摘要
反义寡核苷酸(antisense oligonucleotides,ASONs)作为新型的基因治疗药物受到国内外科研人员及各大制药公司的普遍关注。但此类药物生物学稳定性较差,易被体内核酸酶降解;另外由于ASONs分子为多聚阴离子大分子,生理条件下很难通过带负电荷的细胞膜,因此生物利用度低、靶组织内聚集浓度低。目前,可通过对天然核酸序列经过各类化学修饰(如:硫代修饰、混合骨架核酸)或构建新的核酸类似物(如:肽核酸)等来增加ASONs的稳定性;而如何提高ASONs的胞内摄取和靶向递送仍是目前反义药物开发急需解决的问题。近年来研究人员对多种药物递送方式进行了研究,包括使用脂质体类递送载体[6-8]、阳离子多聚物类载体[9-11]、多肽蛋白类载体[12-15]以及一些特异性受体的配体[16-17]。其中,多肽蛋白类修饰是目前ASONs靶向递送研究的热点。靶向肽具有分子量小、组织穿透能力更强、低免疫原性和高亲和力、容易大量合成等优点,在药物靶向导入系统(drugdelivery system,DDS)中具有广阔的应用前景。
在前期工作中,本室筛选获得多条抗流感病毒(IV)效果较好的反义核酸序列(Prop5,专利申请号:201010179175.5,公开号:CN101864421A;Flutide,专利号ZL97120355.5)[18]。同时还针对几种重要的乙肝病毒(HBV)感染关键分子,如ASGPR、fibronectin、EREG、ABHD2,筛选得到4条特异性好的ASONs(专利分别为ZL2004100583001.1、 ZL200410073618.7、 ZL200610075976.0和ZL200610075977.5),在体内外均具有较强的抗HBV活性[19-21]。越来越多的研究证明了ASONs是病毒感染性疾病的有效治疗手段之一。由于病毒是一类严格的细胞内寄生病原体,病毒基因组只有进入到宿主细胞内才能进行子代病毒的复制和进一步的感染,而ASONs要发挥反义抑制作用必须要进入靶细胞,因此能否寻找到一条途径将抗病毒ASONs直接导入到病毒感染的细胞内至关重要。目前ASONs的靶向转运研究中多选取各类细胞表面特异性受体的配体作为靶向分子,而针对病毒表面特异性抗原或蛋白的特异结合配体作为ASONs靶向转运的靶向分子尚未发现相关报道。本室提出一种基于病毒颗粒介导的ASONs靶向转运策略,期望借助病毒颗粒感染入胞过程将ASONs直接导入病毒感染细胞内,解决ASONs胞内摄取有限和靶向性差的问题,最终起到增强抗病毒活性、降低给药量和毒副作用的目的。
本研究主要以抗流感病毒的ASONs(Prop5)为被修饰对象。首先采用原核表达和噬菌体展示技术进行流感病毒表面蛋白HA和NA特异性结合多肽的筛选,各得到一条特异性结合肽H17和N3,通过荧光显微镜及流式细胞仪等对这两条结合肽在病毒感染细胞内的摄取与分布进行考察,发现流感病毒感染可显著增加多肽H17在病毒感染细胞中的摄取,随着感染时间的延长摄取率逐渐增多。在激光共聚焦显微镜下观察发现H17可与病毒共同分布于细胞质中。虽然多肽N3在病毒感染细胞中的摄取率也有所提升,但增加速度较慢。另外,对H17的抗病毒活性检测发现其对流感病毒所致细胞病变没有显著地抑制作用,且在给药100μM不会对细胞增殖产生影响。因此,H17将作为ASONs递送系统中的靶向分子用于后续递送系统的合成与构建。随后我们通过两种不同的合成路线对多肽及ASONs进行共价偶联。最终合成并制备出包括荧光标记物在内的4种多肽-ASONs缀合物,通过RP-HPLC分析及MALDI-TOF-MS鉴定缀合物结构及分子量正确。通过对缀合物的理化性质考察发现多肽偶联不影响ASONs与互补链的结合,也不影响ASONs的血清稳定性。最后我们初步确定了缀合物的保存条件。接下来通过激光共聚焦显微镜、流式细胞术及活体成像技术对缀合物进行胞内摄取、分布及组织内分布的考察,发现流感病毒结合肽的修饰对ASONs入胞过程具有选择特异性,病毒靶向肽修饰可显著提高病毒感染细胞对ASONs的摄取,且修饰化合物(prop5-HABP)的透膜率具有显著的时间依赖性和浓度依赖性。病毒靶向肽修饰还可使ASONs在病毒感染组织内快速蓄积。采用Western blot及荧光定量PCR法考察了不同浓度下prop5修饰前后对细胞内靶基因的抑制作用及对流感病毒复制的抑制作用,检测结果均显示多肽修饰后可显著提高prop5对靶基因PDCD5的抑制作用和抗IV活性,且不影响细胞的增殖。上述结果证明病毒靶向肽修饰可实现对ASONs的定向转运。
为进一步验证病毒靶向递送系统的可行性,我们选择前期筛选得到的具有抗HBV活性的靶向Fibronetin的FN1(序列:5’-GCTCATCTCCCTCCTCACTC-3’)为靶向修饰研究的目标ASON。根据文献(J Microbiol,2007,45:528-533)报道,确定HBV前S1抗原特异性结合肽B3作为靶向到HBV颗粒的分子,二者偶联后得到多肽-ASONs缀合物FN1-B3。通过对其Tm值进行测定,发现多肽缀合不会影响FN1与互补链的结合。之后采用HepG2.2.15细胞和HBV阳性血清感染人原代肝细胞模型对多肽修饰后FN1的靶向性、透膜性及抗病毒活性进行考察,结果表明HBV结合肽偶联到FN1上同样可提高其在HBV感染细胞中的摄取及抗病毒活性。
综上所述,通过在两种不同病毒感染细胞模型上的验证实验,均证明所提出的通过病毒颗粒介导的寡核苷酸靶向转运策略是可行的,病毒特异性结合肽与抗病毒ASONs偶联后,可在病毒感染入胞过程中,通过其特异性地结合病毒颗粒将ASONs带入被感染细胞及组织内,实现对ASONs的定向转运,发挥抗病毒疗效。本课题提出的基于病毒靶向肽修饰的ASONs靶向转运与以往的靶向递送系统作用机制完全不同,其研究结果可为其他抗病毒ASONs药物新型靶向给药体系的建立提供启示和促进作用;还可为流感病毒及乙肝病毒的预防或治疗提供特异性好、作用机制明确、靶向性佳的候选药物。
As a new kind of drug for gene therapy, antisense oligonucleotide (ASONs) iswidespread concern by researchers at home and abroad and the major pharmaceuticalcompanies. But this kind of drug suffers from biological instability, and is easy to bedegraded by nuclease in vivo. In addition, because most of the molecules of ASONs arepoly anionic macromolecules and they are difficult to diffuse through the negtativelycharged cell membrane bilayers under physiological conditions. Their biologicalutilization and denseness in target tissues are low. These properties mean thebioavailability of ASONs is low and concentration in target tissue difficult toaccumulate. At present, the stability of ASONs is often enhanced by various types ofchemical modification of natural nucleic acid sequence (such as: phosphorothioate,mixed skeleton nucleic acid) or to build new nucleic acid analogs (such as: peptidenucleic acid). However, how to improve the ASONs intracellular uptake and targeteddelivery are still urgent problems of the antisense drug development. Researchersstudied a variety of drug delivery way, including the use of a liposome deliveryvehicle[6-8], the cationic polymer vectors[9-11]peptides or proteins carriers[12-15], and somespecific receptor ligands[16-17]. Peptide or protein modifications are currently ASONstargeted delivery research hotspot. Targeting peptide has the superiorities of smallmolecular weight, tissue penetration ability, low immunogenicity, high affinity andeasily synthesized in large quantities, etc., so it has broad application prospects in drugtargeting system (DDS).
