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A delivery system targeting haemagglutinin of influenza virus A to facilitate antisense-based anti-H1N1 therapy Xiaoran Ding, Jing Yang, Dandan Lu, Qingjun Li, Zhaoyan Zhang, Zhe Zhou, and Shengqi Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00124 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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Bioconjugate Chemistry

A delivery system targeting haemagglutinin of influenza virus A to facilitate antisense-based anti-H1N1 therapy

XiaoRan Ding, Jing Yang, DanDan Lu, QingJun Li, ZhaoYan Zhang, Zhe Zhou*, ShengQi Wang*

Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850, P.R.China.

* To whom correspondence should be addressed: Zhe Zhou, Shengqi Wang Lab of Biotechnology, Beijing Institute of Radiation Medicine, No. 27, Tai Ping Road, Beijing 100850, P. R. China. Phone/Fax: +86-10-66932211 E-mail: [email protected], [email protected]

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ABSTRACT: Antisense oligonucleotides (ODNs) are therapeutic molecules that hybridize to complementary target mRNA sequences. To further overcome the poor cellular uptake of ODNs, we proposed a novel strategy to deliver ODNs by conjugating the anti-influenza A virus (IAV) ODN with a peptide showing high affinity to the haemagglutinin (HA) on the surface of IAV particles or the IAV infected host cells. The HA-specific binding peptides were selected by phage display and the individual binding clones are characterized by DNA sequencing, and the selected phage was further assayed by ELISA. The final selected HA-binding peptide, SHGRITFAYFAN, was conjugated to an anti-IAV ODN. The delivery efficiency and the anti-IAV effects of the conjugated molecule were evaluated in a cell culture and a mouse infection model. The conjugated molecule was successfully delivered into IAV-infected host cells more efficiently than the anti-IAV ODN in vitro and in vivo. Furthermore, the conjugated molecule protected 80% of the mice from lethal challenge and inhibited the plaque count by 75%, compared to the un-conjugated molecule (60% and 40%). These findings demonstrate that the delivery of antisense oligodeoxynucleotides to infected tissues by a virus-binding peptide-mediated system is a potential therapeutic strategy against IAV.

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INTRODUCTION Antisense oligonucleotides (ODNs) are a class of therapeutic molecules that can hybridize to their complementary target mRNA sequences in a selective and sequence-specific manner, thereby inhibiting protein expression1, 2. The severe enzymatic degradation by cellular nucleases and poor cellular uptake are the two main obstacles to the practical application of ODNs as a therapeutic agent. In the past decade, the enzymatic susceptibility of ODN has been partially addressed by chemical modification, e.g. phosphorothioate, and by employing new ODN analogues such as PMO, LNA, and peptide nucleic acids (PNAs)3-6. The first successful antisense ODN, Vitravene (ISIS Pharmaceuticals Inc.), was approved by the U.S. FDA for retinitis induced by cytomegalovirus in 19987, 8. In 2013, the second antisense drug Kynamro, an oligonucleotide inhibitor for homozygous familial hypercholesterolemia (HoFH), was approved to treat inherited cholesterol disorder9-11. Most recently, the FDA allowed limited use of an ODN anti-Ebola drug in 201412, 13. Although, the stability and specificity of antisense ODN has been improved by various chemical modifications, the issue of poor and unspecific cellular uptake exists and numerous delivery systems have been proposed, including cationic liposomes, cationic lipids, cationic polymers, complexation with peptide-containing antibodies and nucleic acid binding domains14-17. Influenza virus replication is initiated by haemagglutinin (HA), which mediates entry into the target cell through virion binding to sialic acid–containing receptors at the cell surface. The virus-receptor complex is taken into cells by endocytosis. Following entry, the acidic environment of the late endosomal vesicles containing the virus particles triggers a conformational change of HA which mediates the fusion of the viral envelope with the endosomal membrane. Then, the M2 3



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protein pumps protons (H+) flow from the endosome into the virion resulting in the dissociation of M1 from the viral ribonucleoprotein complexes (vRNPs), which triggers the release of viral genomic RNA into the cytoplasm. After that, vRNA is transported into the cell nucleus where vRNA replication occurs18-20. As HA plays an important role in the entry of the influenza A virus (IAV) into host cells, we proposed a novel strategy to deliver ODN in vivo by conjugating the ODN with a peptide that has a high affinity to HA. We obtained a peptide showing high affinity to HA by performing an in vitro phage display library screening experiment and then studied the uptake of the ODN conjugated with the HA-binding peptide (HABP) in cultured cells and mice infected with IAV. We found that effective delivery of the HABP conjugated with the ODN inhibited the replication of IAV both in vitro and in vivo. The ODN delivery strategy used in this study presents a novel approach to delivering anti-viral ODN into cells.

