Study On The Delivery System Of Antisense Oligonucleotides Using Virus As A Carrier

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.