In previous work, the laboratory screened a number of anti-influenza viruses (IV)ASONs (Prop5patent application number:201010179175.5, Publication Number:CN101864421A; Flutide Patent No. ZL97120355.5)[18]. Four good specificity ASONs(Patent ZL2004100583001.1, ZL200410073618.7, ZL200610075976.0,ZL200610075977.5) which targeting the key molecules of Hepatitis B Virus (HBV)infection (ASGPR, fibronectin, EREG, ABHD2) are also gotten. They all have stronganti-HBV activity in vitro and in vivo.[19-21]More and more studies have shown thatASONs is one of the effective treatments of viral infection. Because the virus is a strictintracellular parasitic pathogens, the viral genome is only entered into the host cell canbe carried out within the progeny virus replication and further infection. ASONs to playthe inhibition of the target gene must enter target cells. Therefore, it is very important that a way should be found to delivery the anti-virus ASONs into the cells infected byvirus. At present, in the researches of the targeting delivery of ASONs, kinds of specificcell surface receptor ligands are often chosen as the targeting molecules. However, therehave been no reports about the virus surface antigen or protein specifically bindingligand as a delivery system targeting molecules. Our laboratory proposed a novelASONs targeting delivery strategy based on virus particles mediated to increase themembrane permeability, targeting ability, specificity, antiviral activity, and reduce thetoxicity, which can directly delivery ASONs into the virus-infected cells by theinfection process of viral particles.
In this study, anti-flu ASONs (Prop5) was modified. Firstly, the recombinantinfluenza virus surface proteins HA and NA-specific binding peptides were screened byphage display technology. And then a specific binding peptide H17and N3are gotten,respectively. By investigating the uptake and distribution of the two binding peptides invirus-infected cells using fluorescence microscope and flow cytometry, we found thatflu virus could significantly increase the uptake of H17in virus-infected cells and theabsorption uptake rate increased with the extension of the infection time. By using laserscanning confocal microscope, we found that H17and the virus distributed in thecytoplasm at the same time. Although the uptake rate of N3in virus-infected cells alsoimproved, but slow increase. In addition, the H17anti-viral activity detected nosignificant inhibition of the cytopathic effect caused by influenza virus and in theadministration100μM no proliferation impact. Thus, H17will be used in the synthesisand construction of the following targeting delivery system as the targeting molecules.And then, we carried out covalent coupling on peptide and ASONs in two differentsynthesis routes. Finally, four peptide-ASONs conjugates were synthesized andproduced, including fluorescently labeled conjugate. The structure and molecularweight of conjugates are proved correct by the RP-HPLC analysis and theMALDI-TOF-MS identification. By investigating the physical and chemical propertiesof conjugate, we found that peptide modification had no influence on the combinationof ASONs and complementary strand, and also had no influence on the serum stabilityof ASONs. At last, we preliminarily determined the storage conditions of conjugate.Next, conjugates intracellular uptake, distribution and tissue distribution by laserscanning confocal microscopy, flow cytometry and in vivo imaging techniques study,we found that ASONs modified by flu virus binding peptide had select specific in the process of endocytosis. Virus targeted peptide modification can significantly improvethe uptake of the virus-infected cells. And the membrane permeability of Prop5-HABPhad significant time-and density-dependency. The virus targeting peptide modificationcan also allows ASONs rapid accumulation of virus infected tissue.