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RESULTS Confirmation of in vitro binding by viral ELISA and cellular co-staining. After the fourth round of panning, 60 phage clones were randomly selected and sequenced. Twenty-three different phage clones targeting HA and 16 different phage clones targeting NA (neuraminidase) were obtained (S-Table 2). Multiple sequence alignment analyses did not reveal strong homology among the different peptide sequences. An ELISA was performed to determine the affinity of the 39 phage clones for HA or NA and exclude false-positives and clones that bound with equal affinity to HA or NA. The results showed that 13 phages significantly bound to HA and 4 phages bound to NA (Fig. 1a). Among the 17 positive phage clones, H17 (SHGRITFAYFAN) showing the most effective binding. Therefore, the H17 phage clone’s peptide, which was renamed as HABP, needed further investigation. To determine the ability of HABP to enter the cells, we incubated A549 cells with binding peptides together with IAV. A549 cells were treated with PBS (mock), cy3-labelled IAV, FITC-labelled HABP, cy3-labelled IAV plus FITC-labelled HABP and imaged by a confocal microscope at 6 h after the treatment (Fig. 1b). The signal of the FITC-labelled peptide overlapped with the signal of cy3-labelled IAV in the cy3-labelled IAV plus FITC-labelled peptide group, while there was no significant signal overlap in the FITC-labelled peptide group and the mock group. Synthesis and identification of the HABP-ligated ODN. The anti-IAV ODN sequence (flutide, FT), which specific targets the 5'-terminal conserved region of influenza A virus RNA segments21, was modified it with a thiol C6 S-S linker at the 5′-end. The HABP was coupled with an N-succinimidyl 6-maleimidohexanoate that could be reacted with 5′-thiol oligonucleotide via the Michael addition reaction as shown in Figure (Fig. 2a). Then, the ODN was conjuated to the HABP. We After purification by reverse-phase HPLC, the identity of the final products were confirmed by MALDI-TOF mass spectroscopy (FT-HABP: calculated mass =5848.3 (M+H)+ and mass found =5849.18 (M+H)+) (Fig. 2b). Conjugation was performed with a hetero-bifunctional linker (EMCS). The product was verified by MALDI-TOF mass spectroscopy and digestion with proteinase K (Fig. 2c). FT-HABP showed remarkable stability in 10% or 50% serum relative to 5



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unmodified antisense ODNs, similar to gap-mer alone (Fig. 2d, supplemental fig. 1). HABP enhances the delivery of anti-IAV ODNs in lung cells infected with IAV in vivo. To evaluate whether the HABP enhances the lung deposition of ODNs in vivo, mice were intranasally administered with 14.4 mg/kg FAM-labelled HABP-ligated ODN or molar matched control chemicals. As shown in Fig. 3a, 2.4-fold greater fluorescence was observed in HABP-ligated ODNs than in either ODNs or HABP in infected lungs at 4 h after the infection. Furthermore, the expression of the NP protein of the IAV in the lungs of infected mice was detected by immuno-staining. Fluorescence microscopy was performed using the lungs of IAV-infected mice that were administered a fluorescence-labelled HABP-ligated ODNs. Compared to the control chemicals and the uninfected mice, HABP visibly increased ODNs in the lungs of the infected mice (Fig. 3b). HABP enhances the antiviral effects of anti-influenza ODNs in vivo. The results from the plaque assay supported the hypothesis that HABP enhances the antiviral effects of anti-IAV ODNs (Fig. 4a). A significant inhibitory effect on viral replication was observed when cells were treated with ODN or HABP-ligated ODNs. The higher (35%) inhibitory effect was observed when the HABP-ligated ODNs were used. An A/FM/1/47 (H1N1)-infected murine model was used to test the anti-IAV activity of HABP-ligated ODNs in vivo. The results showed that HABP-ligated ODNs (14.4 mg/kg/d, intranasal administration) could protect 80% of the mice from lethal challenge with the mouse-adapted variant of IAV. Six out of ten mice survived when ODNs were intranasally administered. Meanwhile, zanamivir at a dosage of 10 mg/kg/d prevented 70% lethality in the infected mice (Fig. 4b). Observation of average weight changes in each group showed that HABP-ligated ODNs could reduce infected mice weight loss in a manner similar to that of

zanamivir (Fig. 4b). The copies of the IAV vRNA were measured by detecting the viral M1 gene expression level in the lung tissue by using real-time PCR. The quantity of M1 vRNA was analysed at 6 days after infection. As shown in Fig. 4c, the level of M1 vRNA significantly reduced in the lungs of the mice treated with 0.58, 2.88 or 14.4 mk/kg HABP-ligated ODNs in a dose-dependent manner. 6


