMO-DMD44-1 was administered to NHPs, and an extended circulation half-life of EEV-PMO-DMD44-1 of up to 50 hours was observed in the plasma of NHPs (data not shown). This pharmacokinetic profile suggests the opportunity for expected tissue exposure, target binding, and pharmacodynamic effects. Seven days after IV infusion, robust exon 44 skipping was observed in different muscle groups isolated from NHPs treated with EEV-PMO-DMD44-1 (Figure 11B). Therefore, extended half-life and high levels (almost 90% in biceps) of exon skipping were observed in NHPs administered EEV-PMO-DMD44-1.
图27示出了在NHP中观察到EEV-PMO-DMD44-1的循环半衰期延长。FIG. 27 shows that an increase in the circulating half-life of EEV-PMO-DMD44-1 was observed in NHPs.
图28示出了单一30mg/kg IV剂量的EEV-PMO-DMD44-1导致在NHP的骨骼肌和心脏两者中有意义水平的外显子跳跃，这提供了翻译潜力的置信度。在以30mg/kg IV输注1小时后7天，在分离自EEV-PMO-DMD44-1处理的NHP的不同肌肉组中观察到稳健的外显子44跳跃。Figure 28 shows that a single 30 mg/kg IV dose of EEV-PMO-DMD44-1 resulted in meaningful levels of exon skipping in both skeletal muscle and heart of NHPs, providing confidence in translational potential. Robust exon 44 skipping was observed in different muscle groups isolated from EEV-PMO-DMD44-1 treated NHPs 7 days after 1 hour IV infusion at 30 mg/kg.
这些结果代表一组稳健的翻译数据。外显子跳跃转化为在心脏和骨骼肌中有希望的抗肌萎缩蛋白产生。抗肌萎缩蛋白产生足以导致功能改善。抗肌萎缩蛋白产生在单次注射后持续4+周。These results represent a robust set of translational data. Exon skipping translates into promising dystrophin production in cardiac and skeletal muscle. Dystrophin production is sufficient to result in functional improvements. Dystrophin production persists for 4+ weeks following a single injection.
hDMD小鼠：向人营养不良小鼠IV给药15mg/kg的EEV-PMO-DMD44-1或与同一外显子44跳跃PMO缀合的R6(聚精氨酸)线性肽。在用EEV-PMO-DMD-44治疗的小鼠中，心脏(图29A)、横膈膜(图29B)和三头肌(图29C)中的外显子跳跃在60％至大约95％之间，而在施用R6-PMO的小鼠中，外显子跳跃小于20％。hDMD mice: Human dystrophic mice were IV dosed with 15 mg/kg of EEV-PMO-DMD44-1 or R6 (polyarginine) linear peptide conjugated to the same exon 44 skipping PMO. In mice treated with EEV-PMO-DMD-44, exon skipping ranged from 60% to approximately 95% in the heart ( FIG. 29A ), diaphragm ( FIG. 29B ), and triceps ( FIG. 29C ), whereas in mice administered R6-PMO, exon skipping was less than 20%.
实施例7.EEV-PMO-DMD44-1在CD1小鼠中的药代动力学研究Example 7. Pharmacokinetic study of EEV-PMO-DMD44-1 in CD1 mice
使用CD1小鼠模型来研究EEV-PMO-DMD44-1和PMO-DMD44-1(EEV-PMO-DMD44-1的主要代谢物)的血浆、肾脏和胫骨前肌药物暴露(AUC)。The CD1 mouse model was used to study plasma, kidney, and tibialis anterior muscle drug exposure (AUC) of EEV-PMO-DMD44-1 and PMO-DMD44-1 (the major metabolite of EEV-PMO-DMD44-1).
经由静脉内注射80mpk的EEV-PMO-DMD44-1或PMO-DMD44-1对五至七周龄的小鼠进行治疗。在不同时间点对小鼠放血和/或处死。Five to seven week old mice were treated via intravenous injection of 80 mpk of EEV-PMO-DMD44-1 or PMO-DMD44-1. Mice were bled and/or sacrificed at various time points.