The inhibition of intracellular target genes and influenza virus replication afterProp5different concentrations modified with peptide were investigated by Western blotand quantitative PCR. All the test results demonstrated that the peptide modificationcould significantly increase the inhibition of the target gene PDCD5and the anti-IVactivity, without affecting cell proliferation. The above results show that virus targetingpeptide modification can achieve directional delivery of ASONs.
To further verify the feasibility of virus targeted delivery system, we have chosenthe pre-screened targeting Fibronetin of FN1(sequence: the5'-GCTCATCTCCCTCCTCACTC-3') which has an anti-HBV activity as the objective in targetingmodification. According to the literature (J Microbiol,2007,45:528-533), wedetermined to choose the HBV pre-S1antigen-specific binding peptide B3as amolecular targeted HBV particles. Conjugate FN1-B3was gotten by coupling of them.By identifying its Tm value, we found that peptide coupling had no influence on thecombination of FN1and its complementary strand. Then, we investigated the targetingability and membrane permeability of peptide modified FN1and anti-viral activity byusing the HepG2.2.15cells and HBV-positive serum infected primary human liver cellmodel. The results demonstrated that HBV binding peptide modification can alsoincrease the uptake and anti-viral activity in HBV-infected cells.
In summary, the experiments carried out on two different virus-infected cellsdemonstrated that the viral particles mediated targeting delivery strategy is feasible.After the coupling of virus binding peptides, the ASONs can be delivered into theinfected cells and tissues by the specific binding of viral particles when the virus infectcells, realizing the targeting delivery for ASONs and playing their antiviral activity. Thevirus mediated targeting delivery proposed in this dissertation is different from theformer ones in the mechanism. The findings can be provided for the establishment ofnew targeted drug delivery system of other antiviral ASONs drugs inspiration and role;can also provide good specificity, a clear mechanism of action and good targetingability candidate drugs for the prevention or treatment of influenza virus and hepatitis Bvirus.
引文
[1] Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNAtranslation by a specific oligodeoxyribonucleotide[J]. Proc Natl Acad Sci U S A,1978,75(1):285-288.
[2] Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication andcell transformation by a specific oligodeoxynucleotide[J]. Proc Natl Acad Sci US A,1978,75(1):280-284.
[3] Braasch DA, Corey DR. Novel antisense and peptide nucleic acid strategies forcontrolling gene expression[J]. Biochemistry,2002,41(14):4503-4510.
[4] Crooke ST. Therapeutic applications of oligonucleotides[J]. Annu RevPharmacol Toxicol,1992,32:329-376.
[5] Agrawal S, Iyer RP. Perspectives in antisense therapeutics[J]. Pharmacol Ther,1997,76(1-3):151-160.
[6] Drummond DC, Meyer O, Hong K, et al. Optimizing liposomes for delivery ofchemotherapeutic agents to solid tumors[J]. Pharmacol Rev,1999,51(4):691-743.
[7] Meyer O, Kirpotin D, Hong K, et al. Cationic liposomes coated withpolyethylene glycol as carriers for oligonucleotides[J]. J Biol Chem,1998,273(25):15621-15627.
[8] Fresta M, Chillemi R, Spampinato S, et al. Liposomal delivery of a30-merantisense oligodeoxynucleotide to inhibit proopiomelanocortin expression[J]. JPharm Sci,1998,87(5):616-625.
[9] Sundaram S, Viriyayuthakorn S, Roth CM. Oligonucleotide structure influencesthe interactions between cationic polymers and oligonucleotides[J].Biomacromolecules,2005,6(6):2961-2968.
[10] DeLong RK, Yoo H, Alahari SK, et al. Novel cationic amphiphiles as deliveryagents for antisense oligonucleotides[J]. Nucleic Acids Res,1999,27(16):3334-3341.
[11] Gonzalez Ferreiro M, Crooke RM, Tillman L, et al. Stability of polycationiccomplexes of an antisense oligonucleotide in rat small intestine homogenates[J].Eur J Pharm Biopharm,2003,55(1):19-26.
[12] Lochmann D, Jauk E, Zimmer A. Drug delivery of oligonucleotides bypeptides[J]. Eur J Pharm Biopharm,2004,58(2):237-251.
[13] Kubo T, Kanno K, Ohba H, et al. Control of intracellular delivery ofoligonucleotides by signal peptides and genetic expression in human cells[J].Nucleic Acids Symp Ser (Oxf),2004(48):303-304.
[14] Abes S, Moulton H, Turner J, et al. Peptide-based delivery of nucleic acids:design, mechanism of uptake and applications to splice-correctingoligonucleotides[J]. Biochem Soc Trans,2007,35(Pt1):53-55.
[15] Wartlick H, Spankuch-Schmitt B, Strebhardt K, et al. Tumour cell delivery ofantisense oligonuclceotides by human serum albumin nanoparticles[J]. J ControlRelease,2004,96(3):483-495.
[16] Citro G, Perrotti D, Cucco C, et al. Inhibition of leukemia cell proliferation byreceptor-mediated uptake of c-myb antisense oligodeoxynucleotides[J]. ProcNatl Acad Sci U S A,1992,89(15):7031-7035.
[17] Leamon CP, Cooper SR, Hardee GE. Folate-liposome-mediated antisenseoligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo[J].Bioconjug Chem,2003,14(4):738-747.
[18] Duan M, Zhou Z, Lin RX, et al. In vitro and in vivo protection against the highlypathogenic H5N1influenza virus by an antisense phosphorothioateoligonucleotide[J]. Antivir Ther,2008,13(1):109-114.