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DISCUSSION In the present study, we designed virus-binding peptide-conjugated ODNs to interfere with the replication of the vRNA of IAV. We found that the delivery of the ODNs can be improved by using the HABP. Furthermore, the HABP effectively inhibited virus propagation both in vitro and in vivo, indicating that the antisense ODN could be successfully delivered in the virus-infected cells by binding it to the HA peptides on the surface of the IAV, thereby effectively inhibiting the replication of the virus in the plasma of the cells. A number of studies have attempted to develop cell-penetrating peptides (CPPs) or proteins as delivery vehicles for antisense ODNs. CPPs are short basic amino acid-rich peptides that can not only enter cells themselves but also facilitate the transport of molecular cargos across the plasma membrane. The first generation of CPPs contained prototypical CPPs such as TAT and ANT, which were derived from natural transcription factors. In a pioneering study, the phosphorothioate oligonucleotide component of a conjugate was found to be complementary to a site flanking the AUG codon for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumour cells. Two types of peptide-antisense oligonucleotide conjugates caused substantial inhibition of the cell surface expression of P-glycoprotein at concentrations < 1 µM, demonstrating the successful application of this strategy23. The next generation of CPPs were designed with extra characters through ligation with other peptides. For example, MPG derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen forms stable non-covalent complexes with nucleic acids and improves antisense ODN delivery24. Furthermore, many peptides and proteins have been used to deliver antisense ODN by targeting the receptors expressed in tumours, for example, integrins, bombesin receptors, transferrin receptors25 and EGFR26. Antibodies have also been used for ODN delivery with success. Antibodies targeting membrane markers of tumours have been used to target siRNAs to the tumours, which led to successful outcomes of RNAi after systemic administration27. Although all these three approaches have demonstrated functional delivery of ODNs in experimental models, or even clinical trials, specific and effective exposure of the conjugated ODNs remains to be addressed. Development of tissue or cell type targeted delivery systems will improve uptake and thereafter the effectiveness of ODNs, which will lead to broader distribution 7



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and wider applications of therapeutic ODNs. We designed an IAV HABP as a delivery molecule to interfere with the replication of IAV based on its life cycle. The IAV attaches to sialic acid–containing receptors at the host cell surface. Then, the virus-receptor complex is taken into cells by endocytosis. When the endosomal vesicles containing the virus particles move towards the cell nucleus, the pH in the endosomal vesicles drops. When the pH reaches 5.0, the viral HA protein undergoes a conformational rearrangement. This change exposes a fusion peptide on the HA, a short, hydrophobic sequence that inserts into the endosomal membrane and causes it to fuse with the viral envelope. When this occurs, the viral RNAs are released into the cytoplasm, and then, transported into the cell nucleus where viral RNA replication occurs. Firstly, we screened HABPs using a phage display library method. Then, we evaluated the binding activity of these peptides on HA. An ODN sequence, which is known to bind to the viral sequence of the IAV21, was conjugated to the selected HABP. The conjugated molecule was successfully delivered into host cells together with the IAV. Furthermore, another ODN molecule, which showed anti-IAV effects both in vivo and in vitro by targeting the host PDCD5 mRNA28, was also conjugated to the selected HABP. This conjugate also showed a significant inhibitory effect on IAV replication both in vitro and in vivo (supplemental fig. 2-3). These evidences indicate that the delivery of ODN by an HABP was successfully achieved in this study. Unlike the nueraminidase inhibitor such as zanamirvir, the conjugate we designed sequence specifically target the conserve region of the vRNA and lower un-target effect may be predicted. The virus-binding peptide was introduced to this new type conjugat, which can enter the target cell and inbit the replication of the virus more efficiently as compared to the traditional ODNs. The delivery of antisense ODNs to the lung by a virus-binding peptide system offers the prospect of obtaining increased intracellular concentrations in association with more selective targeting of this strategy. It may prove to be a potential therapeutic strategy for use against IAV or viruses with a similar life cycle. EXPERIMENTAL SECTION Purification of recombinant HA and NA proteins. The DNA was amplified from the influenza A A/california/05/2009(H1N1) which provide by the Dr, Wang Yue from the CDC of China by the 8