表14、表15和表16示出了分别在血浆、肾脏和胫骨前肌中观察到的药代动力学特性。对于表：AUClast＝从零到最后可定量浓度的曲线下面积；D＝剂量；Cmax＝最大血清或血浆浓度；Tmax＝达到Cmax的时间；CL＝总血浆、血清或血液清除率；t1/2＝消除半衰期；Vss＝平衡时的表观分布容积；Qh＝肝血流量(ml/min/kg)。Tables 14, 15 and 16 show the pharmacokinetic properties observed in plasma, kidney and tibialis anterior muscle, respectively. For the tables: AUClast = area under the curve from zero to the last quantifiable concentration; D = dose; Cmax = maximum serum or plasma concentration; Tmax = time to reach Cmax ; CL = total plasma, serum or blood clearance; t1/2 = elimination half-life; Vss = apparent volume of distribution at equilibrium; Qh = liver blood flow (ml/min/kg).
与肾脏相比，胫骨前肌中代谢物的AUC值低约1000倍。血浆中的代谢物平均停留时间(MRT)值可能与组织MRT值直接相关，因为在尿液排泄之前从组织转移到血浆。The AUC values of metabolites in the tibialis anterior muscle were approximately 1000-fold lower compared to the kidney. Metabolite mean residence time (MRT) values in plasma may be directly related to tissue MRT values due to transfer from tissues to plasma prior to urinary excretion.
表14.血浆药代动力学特性Table 14. Plasma pharmacokinetic properties
表15.肾脏药代动力学特性Table 15. Renal pharmacokinetic properties
表16.胫骨前肌药代动力学特性Table 16. Pharmacokinetic properties of tibialis anterior muscle
实施例8：hDMD的功效研究Example 8: Efficacy study of hDMD
方法：用80mpk的EEV-PMO(EEV-PMO-DMD44-1)治疗6组(n＝5)雄性hDMD小鼠。在给药后1周、2周和4周收集组织。通过RT-PCR测定5种组织中的外显子跳跃：三头肌、TA、横膈膜、心脏和腓肠肌。Methods: Six groups (n=5) of male hDMD mice were treated with 80 mpk of EEV-PMO (EEV-PMO-DMD44-1). Tissues were collected 1, 2, and 4 weeks after administration. Exon skipping was determined by RT-PCR in five tissues: triceps, TA, diaphragm, heart, and gastrocnemius.
结果：在横膈肌、TA、腓肠肌和三头肌中，外显子跳跃维持至少12周(图30B至图30E)。在4周时观察到心脏中外显子跳跃下降(图30A)。Results: Exon skipping was maintained for at least 12 weeks in the diaphragm, TA, gastrocnemius, and triceps (Figures 30B to 30E). A decrease in exon skipping was observed in the heart at 4 weeks (Figure 30A).
实施例9：对NHP的持续时间效应Example 9: Duration Effect on NHP
方法：经由1小时IV输注以45mpk单剂量的EEV-PMO-DMD44-1对6个NHP进行给药。在图31中所示的时间点进行二头肌活检。通过RT-PCR测定外显子跳跃Methods: 6 NHPs were dosed with a single dose of 45 mpk of EEV-PMO-DMD44-1 via 1 hour IV infusion. Bicep biopsies were performed at the time points indicated in Figure 31. Exon skipping assay by RT-PCR
结果：如图31中所示，2天后观察到外显子跳跃，并在1周左右达到峰值。单一IV剂量后，外显子跳跃持续长达12周(明显的t1/2＝14天)。给药后2天观察到接近峰值功效。Results: As shown in Figure 31, exon skipping was observed after 2 days and peaked around 1 week. Exon skipping persisted for up to 12 weeks after a single IV dose (apparent t1/2 = 14 days). Near peak efficacy was observed 2 days after dosing.
实施例10：定位Example 10: Positioning
确定了三种构建体的亚细胞定位：单独的PMO(PMO)、与EEV缀合的PMO(EEV-PMO)以及与EEV缀合的PMO和核定位信号(EEV-NLS-PMO)。这些构建体的序列信息如表17中所示。The subcellular localization of three constructs was determined: PMO alone (PMO), PMO conjugated to EEV (EEV-PMO), and PMO conjugated to EEV and a nuclear localization signal (EEV-NLS-PMO). The sequence information of these constructs is shown in Table 17.