[19] Yang J, Bo XC, Ding XR, et al. Antisense oligonucleotides targeted againstasialoglycoprotein receptor1block human hepatitis B virus replication[J]. JViral Hepat,2006,13(3):158-165.
[20] Yang J, Ding X, Zhang Y, et al. Fibronectin is essential for hepatitis B viruspropagation in vitro: may be a potential cellular target?[J]. Biochem BiophysRes Commun,2006,344(3):757-764.
[21] Ding X, Wang F, Duan M, et al. Epiregulin as a key molecule to suppresshepatitis B virus propagation in vitro[J]. Arch Virol,2009,154(1):9-17.
[22] Kurreck J. Antisense technologies. Improvement through novel chemicalmodifications[J]. Eur J Biochem,2003,270(8):1628-1644.
[23] Rayburn ER, Zhang R. Antisense, RNAi, and gene silencing strategies fortherapy: mission possible or impossible?[J]. Drug Discov Today,2008,13(11-12):513-521.
[24] Ricotta DN, Frishman W. Mipomersen: a safe and effective antisense therapyadjunct to statins in patients with hypercholesterolemia[J]. Cardiol Rev,2012,20(2):90-95.
[25] Chi KN, Eisenhauer E, Fazli L, et al. A phase I pharmacokinetic andpharmacodynamic study of OGX-011, a2'-methoxyethyl antisenseoligonucleotide to clusterin, in patients with localized prostate cancer[J]. J NatlCancer Inst,2005,97(17):1287-1296.
[26] Vlassov VV, Balakireva LA, Yakubov LA. Transport of oligonucleotides acrossnatural and model membranes[J]. Biochim Biophys Acta,1994,1197(2):95-108.
[27] Sato N, Kobayashi H, Saga T, et al. Tumor targeting and imaging ofintraperitoneal tumors by use of antisense oligo-DNA complexed withdendrimers and/or avidin in mice[J]. Clin Cancer Res,2001,7(11):3606-3612.
[28] Pridgen EM, Langer R, Farokhzad OC. Biodegradable, polymeric nanoparticledelivery systems for cancer therapy[J]. Nanomedicine (Lond),2007,2(5):669-680.
[29] Margus H, Padari K, Pooga M. Cell-penetrating peptides as versatile vehicles foroligonucleotide delivery[J]. Mol Ther,2012,20(3):525-533.
[30] Juliano R, Alam MR, Dixit V, et al. Mechanisms and strategies for effectivedelivery of antisense and siRNA oligonucleotides[J]. Nucleic Acids Res,2008,36(12):4158-4171.
[31] Bottcher-Friebertshauser E, Stein DA, Klenk HD, et al. Inhibition of influenzavirus infection in human airway cell cultures by an antisense peptide-conjugatedmorpholino oligomer targeting the hemagglutinin-activating proteaseTMPRSS2[J]. J Virol,2011,85(4):1554-1562.
[32] Lai SH, Stein DA, Guerrero-Plata A, et al. Inhibition of respiratory syncytialvirus infections with morpholino oligomers in cell cultures and in mice[J]. MolTher,2008,16(6):1120-1128.
[33] Opriessnig T, Patel D, Wang R, et al. Inhibition of porcine reproductive andrespiratory syndrome virus infection in piglets by a peptide-conjugatedmorpholino oligomer[J]. Antiviral Res,2011,91(1):36-42.
[34] Burrer R, Neuman BW, Ting JP, et al. Antiviral effects of antisense morpholinooligomers in murine coronavirus infection models[J]. J Virol,2007,81(11):5637-5648.
[35] Deas TS, Bennett CJ, Jones SA, et al. In vitro resistance selection and in vivoefficacy of morpholino oligomers against West Nile virus[J]. Antimicrob AgentsChemother,2007,51(7):2470-2482.
[36] Del Vecchio CA, Li G, Wong AJ. Targeting EGF receptor variant III:tumor-specific peptide vaccination for malignant gliomas[J]. Expert RevVaccines,2012,11(2):133-144.
[37] Wu C, Lo SL, Boulaire J, et al. A peptide-based carrier for intracellular deliveryof proteins into malignant glial cells in vitro[J]. J Control Release,2008,130(2):140-145.
[38] Tang B, Li Z, Huang D, et al. Screening of a specific peptide binding to VPAC1receptor from a phage display peptide library[J]. PLoS One,2013,8(1):e54264.
[39] Koivistoinen A, Ilonen, II, Punakivi K, et al. A novel peptide (Thx) homing tonon-small cell lung cancer identified by ex vivo phage display[J]. Clin TranslOncol,2012.
[40] Desimmie BA, Humbert M, Lescrinier E, et al. Phage display-directed discoveryof LEDGF/p75binding cyclic peptide inhibitors of HIV replication[J]. Mol Ther,2012,20(11):2064-2075.
[41] Molek P, Strukelj B, Bratkovic T. Peptide phage display as a tool for drugdiscovery: targeting membrane receptors[J]. Molecules,2011,16(1):857-887.
[42] McCauley JW, Mahy BW. Structure and function of the influenza virusgenome[J]. Biochem J,1983,211(2):281-294.
[43] Wiley DC, Skehel JJ. The structure and function of the hemagglutinin membraneglycoprotein of influenza virus[J]. Annu Rev Biochem,1987,56:365-394.
[44] Shtyrya YA, Mochalova LV, Bovin NV. Influenza virus neuraminidase: structureand function[J]. Acta Naturae,2009,1(2):26-32.
[45] Singh Y, Murat P, Defrancq E. Recent developments in oligonucleotideconjugation[J]. Chem Soc Rev,2010,39(6):2054-2070.