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polymerase reaction (PCR) using the primer pairs (Supplemental Table 1). Subsequently, the gene NA and HA was cloned into the pGEX-6p-1 to construct the recombinant plasmid pGEX-6p-NA and pGEX-6p-HA at EcoRI and XhoRI sites. The cloned plasmid was transformed into E. coli strain BL21 and induced using 1mM IPTG at 37℃.The fraction containing the rHA and rNA were isolated and purified. The purified rHA and rNA could be confirmed by anti-HA antibody, anti-NA antibody and anti-GST antibody. Selection of HA and NA-specific binding peptides by phage display. Ph.D.-12 phage display peptide library was purchased from New England BioLabs (Ipswich, MA), and the panning procedures were performed as described in the instruction manual. The first round of panning was carried out by incubating 1 × 1011 plaque-forming units (pfu) of phage library with the coated plate at room temperature for 2 h with gentle shaking, washing away the unbound phage for five times with PBS. Bound phage was eluted using elution buffer (0.2 M glycine–HCl, pH 2.2, 1 mg/ml BSA) (Sigma-Aldrich) at room temperature for 10 min and then neutralized with 1 M NaCl (pH 9.1) (Sigma-Aldrich). Eluted phage were titered by adding 10 µl of eluate to 200 µl of E. coli ER2738 and placed in melted LB top agar (7 g agarose/100 ml LB agar medium) on IPTG/X-Gal (Fermentas, Glen Burnie, MD) LB agar plates. The residual eluate was then amplified and taken through two additional binding/amplification cycles, at a fixed phage input of 2 × 1011 pfu. After 4 rounds, individual binding clones are characterized by DNA sequencing, and the binding abilities of selected phages are assayed by ELISA. Peptide-oligonucleotide

conjugate

synthesis.

The

anti-IAV

ODN

sequence,

flutide

(5′-CCTTGTTTCTACT-3′), was derived from a previous work21. The phosphorothioate oligonucleotide prop5 (5’-CCCTGTGCTTTGCTTCCTGT-3’) was synthesized using an ABI8909 nucleic acid synthesis system (Applied Biosystems, Foster City, CA, USA). To prepare peptide conjugates, a thiol C6 S-S linker (Glen Research, Sterling, VA, USA) was introduced at the 5’-end of the oligonucleotides. After normal deprotection, the disulfide can be cleaved at 55℃in 2 hours with 100 mM DTT in 0.08M BBS buffer. The reaction mixture was desalted through Sephadex G25 (GE Healthcare, Uppsala, Sweden), and directly used for the conjugation reaction. The mismatched sequence with the same modified process was used as a control oligonucleotide. For uptake and homing studies, oligonucleotides were obtained from the supplier with a 3’-end FAM 9



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or Rhodamine red label (Glen Research, Sterling, VA, USA). The peptide HABP (SHGRITFAYFAN) was reacted with maleimide NHS ester in BBS buffer at room temperature. Then, maleimide-HABP was isolated and analysed by reverse-phase HPLC on Alliance 2695 separation module with a 996 photodiode array detector (Waters, Massachusetts, USA) and confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI–TOF) mass spectroscopy. Finally, thiol oligonucleotides were reacted with the maleimide-HABP in a reaction buffer (final salt concentration adjusted to 200mM NaCl, 30% DMF). The reaction mixture was vortexed and allowed to stand for 30 min at room temperature. Then, the supernate was purified by reverse-phase HPLC using an XBridgeTM OST C18 column (4.6 × 50 mm, 2.5 µm particle size; Waters, Massachusetts, USA). The purified conjugates were confirmed by MALDI–TOF. Cells, virus, and in vitro antiviral assays. A549 and MDCK cells were purchased from the American Type Cell Collection (ATCC, Manassas, USA) and were cultured in 1640 medium supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin, and 100 lg/mL streptomycin under standard conditions in a humidified atmosphere containing 5% CO2. Influenza A/FM/1/86 (H1N1) virus was grown in the allantoic cavity of 10-d-old embryonated chicken eggs (Specific Pathogen Free, SCXK-BJ-2009-0003, Merial Vital Laboratory Animal Technology, Beijing, China) for 48 h at 35 °C. The allantoic fluid was cleared by centrifugation at 6,000 g for 15 min and stored at 80 °C until use. The experiments were reviewed and approved by the Animal Ethics Committee of the Beijing Institute of Radiation Medicine in accordance with the regulations of Beijing Administration Office of Laboratory Animal. The antiviral efficiency of HABP in cultured A549 cells was determined based on the inhibition of virus-induced cytopathic effects (CPEs) and plaque assay results. In order to avoid the quick occurrence of CPEs while achieving high infectivity, the H1N1 virus was inoculated at a multiplicity of infection (MOI) of 1.38 (75% tissue culture infective dose) in 1640 medium supplemented with 1 µg/ml trypsin in the absence of FBS