表17.构建体Table 17. Constructs
简而言之，使THP-1单核细胞与3μM的PMO、EEV-PMO或EEV-NLS-PMO接触并孵育24小时，并且通过LC-MS进行检查。Briefly, THP-1 monocytes were exposed to 3 μM PMO, EEV-PMO, or EEV-NLS-PMO and incubated for 24 h and examined by LC-MS.
图32A示出了PMO与EEV-PMO与EEV-NLS-PMO的全细胞摄取。与单独的PMO相比，EEV-PMO和EEV-NLS-PMO均显示出细胞摄取的显著增加。Figure 32A shows the whole cell uptake of PMO with EEV-PMO and EEV-NLS-PMO. Both EEV-PMO and EEV-NLS-PMO showed a significant increase in cellular uptake compared to PMO alone.
·EEV-PMO与PMO：约3倍EEV-PMO vs. PMO: about 3 times
·EEV-NLS-PMO与PMO：约58倍EEV-NLS-PMO vs. PMO: about 58 times
·EEV-NLS-PMO与EEV-PMO：约19倍EEV-NLS-PMO vs. EEV-PMO: about 19 times
图32B示出了使用LC-MS/MS测定的THP细胞中PMO与EEV-PMO与EEV-NLS-PMO的亚细胞定位。如图32B中所示，与单独PMO相比，EEV-PMO表现出改进的细胞通透性。NLS的添加进一步提高了细胞通透性。图32C示出了三种构建体的核摄取。Figure 32B shows the subcellular localization of PMO vs. EEV-PMO vs. EEV-NLS-PMO in THP cells determined using LC-MS/MS. As shown in Figure 32B, EEV-PMO exhibits improved cell permeability compared to PMO alone. The addition of NLS further improves cell permeability. Figure 32C shows the nuclear uptake of the three constructs.
权利要求书(按照条约第19条的修改)Claims (as amended under Article 19)
1.一种化合物，所述化合物包含式(C)：1. A compound comprising formula (C):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
其中R1、R2、R3和R4中的两者包含苯丙氨酸的侧链wherein two of R1 , R2 , R3 and R4 contain a side chain of phenylalanine
EP是包含4至8个氨基酸残基的环外肽，所述氨基酸残基包含1或2个包含含有胍基或其质子化形式的侧链的氨基酸残基以及2、3或4个赖氨酸残基；EP is an exocyclic peptide comprising 4 to 8 amino acid residues, wherein the amino acid residues include 1 or 2 amino acid residues containing a side chain containing a guanidine group or its protonated form and 2, 3 or 4 lysine residues;
每个m独立地是0-3的整数；Each m is independently an integer from 0 to 3;
n是0-2的整数；n is an integer from 0 to 2;
x’是2-20的整数；x’ is an integer from 2 to 20;
y是1-5的整数；y is an integer from 1 to 5;
q是1-4的整数；q is an integer from 1 to 4;
z’是2-20的整数；z’ is an integer between 2 and 20;
AC是与包含前mRNA序列中的DMD基因的外显子44的至少一部分的靶序列互补的反义化合物。AC is an antisense compound that is complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence.
2.如权利要求1所述的化合物，其中q是1。2. A compound as claimed in claim 1, wherein q is 1.
3.如权利要求1或2所述的化合物，其中所述EP具有以下结构：Ac-PKKKRKV。3. A compound as described in claim 1 or 2, wherein the EP has the following structure: Ac-PKKKRKV.
4.如权利要求1-3中任一项所述的化合物，其中所述环肽包含FGFGRGRQ。4. A compound as described in any one of claims 1-3, wherein the cyclic peptide comprises FGFGRGRQ.
5.如权利要求1-4中任一项所述的化合物，所述化合物包含：Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OH。5. The compound of any one of claims 1-4, comprising: Ac-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH.
6.如权利要求1-5中任一项所述的化合物，其中所述AC包含以下序列：5’-AAACGCCGCCATTTCTCAACAGATC-3’。6. A compound as described in any one of claims 1-5, wherein the AC comprises the following sequence: 5’-AAACGCCGCCATTTCTCAACAGATC-3’.