[46] Lu K, Duan QP, Ma L, et al. Chemical strategies for the synthesis ofpeptide-oligonucleotide conjugates[J]. Bioconjug Chem,2010,21(2):187-202.
[47] Konate K, Crombez L, Deshayes S, et al. Insight into the cellular uptakemechanism of a secondary amphipathic cell-penetrating peptide for siRNAdelivery[J]. Biochemistry,2010,49(16):3393-3402.
[48] Futaki S, Ohashi W, Suzuki T, et al. Stearylated arginine-rich peptides: a newclass of transfection systems[J]. Bioconjug Chem,2001,12(6):1005-1011.
[49] Pooga M, Soomets U, Hallbrink M, et al. Cell penetrating PNA constructsregulate galanin receptor levels and modify pain transmission in vivo[J]. NatBiotechnol,1998,16(9):857-861.
[50] Lundin P, Johansson H, Guterstam P, et al. Distinct uptake routes ofcell-penetrating peptide conjugates[J]. Bioconjug Chem,2008,19(12):2535-2542.
[51] Rapozzi V, Cogoi S, Xodo LE. Antisense locked nucleic acids efficientlysuppress BCR/ABL and induce cell growth decline and apoptosis in leukemiccells[J]. Mol Cancer Ther,2006,5(7):1683-1692.
[52] Fattal E, Bochot A. Antisense oligonucleotides, aptamers and SiRNA: promisesfor the treatment of ocular diseases[J]. Arch Soc Esp Oftalmol,2006,81(1):3-4,5-6.
[53] Fuessel S, Herrmann J, Ning S, et al. Chemosensitization of bladder cancer cellsby survivin-directed antisense oligodeoxynucleotides and siRNA[J]. Cancer Lett,2006,232(2):243-254.
[54] Maier MA, Esau CC, Siwkowski AM, et al. Evaluation of basic amphipathicpeptides for cellular delivery of antisense peptide nucleic acids[J]. J Med Chem,2006,49(8):2534-2542.
[55] Wang CY, Feng SQ, Liu Y, et al.[Study of TAT-PEG loaded magnetic liposomescrossing blood spinal cord barrier after spinal cord injury in rats][J]. ZhonghuaYi Xue Za Zhi,2011,91(3):193-197.
[56] Bartlett RL,2nd, Panitch A. Thermosensitive nanoparticles with pH-triggereddegradation and release of anti-inflammatory cell-penetrating peptides[J].Biomacromolecules,2012,13(8):2578-2584.
[57] Gooding M, Browne LP, Quinteiro FM, et al. siRNA delivery: from lipids tocell-penetrating peptides and their mimics[J]. Chem Biol Drug Des,2012,80(6):787-809.
[58] Wang HY, Wang RF. Enhancing cancer immunotherapy by intracellular deliveryof cell-penetrating peptides and stimulation of pattern-recognition receptorsignaling[J]. Adv Immunol,2012,114:151-176.
[59] Mager I, Langel K, Lehto T, et al. The role of endocytosis on the uptake kineticsof luciferin-conjugated cell-penetrating peptides[J]. Biochim Biophys Acta,2012,1818(3):502-511.
[60] Chaubey B, Tripathi S, Pandey VN. Single acute-dose and repeat-doses toxicityof anti-HIV-1PNA TAR-penetratin conjugate after intraperitonealadministration to mice[J]. Oligonucleotides,2008,18(1):9-20.
[61] Yang GH, Yoon SO, Jang MH, et al. Affinity maturation of an anti-hepatitis Bvirus PreS1humanized antibody by phage display[J]. J Microbiol,2007,45(6):528-533.
[62] Ding X, Yang J, Wang S. Antisense oligonucleotides targeting abhydrolasedomain containing2block human hepatitis B virus propagation[J].Oligonucleotides,2011,21(2):77-84.
[63] Alberti A, Cavalletto D, Chemello L, et al. Fine specificity of human antibodyresponse to the PreS1domain of hepatitis B virus[J]. Hepatology,1990,12(2):199-203.
[64] Meier A, Mehrle S, Weiss TS, et al. Myristoylated PreS1-domain of the hepatitisB virus L-protein mediates specific binding to differentiated hepatocytes[J].Hepatology,2012.
[65] Zhang X, Lin SM, Chen TY, et al. Asialoglycoprotein receptor interacts with thepreS1domain of hepatitis B virus in vivo and in vitro[J]. Arch Virol,2011,156(4):637-645.
[66] Bremer CM, Sominskaya I, Skrastina D, et al. N-terminalmyristoylation-dependent masking of neutralizing epitopes in the preS1attachment site of hepatitis B virus[J]. J Hepatol,2011,55(1):29-37.
[67] Miyata R, Ueda M, Jinno H, et al. Selective targeting by preS1domain ofhepatitis B surface antigen conjugated with phosphorylcholine-basedamphiphilic block copolymer micelles as a biocompatible, drug delivery carrierfor treatment of human hepatocellular carcinoma with paclitaxel[J]. Int J Cancer,2009,124(10):2460-2467.
[68] Yang X, Hu W, Li F, et al. Gene cloning, bacterial expression, in vitro refolding,and characterization of a single-chain Fv antibody against PreS1(21-47)fragment of HBsAg[J]. Protein Expr Purif,2005,41(2):341-348.
[69] Tan WS, Tan GH, Yusoff K, et al. A phage-displayed cyclic peptide that interactstightly with the immunodominant region of hepatitis B surface antigen[J]. J ClinVirol,2005,34(1):35-41.
