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
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