7.如权利要求1-5中任一项所述的化合物，其中所述AC包含以下序列：-ACTGTTCAGCTTCTGTTAGCCACTG-3’。7. A compound as described in any one of claims 1-5, wherein the AC comprises the following sequence: -ACTGTTCAGCTTCTGTTAGCCACTG-3’.
8.如权利要求1-5中任一项所述的化合物，其中所述AC包含以下序列：5’-AACGCCGCCATTTCTCAACAGATCT-3’。8. A compound as described in any one of claims 1-5, wherein the AC comprises the following sequence: 5’-AACGCCGCCATTTCTCAACAGATCT-3’.
9.如权利要求1-5中任一项所述的化合物，其中所述AC包含从SEQ ID No：1的59位开始的25个连续核苷酸，由以下序列组成：5’-TGTTCAGCTTCTGTTAGCCACTGAT-3’。9. A compound as described in any one of claims 1 to 5, wherein the AC comprises 25 consecutive nucleotides starting from position 59 of SEQ ID No: 1, consisting of the following sequence: 5’-TGTTCAGCTTCTGTTAGCCACTGAT-3’.
10.如权利要求1-5中任一项所述的化合物，其中所述AC包含从SEQ ID No：1的6位开始的25个连续核苷酸，由以下序列组成：5’-CCGCCATTTCTCAACAGATCTGTCA-3’5’。10. A compound as described in any one of claims 1 to 5, wherein the AC comprises 25 consecutive nucleotides starting from position 6 of SEQ ID No: 1, consisting of the following sequence: 5’-CCGCCATTTCTCAACAGATCTGTCA-3’5’.
11.如权利要求1所述的化合物，其中所述AC包含至少一种修饰的核苷酸或核酸，所述修饰的核苷酸或核酸包含硫代磷酸酯(PS)核苷酸、二氨基磷酸酯吗啉代核苷酸(PMO)、锁核酸(LNA)、肽核酸(PNA)、包含2′-O-甲基(2′-OMe)修饰的主链的核苷酸、2′O-甲氧基-乙基(2′-MOE)核苷酸、2′，4′约束乙基(cEt)核苷酸或2′-脱氧-2′-氟-β-D-阿糖核酸(2′F-ANA)。11. The compound of claim 1, wherein the AC comprises at least one modified nucleotide or nucleic acid, the modified nucleotide or nucleic acid comprising a phosphorothioate (PS) nucleotide, a phosphorodiamidate morpholino nucleotide (PMO), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a nucleotide comprising a 2′-O-methyl (2′-OMe) modified backbone, a 2′O-methoxy-ethyl (2′-MOE) nucleotide, a 2′,4′ constrained ethyl (cEt) nucleotide, or a 2′-deoxy-2′-fluoro-β-D-arabino nucleic acid (2′F-ANA).
12.如权利要求1所述的化合物，其中所述AC包含15-30个核酸。12. A compound as described in claim 1, wherein the AC contains 15-30 nucleic acids.
13.如权利要求1所述的化合物，其中所述环肽与所述AC的3′端缀合。13. The compound as described in claim 1, wherein the cyclic peptide is conjugated to the 3′ end of the AC.
14.如权利要求1所述的化合物，其中所述环肽与所述AC的5′端缀合。14. The compound as described in claim 1, wherein the cyclic peptide is conjugated to the 5′ end of the AC.
15.如权利要求1所述的化合物，其中所述环肽与所述AC的主链缀合。15. The compound of claim 1, wherein the cyclic peptide is conjugated to the backbone of the AC.
16.如权利要求1-15中任一项所述的化合物，所述化合物还包含将所述环肽缀合至所述AC的接头。16. A compound as described in any one of claims 1-15, further comprising a linker for conjugating the cyclic peptide to the AC.
17.如权利要求16所述的化合物，其中所述接头共价结合到所述AC的5′端。17. A compound as described in claim 16, wherein the linker is covalently bound to the 5′ end of the AC.