[70] Park SG, Jeong YJ, Lee YY, et al. Hepatitis B virus-neutralizing anti-pre-S1human antibody fragments from large naive antibody phage library[J]. AntiviralRes,2005,68(3):109-115.
[71] Zhang ZC, Hu XJ, Yang Q. Generation of high affinity human single-chainantibody against PreS1of hepatitis B virus from immune phage-displayantibody library[J]. Hepatobiliary Pancreat Dis Int,2004,3(1):77-81.
[72] Tang H, McLachlan A. Transcriptional regulation of hepatitis B virus by nuclearhormone receptors is a critical determinant of viral tropism[J]. Proc Natl AcadSci U S A,2001,98(4):1841-1846.
[73] Sells MA, Zelent AZ, Shvartsman M, et al. Replicative intermediates of hepatitisB virus in HepG2cells that produce infectious virions[J]. J Virol,1988,62(8):2836-2844.
[74] Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2cells transfected with cloned hepatitis B virus DNA[J]. Proc Natl Acad Sci U SA,1987,84(4):1005-1009.
[1] Agrawal S, Iyer RP. Perspectives in antisense therapeutics[J]. Pharmacol Ther,1997,76(1-3):151-160.
[2] Kurreck J. Antisense technologies. Improvement through novel chemical modifications[J].Eur J Biochem,2003,270(8):1628-1644.
[3] Rayburn ER, Zhang R. Antisense, RNAi, and gene silencing strategies for therapy: missionpossible or impossible?[J]. Drug Discov Today,2008,13(11-12):513-521.
[4] Ricotta DN, Frishman W. Mipomersen: a safe and effective antisense therapy adjunct tostatins in patients with hypercholesterolemia[J]. Cardiol Rev,2012,20(2):90-95.
[5] Chi KN, Eisenhauer E, Fazli L, et al. A phase I pharmacokinetic and pharmacodynamicstudy of OGX-011, a2'-methoxyethyl antisense oligonucleotide to clusterin, in patients withlocalized prostate cancer[J]. J Natl Cancer Inst,2005,97(17):1287-1296.
[6] Burnett JC, Rossi JJ. RNA-based therapeutics: current progress and future prospects[J].Chem Biol,2012,19(1):60-71.
[7] Meyer O, Kirpotin D, Hong K, et al. Cationic liposomes coated with polyethylene glycol ascarriers for oligonucleotides[J]. J Biol Chem,1998,273(25):15621-15627.
[8] Sundaram S, Viriyayuthakorn S, Roth CM. Oligonucleotide structure influences theinteractions between cationic polymers and oligonucleotides[J]. Biomacromolecules,2005,6(6):2961-2968.
[9] Sato N, Kobayashi H, Saga T, et al. Tumor targeting and imaging of intraperitoneal tumorsby use of antisense oligo-DNA complexed with dendrimers and/or avidin in mice[J]. ClinCancer Res,2001,7(11):3606-3612.
[10] Pridgen EM, Langer R, Farokhzad OC. Biodegradable, polymeric nanoparticle deliverysystems for cancer therapy[J]. Nanomedicine (Lond),2007,2(5):669-680.
[11] Leamon CP, Cooper SR, Hardee GE. Folate-liposome-mediated antisenseoligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo[J]. BioconjugChem,2003,14(4):738-747.
[12] Abes S, Moulton H, Turner J, et al. Peptide-based delivery of nucleic acids: design,mechanism of uptake and applications to splice-correcting oligonucleotides[J]. BiochemSoc Trans,2007,35(Pt1):53-55.
[13] Margus H, Padari K, Pooga M. Cell-penetrating peptides as versatile vehicles foroligonucleotide delivery[J]. Mol Ther,2012,20(3):525-533.
[14] Lochmann D, Jauk E, Zimmer A. Drug delivery of oligonucleotides by peptides[J]. Eur JPharm Biopharm,2004,58(2):237-251.
[15] Fawell S, Seery J, Daikh Y, et al. Tat-mediated delivery of heterologous proteins intocells[J]. Proc Natl Acad Sci U S A,1994,91(2):664-668.
[16] Juliano RL, Ming X, Nakagawa O. Cellular uptake and intracellular trafficking of antisenseand siRNA oligonucleotides[J]. Bioconjug Chem,2012,23(2):147-157.
[17] Juliano RL. Peptide-oligonucleotide conjugates for the delivery of antisense and siRNA[J].Curr Opin Mol Ther,2005,7(2):132-136.
[18] Singh Y, Murat P, Defrancq E. Recent developments in oligonucleotide conjugation[J].Chem Soc Rev,2010,39(6):2054-2070.
[19] Lu K, Duan QP, Ma L, et al. Chemical strategies for the synthesis of peptide-oligonucleotideconjugates[J]. Bioconjug Chem,2010,21(2):187-202.
[20] Konate K, Crombez L, Deshayes S, et al. Insight into the cellular uptake mechanism of asecondary amphipathic cell-penetrating peptide for siRNA delivery[J]. Biochemistry,2010,49(16):3393-3402.
[21] Del Vecchio CA, Li G, Wong AJ. Targeting EGF receptor variant III: tumor-specific peptidevaccination for malignant gliomas[J]. Expert Rev Vaccines,2012,11(2):133-144.
[22] Bongartz JP, Aubertin AM, Milhaud PG, et al. Improved biological activity of antisenseoligonucleotides conjugated to a fusogenic peptide[J]. Nucleic Acids Res,1994,22(22):4681-4688.
[23] Allinquant B, Hantraye P, Mailleux P, et al. Downregulation of amyloid precursor proteininhibits neurite outgrowth in vitro[J]. J Cell Biol,1995,128(5):919-927.
[24] Pooga M, Soomets U, Hallbrink M, et al. Cell penetrating PNA constructs regulate galaninreceptor levels and modify pain transmission in vivo[J]. Nat Biotechnol,1998,16(9):857-861.
[25] Abes R, Moulton HM, Clair P, et al. Delivery of steric block morpholino oligomers by(R-X-R)4peptides: structure-activity studies[J]. Nucleic Acids Res,2008,36(20):6343-6354.
[26] Henke E, Perk J, Vider J, et al. Peptide-conjugated antisense oligonucleotides for targetedinhibition of a transcriptional regulator in vivo[J]. Nat Biotechnol,2008,26(1):91-100.
[27] Morris MC, Gros E, Aldrian-Herrada G, et al. A non-covalent peptide-based carrier for invivo delivery of DNA mimics[J]. Nucleic Acids Res,2007,35(7):e49.
[28] Meng S, Wei B, Xu R, et al. TAT peptides mediated small interfering RNA delivery toHuh-7cells and efficiently inhibited hepatitis C virus RNA replication[J]. Intervirology,2009,52(3):135-140.
[29] Chaubey B, Tripathi S, Pandey VN. Single acute-dose and repeat-doses toxicity ofanti-HIV-1PNA TAR-penetratin conjugate after intraperitoneal administration to mice[J].Oligonucleotides,2008,18(1):9-20.
[30] Bottcher-Friebertshauser E, Stein DA, Klenk HD, et al. Inhibition of influenza virusinfection in human airway cell cultures by an antisense peptide-conjugated morpholinooligomer targeting the hemagglutinin-activating protease TMPRSS2[J]. J Virol,2011,85(4):1554-1562.
[31] Lai SH, Stein DA, Guerrero-Plata A, et al. Inhibition of respiratory syncytial virus infectionswith morpholino oligomers in cell cultures and in mice[J]. Mol Ther,2008,16(6):1120-1128.
[32] Opriessnig T, Patel D, Wang R, et al. Inhibition of porcine reproductive and respiratorysyndrome virus infection in piglets by a peptide-conjugated morpholino oligomer[J].Antiviral Res,2011,91(1):36-42.
[33] Burrer R, Neuman BW, Ting JP, et al. Antiviral effects of antisense morpholino oligomers inmurine coronavirus infection models[J]. J Virol,2007,81(11):5637-5648.
[34] Deas TS, Bennett CJ, Jones SA, et al. In vitro resistance selection and in vivo efficacy ofmorpholino oligomers against West Nile virus[J]. Antimicrob Agents Chemother,2007,51(7):2470-2482.
[35] Wagner E. Polymers for siRNA delivery: inspired by viruses to be targeted, dynamic, andprecise[J]. Acc Chem Res,2012,45(7):1005-1013.
[36] Ogris M. Nucleic acid based therapeutics for tumor therapy[J]. Anticancer Agents MedChem,2006,6(6):563-570.
[37] Ogris M, Wagner E. To be targeted: is the magic bullet concept a viable option for syntheticnucleic acid therapeutics?[J]. Hum Gene Ther,2011,22(7):799-807.
[38] Kilk K, El-Andaloussi S, Jarver P, et al. Evaluation of transportan10in PEI mediatedplasmid delivery assay[J]. J Control Release,2005,103(2):511-523.
[39] Kleemann E, Dailey LA, Abdelhady HG, et al. Modified polyethylenimines as non-viralgene delivery systems for aerosol gene therapy: investigations of the complex structure andstability during air-jet and ultrasonic nebulization[J]. J Control Release,2004,100(3):437-450.
[40] Sirsi SR, Schray RC, Guan X, et al. Functionalized PEG-PEI copolymers complexed toexon-skipping oligonucleotides improve dystrophin expression in mdx mice[J]. Hum GeneTher,2008,19(8):795-806.
[41] Schiffelers RM, Ansari A, Xu J, et al. Cancer siRNA therapy by tumor selective deliverywith ligand-targeted sterically stabilized nanoparticle[J]. Nucleic Acids Res,2004,32(19):e149.
[42] Tietze N, Pelisek J, Philipp A, et al. Induction of apoptosis in murine neuroblastoma bysystemic delivery of transferrin-shielded siRNA polyplexes for downregulation of Ran[J].Oligonucleotides,2008,18(2):161-174.
[43] Dohmen C, Wagner E. Multifunctional CPP polymer system for tumor-targeted pDNA andsiRNA delivery[J]. Methods Mol Biol,2011,683:453-463.
[44] Scheule RK, St George JA, Bagley RG, et al. Basis of pulmonary toxicity associated withcationic lipid-mediated gene transfer to the mammalian lung[J]. Hum Gene Ther,1997,8(6):689-707.
[45] Filion MC, Phillips NC. Toxicity and immunomodulatory activity of liposomal vectorsformulated with cationic lipids toward immune effector cells[J]. Biochim Biophys Acta,1997,1329(2):345-356.
[46] Torchilin VP. Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers[J].Adv Drug Deliv Rev,2008,60(4-5):548-558.
[47] Hatakeyama H, Akita H, Harashima H. A multifunctional envelope type nano device(MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcomingthe PEG dilemma[J]. Adv Drug Deliv Rev,2011,63(3):152-160.
[48] Ambegia E, Ansell S, Cullis P, et al. Stabilized plasmid-lipid particles containingPEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective geneexpression[J]. Biochim Biophys Acta,2005,1669(2):155-163.
[49] Hayashi Y, Yamauchi J, Khalil IA, et al. Cell penetrating peptide-mediated systemic siRNAdelivery to the liver[J]. Int J Pharm,2011,419(1-2):308-313.