18.如权利要求16所述的化合物，其中所述接头共价结合到所述AC的3′端。18. A compound as described in claim 16, wherein the linker is covalently bound to the 3′ end of the AC.
19.如权利要求16所述的化合物，其中所述接头共价结合到所述AC的主链。19. A compound as described in claim 16, wherein the linker is covalently bonded to the backbone of the AC.
20.如权利要求16-19中任一项所述的化合物，其中所述接头共价结合到所述环肽上的氨基酸残基的侧链。20. A compound as described in any one of claims 16-19, wherein the linker is covalently bound to the side chain of the amino acid residue on the cyclic peptide.
21.如权利要求16-20中任一项所述的化合物，其中所述接头是二价或三价C1-C50亚烷基，其中1-25个亚甲基任选且独立地被-N(H)-、-N(C1-C4烷基)-、-N(环烷基)-、-O-、-C(O)-、-C(O)O-、-S-、-S(O)-、-S(O)2-、-S(O)2N(C1-C4烷基)-、-S(O)2N(环烷基)-、-N(H)C(O)-、-N(C1-C4烷基)C(O)-、-N(环烷基)C(O)-、-C(O)N(H)-、-C(O)N(C1-C4烷基)、-C(O)N(环烷基)、芳基、杂芳基、环烷基或环烯基替换。21. The compound of any one of claims 16-20, wherein the linker is a divalent ortrivalent C1-C50 alkylene group, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1 -C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)- , -C(O)O-, -S-, -S(O)-, -S(O)2-, -S (O)2N (C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1 -C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1 -C4 alkyl), -C(O)N(cycloalkyl), aryl, heteroaryl, cycloalkyl, or cycloalkenyl.
22.如权利要求16-21中任一项所述的化合物，其中所述接头包含：22. A compound as described in any one of claims 16-21, wherein the linker comprises:
(i)一个或多个D或L氨基酸残基，其每一者任选地被取代；(i) one or more D or L amino acid residues, each of which is optionally substituted;
(ii)任选取代的亚烷基；(ii) optionally substituted alkylene;
(iii)任选取代的亚烯基；(iii) optionally substituted alkenylene;
(iv)任选取代的亚炔基；(iv) optionally substituted alkynylene;
(v)任选取代的碳环基；(v) an optionally substituted carbocyclic group;
(vi)任选取代的杂环基；(vi) optionally substituted heterocyclic group;
(vii)一个或多个-(R1-J-R2)z”-亚基，其中R1和R2中的每一者在每种情况下独立地选自亚烷基、亚烯基、亚炔基、碳环基和杂环基，每个J独立地是C、NR3、-NR3C(O)-、S和O，其中R3独立地选自H、烷基、烯基、炔基、碳环基和杂环基，其每一者任选地被取代，并且z”是1至50的整数；(vii) one or more -(R1- JR2 )z"-ene groups, wherein each of R1 and R2 is independently selected at each occurrence from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S, and O, wherein R3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50;
(viii)-(R1-J)z”-或-(J-R1)z”-，其中R1中的每一者在每种情况下独立地是亚烷基、亚烯基、亚炔基、碳环基或杂环基，每个J独立地是C、NR3、-NR3C(O)-、S或O，其中R3是H、烷基、烯基、炔基、碳环基或杂环基，其每一者任选地被取代，并且z”是1至50的整数；或(ix)它们的组合。(viii) -(R1- J)z"- or -(JR1 )z"-, wherein each of R1 is independently at each occurrence alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; or (ix) combinations thereof.
23.如权利要求22所述的化合物，其中所述接头包含：23. The compound of claim 22, wherein the linker comprises:
(i)β丙氨酸和赖氨酸残基；(i) β-alanine and lysine residues;
(ii)-(J-R1)z；(ii) -(JR1 )z;
(iii)-(J-R2)x，或(iii) -(JR2 )x, or
(iv)它们的组合。(iv) combinations thereof.
24.如权利要求22或23所述的化合物，其中每个R1和R2独立地是亚烷基，每个J是O，每个x独立地是1至20的整数，并且每个z独立地是1至20的整数。24. The compound of claim 22 or 23, wherein each R1 and R2 are independently alkylene, each J is O, each x is independently an integer from 1 to 20, and each z is independently an integer from 1 to 20.
25.如权利要求16-20中任一项所述的化合物，其中所述接头包含：25. A compound as described in any one of claims 16-20, wherein the linker comprises:
(i)-(OCH2CH2)x-亚基，其中x是1至20的整数；(i) -(OCH2 CH2 )x -subunit, wherein x is an integer from 1 to 20;
(ii)甘氨酸、β-丙氨酸、4-氨基丁酸、5-氨基戊酸或6-氨基戊酸或它们的组合的一个或多个残基；或(ii) one or more residues of glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid or 6-aminovaleric acid or a combination thereof; or
(iii)(i)和(ii)的组合。(iii) A combination of (i) and (ii).
26.如权利要求16-25中任一项所述的化合物，其中所述接头具有以下结构：26. A compound as described in any one of claims 16-25, wherein the linker has the following structure:
其中：in:
x是1-20的整数；x is an integer from 1 to 20;
y是1-5的整数；y is an integer from 1 to 5;
z是1-20的整数；z is an integer from 1 to 20;
M是结合部分；并且M is a binding moiety; and
AASC是所述环肽的氨基酸残基。AASC are the amino acid residues of the cyclic peptide.
27.如权利要求26所述的化合物，其中M是：27. The compound as claimed in claim 26, wherein M is:
-C(O)-、其中R是烷基、烯基、炔基、碳环基或杂环基；或其中M是：-C(O)-, wherein R is alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl; or wherein M is:
其中：R1是亚烷基、环烷基或其中m是0至10，其中每个R独立地是烷基、烯基、炔基、碳环基或杂环基，并且其中每个B独立地选自核碱基。Wherein:R1 is alkylene, cycloalkyl or wherein m is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, and wherein each B is independently selected from a nucleobase.
28.如权利要求27所述的化合物，其中M是-C(O)-。28. A compound as described in claim 27, wherein M is -C(O)-.
29.如权利要求26-28中任一项所述的化合物，其中z是11。29. A compound as described in any one of claims 26-28, wherein z is 11.
30.如权利要求26-29中任一项所述的化合物，其中x是1。30. A compound as described in any one of claims 26-29, wherein x is 1.
31.如权利要求26-30中任一项所述的化合物，所述化合物包含在所述接头的氨基基团处与所述接头缀合的环外肽(EP)。31. A compound as described in any one of claims 26-30, comprising an exocyclic peptide (EP) conjugated to the linker at the amino group of the linker.
32.如权利要求31所述的化合物，其中所述EP包含2至10个氨基酸残基。32. A compound as described in claim 31, wherein the EP comprises 2 to 10 amino acid residues.
33.如权利要求1所述的化合物，其中每个赖氨酸残基的侧链上的氨基基团被三氟乙酰基(-COCF3)基团、烯丙氧基羰基(Alloc)、1-(4，4-二甲基-2，6-二氧代亚环己基)乙基(Dde)或(4，4-二甲基-2，6-二氧代环己-1-亚基-3)-甲基丁基(ivDde)基团取代。33. The compound of claim 1, wherein the amino group on the side chain of each lysine residue is substituted by a trifluoroacetyl (-COCF3 ) group, an allyloxycarbonyl (Alloc), a 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) or a (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) group.
34.如权利要求1-33中任一项所述的化合物，其中所述环外肽(EP)包含具有疏水侧链的至少2个氨基酸残基。34. A compound as described in any one of claims 1 to 33, wherein the exocyclic peptide (EP) comprises at least 2 amino acid residues having hydrophobic side chains.
35.如权利要求34所述的化合物，其中所述具有疏水侧链的氨基酸残基选自缬氨酸、脯氨酸、丙氨酸、亮氨酸、异亮氨酸和甲硫氨酸。35. A compound as described in claim 34, wherein the amino acid residue having a hydrophobic side chain is selected from valine, proline, alanine, leucine, isoleucine and methionine.