[50] Tagalakis AD, He L, Saraiva L, et al. Receptor-targeted liposome-peptide nanocomplexesfor siRNA delivery[J]. Biomaterials,2011,32(26):6302-6315.
[51] Nakamura Y, Kogure K, Yamada Y, et al. Significant and prolonged antisense effect of amultifunctional envelope-type nano device encapsulating antisense oligodeoxynucleotide[J].J Pharm Pharmacol,2006,58(4):431-437.
[52] Nakamura Y, Kogure K, Futaki S, et al. Octaarginine-modified multifunctionalenvelope-type nano device for siRNA[J]. J Control Release,2007,119(3):360-367.
[53] Akita H, Kogure K, Moriguchi R, et al. Reprint of: Nanoparticles for ex vivo siRNAdelivery to dendritic cells for cancer vaccines: Programmed endosomal escape anddissociation[J]. J Control Release,2011,149(1):58-64.
[54] Hatakeyama H, Akita H, Ito E, et al. Systemic delivery of siRNA to tumors using a lipidnanoparticle containing a tumor-specific cleavable PEG-lipid[J]. Biomaterials,2011,32(18):4306-4316.
[55] Valadi H, Ekstrom K, Bossios A, et al. Exosome-mediated transfer of mRNAs andmicroRNAs is a novel mechanism of genetic exchange between cells[J]. Nat Cell Biol,2007,9(6):654-659.
[56] Skog J, Wurdinger T, van Rijn S, et al. Glioblastoma microvesicles transport RNA andproteins that promote tumour growth and provide diagnostic biomarkers[J]. Nat Cell Biol,2008,10(12):1470-1476.
[57] Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, et al. Functional delivery of viralmiRNAs via exosomes[J]. Proc Natl Acad Sci U S A,2010,107(14):6328-6333.
[58] Montecalvo A, Larregina AT, Shufesky WJ, et al. Mechanism of transfer of functionalmicroRNAs between mouse dendritic cells via exosomes[J]. Blood,2012,119(3):756-766.
[59] Alvarez-Erviti L, Seow Y, Yin H, et al. Delivery of siRNA to the mouse brain by systemicinjection of targeted exosomes[J]. Nat Biotechnol,2011,29(4):341-345.
[60] Quah BJ, O'Neill HC. The immunogenicity of dendritic cell-derived exosomes[J]. BloodCells Mol Dis,2005,35(2):94-110.
[61] Lentz TL. Rabies virus binding to an acetylcholine receptor alpha-subunit peptide[J]. J MolRecognit,1990,3(2):82-88.
[62] Maier MA, Esau CC, Siwkowski AM, et al. Evaluation of basic amphipathic peptides forcellular delivery of antisense peptide nucleic acids[J]. J Med Chem,2006,49(8):2534-2542.
[63] Du L, Kayali R, Bertoni C, et al. Arginine-rich cell-penetrating peptide dramaticallyenhances AMO-mediated ATM aberrant splicing correction and enables delivery to brainand cerebellum[J]. Hum Mol Genet,2011,20(16):3151-3160.
[64] Jearawiriyapaisarn N, Moulton HM, Buckley B, et al. Sustained dystrophin expressioninduced by peptide-conjugated morpholino oligomers in the muscles of mdx mice[J]. MolTher,2008,16(9):1624-1629.
[65] Heemskerk H, de Winter C, van Kuik P, et al. Preclinical PK and PD studies on2'-O-methyl-phosphorothioate RNA antisense oligonucleotides in the mdx mouse model[J].Mol Ther,2010,18(6):1210-1217.
[66] Moschos SA, Jones SW, Perry MM, et al. Lung delivery studies using siRNA conjugated toTAT(48-60) and penetratin reveal peptide induced reduction in gene expression andinduction of innate immunity[J]. Bioconjug Chem,2007,18(5):1450-1459.
[67] Wu B, Moulton HM, Iversen PL, et al. Effective rescue of dystrophin improves cardiacfunction in dystrophin-deficient mice by a modified morpholino oligomer[J]. Proc NatlAcad Sci U S A,2008,105(39):14814-14819.
[68] Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restorationin patients with Duchenne muscular dystrophy after systemic phosphorodiamidatemorpholino oligomer treatment: an open-label, phase2, dose-escalation study[J]. Lancet,2011,378(9791):595-605.
[69] Sazani P, Ness KP, Weller DL, et al. Repeat-dose toxicology evaluation in cynomolgusmonkeys of AVI-4658, a phosphorodiamidate morpholino oligomer (PMO) drug for thetreatment of duchenne muscular dystrophy[J]. Int J Toxicol,2011,30(3):313-321.
[70] Sazani P, Ness KP, Weller DL, et al. Chemical and mechanistic toxicology evaluation ofexon skipping phosphorodiamidate morpholino oligomers in mdx mice[J]. Int J Toxicol,2011,30(3):322-333.
[71] Amantana A, Moulton HM, Cate ML, et al. Pharmacokinetics, biodistribution, stability andtoxicity of a cell-penetrating peptide-morpholino oligomer conjugate[J]. Bioconjug Chem,2007,18(4):1325-1331.
[72] Moulton HM, Moulton JD. Morpholinos and their peptide conjugates: therapeutic promiseand challenge for Duchenne muscular dystrophy[J]. Biochim Biophys Acta,2010,1798(12):2296-2303.
[73] Wancewicz EV, Maier MA, Siwkowski AM, et al. Peptide nucleic acids conjugated to shortbasic peptides show improved pharmacokinetics and antisense activity in adipose tissue[J]. JMed Chem,2010,53(10):3919-3926.