36.如权利要求1-35中任一项所述的化合物，其中所述环外肽包含以下序列中的一者：36. A compound as described in any one of claims 1-35, wherein the exocyclic peptide comprises one of the following sequences:
PKKKRKV；KR；RR；KKK；KGK；KBK；KBR；KRK；KRR；RKK；RRR；KKKK；KKRK；KRKK；KRRK；RKKR；RRRR；KGKK；KKGK；KKKKK；KKKRK；KBKBK；KKKRKV；PGKKRKV；PKGKRKV；PKKGRKV；PKKKGKV；PKKKRGV；或PKKKRKG。PKKKRKV;KR;RR;KKK;KGK;KBK;KBR;KRK;KRR;RKK;RRR;KKKK;KKRK;KRKK;KRRK;RKKR;RRRR;KGKK;KKGK;KKKKK;KKKRK;KBKBK;KKKRKV;PGKKRKV;PKGKRKV; PKKGRKV; PKKKGKV; PKKKRGV; or PKKKRKG.
37.如权利要求1-36中任一项所述的化合物，所述化合物包含以下结构：37. A compound as described in any one of claims 1 to 36, comprising the following structure:
其中：in:
x是1-20的整数；x is an integer from 1 to 20;
y是1-5的整数；y is an integer from 1 to 5;
z是1-20的整数；z is an integer from 1 to 20;
EP是环外肽；EP is an extracyclic peptide;
M是结合部分；M is the binding part;
AC是反义化合物；并且AC is an antisense compound; and
AASC是所述环肽的氨基酸残基。AASC are the amino acid residues of the cyclic peptide.
38.如权利要求1-37中任一项所述的化合物，其中所述环肽包含4至20个氨基酸残基，其中至少两个氨基酸残基包含含有胍基或其质子化形式的侧链，并且至少两个氨基酸残基独立地包含疏水侧链。38. A compound as described in any one of claims 1-37, wherein the cyclic peptide comprises 4 to 20 amino acid residues, wherein at least two of the amino acid residues comprise a side chain containing a guanidine group or a protonated form thereof, and at least two of the amino acid residues independently comprise a hydrophobic side chain.
39.如权利要求1-38中任一项所述的化合物，其中所述环肽包含含有胍基或其质子化形式的1、2、3或4个酸酸残基。39. A compound as described in any one of claims 1-38, wherein the cyclic peptide comprises 1, 2, 3 or 4 acid residues containing a guanidine group or a protonated form thereof.
40.如权利要求38或39所述的化合物，其中所述环肽包含含有疏水侧链的2、3或4个氨基酸残基。40. A compound as described in claim 38 or 39, wherein the cyclic peptide comprises 2, 3 or 4 amino acid residues containing hydrophobic side chains.
41.如权利要求38-40中任一项所述的化合物，其中所述环肽包含含有侧链的至少一个氨基酸，所述侧链包含或其质子化形式。41. A compound as described in any one of claims 38 to 40, wherein the cyclic peptide comprises at least one amino acid containing a side chain comprising or its protonated form.
42.如权利要求38-41中任一项所述的化合物，其中所述环肽包含含有侧链的1、2、3或4个氨基酸，所述侧链选自或其质子化形式。42. A compound as described in any one of claims 38 to 41, wherein the cyclic peptide comprises 1, 2, 3 or 4 amino acids containing a side chain selected from or its protonated form.
43.如权利要求38-42中任一项所述的化合物，其中所述环肽包含至少一个甘氨酸残基。43. A compound as described in any one of claims 38-42, wherein the cyclic peptide comprises at least one glycine residue.
44.如权利要求38-43中任一项所述的化合物，其中所述环肽包含1、2、3或4个甘氨酸残基。44. A compound as described in any one of claims 38-43, wherein the cyclic peptide comprises 1, 2, 3 or 4 glycine residues.
45.如权利要求1-44中任一项所述的化合物，其中所述环肽包含式(I)：45. A compound as described in any one of claims 1-44, wherein the cyclic peptide comprises formula (I):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R6独立地是H或氨基酸侧链；R4 andR6 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；并且q is 1, 2, 3, or 4; and
每个m独立地是整数0、1、2或3。Each m is independently an integer 0, 1, 2 or 3.
46.如权利要求45所述的化合物，其中包含芳基的所述侧链是苯丙氨酸的侧链。46. A compound as described in claim 45, wherein the side chain containing an aromatic group is a side chain of phenylalanine.
47.如权利要求45或46中任一项所述的化合物，其中R1、R2、R3和R4中的两者是H。47. The compound of any one of claims 45 or 46, wherein two ofR1 ,R2 ,R3 andR4 are H.
48.如权利要求45-47中任一项所述的化合物，其中所述环肽包含式(I-1)、(I-2)、(I-3)或(I-4)的结构：48. A compound as described in any one of claims 45-47, wherein the cyclic peptide comprises a structure of formula (I-1), (I-2), (I-3) or (I-4):
或其质子化形式，or its protonated form,
其中：in:
AASC是氨基酸侧链；并且AASC is an amino acid side chain; and
每个m独立地是0-3的整数。Each m is independently an integer from 0 to 3.
49.如权利要求45-48中任一项所述的化合物，其中所述环肽包含以下结构：49. A compound as described in any one of claims 45-48, wherein the cyclic peptide comprises the following structure:
或其质子化形式，or its protonated form,
其中：in:
AASC是氨基酸侧链；并且AASC is an amino acid side chain; and
每个m独立地是0-3的整数。Each m is independently an integer from 0 to 3.
50.如权利要求45-49中任一项所述的化合物，其中AASC包含天冬酰胺残基、天冬氨酸残基、谷氨酰胺残基、谷氨酸残基、高谷氨酸残基或高谷氨酰胺残基的侧链。50. The compound of any one of claims 45-49, wherein AASC comprises a side chain of an asparagine residue, an aspartic acid residue, a glutamine residue, a glutamic acid residue, a homoglutamic acid residue, or a homoglutamine residue.
51.如权利要求45-50中任一项所述的化合物，其中AASC包含谷氨酰胺的侧链。51. The compound of any one of claims 45-50, wherein AASC comprises a side chain of glutamine.
52.如权利要求1-51中任一项所述的化合物，其中所述AC包含来自表6A-6M的序列、其反向互补序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核酸序列同一性的序列。52. A compound as described in any one of claims 1-51, wherein the AC comprises a sequence from Tables 6A-6M, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto.
53.如权利要求1-51中任一项所述的化合物，其中所述环肽包含GfFGrGrQ。53. A compound as described in any one of claims 1-51, wherein the cyclic peptide comprises GfFGrGrQ.
54.如权利要求1-51中任一项所述的化合物，其中所述环肽包含FfΦGRGRQ。54. A compound as described in any one of claims 1-51, wherein the cyclic peptide comprises FfΦGRGRQ.
55.如权利要求1-53中任一项所述的化合物，所述化合物包含式(C-1)、式(C-2)、式(C-3)或式(C-4)的结构：55. A compound as described in any one of claims 1 to 53, wherein the compound comprises a structure of formula (C-1), formula (C-2), formula (C-3) or formula (C-4):
或其质子化形式；or its protonated form;
其中包含15-30个核酸的序列，所述序列与包含前mRNA序列中的DMD基因的外显子44的至少一部分的靶序列互补。It comprises a sequence of 15-30 nucleic acids that is complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence.
56.一种药物组合物，其包含如权利要求1至55中任一项所述的化合物。56. A pharmaceutical composition comprising a compound as described in any one of claims 1 to 55.
57.一种细胞，其包含如权利要求1至55中任一项所述的化合物。57. A cell comprising a compound as described in any one of claims 1 to 55.
58.一种治疗DMD的方法，所述方法包括向有需要的患者施用如权利要求1至55中任一项所述的化合物。58. A method for treating DMD, comprising administering a compound as described in any one of claims 1 to 55 to a patient in need thereof.
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| PCT/US2022/075691WO2023034817A1 (en) | 2021-09-01 | 2022-08-30 | Compounds and methods for skipping exon 44 in duchenne muscular dystrophy |
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| CN202280066121.9APendingCN118215504A (en) | 2021-09-01 | 2022-08-30 | Compounds and methods for skipping exon 44 in duchenne muscular dystrophy |
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