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. After inoculation (1h), the cells were washed twice in phosphate-buffered

saline (PBS) to remove unabsorbed viruses, and fresh conditional media were added to each well with varying concentrations of chemicals. The cells were then incubated for another 48 h. Cell viability was tested using the CCK-8 assay (Dōjindo Laboratories, Japan). Each experiment was performed in triplicate and repeated three times. 10


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Immunoblotting and real-time RT-PCR (RT-PCR). Cells (6 × 104 cells/well) in 48 wells were recovered with trypsin/EDTA; whole cell lysates were prepared in RIPA buffer for 30 min on ice and clarified by centrifugation, and proteins were resolved using 10% SDS-PAGE. Immunoblot analyses were performed on PVDF membranes blocked in 4% skim milk and PBS-T (0.05% PBS, Tween20) for 1 h at RT, and reacted with anti-IV M1 (Abcam) and anti-GAPDH (Santa Cruz) antibodies overnight at 4°C. They were then washed and exposed to HRP-conjugated anti-IgG (Santa cruz) antibodies and detected by ECL (Pierce). For the in vitro/vivo tests, GAPDH mRNA TaqMan RT-PCR (Applied Biosystems) was performed. cDNA was synthesized using Oligo-dT and GAPDH mRNA expression was detected using Taq-Man probe on 7500 Real time PCR system (Applied Biosystems). Total RNAs from cells or lung tissues at the indicated time points after transfection were isolated and M1 vRNA expression levels were detected using one-step real-time RT-PCR detection kit (SYBR green) according to the method described by Ma et al.22. Immunohistochemistry. Paraffin-embedded lung sections were treated with 3% hydrogen peroxide and blocked with 5% bovine serum albumin. The sections were then incubated sequentially with anti-IAV nucleoprotein antibodies (Dako) and a Texas Red-labelled secondary antibody. Cells were fixed with 4% paraformaldehyde for 30 min at RT, permeabilized in 0.1% Triton X100-PBS for 15 min at RT, blocked in 3% skim milk-PBS for 30 min at RT, and then reacted with Cy3 (GE)-labelled IAV in 0.1% BSA-PBS overnight at 4°C. DNA in cells and tissues was counter-stained with DAPI (Molecular Probes). Cells and tissues were analyzed using a confocal microscope (FV1000, Olympus, Japan). Intranasal administration of antisense ODN-conjugates for in vivo delivery. Mice were randomized into groups (n = 10) and inoculated intranasally and intranasally administered with 20 µl or 20 µl of control peptide (CP, AEYLANPESSEA) or control oligo or PBS:MEM (1:1) (mock) control. Density of fluorescence was monitored by IVIS imaging (NightOWL LB983 with Indigo Software, Berthold Technologies) 4 hours after the administration. Plaque assay. Plaque assays were performed in MDCK cells prepared in 24-well plates. Real infectivity, i.e., infectivity of virions with cleaved HA, was measured by a standard plaque method. 11



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Briefly, infected cells were covered with 1 ml of 1% agarose in DMEM containing 0.3 µg/ml acetylated trypsin (Sigma) in the case of the standard plaque assay and no trypsin in the case of the modified plaque assay. According to the modified assay, after 24 h of incubation at 37 °C, the cells were overlaid additionally with 0.7% agarose (0.5 ml per well) containing 0.8 µg/ml acetylated trypsin. In both plaque assays, cells were stained 3 days after infection with haematoxylin-eosin, and plaques were counted. Statistical analysis. Data are expressed as mean ± SD as indicated and compared by two-tailed t tests or ANOVA. Statistical significance was set at P < 0.05.

ACKNOWLEDGMENTS We thank Ms. XinXiu Deng and Ms. Yuan Gao for their excellent technical supports in ODNs purification and analysis.

ASSOCIATED CONTENT Supporting Information Experimental methods and additional figures are presented in the supporting information.

AUTHOR CONTRIBUTIONS DXR and ZZ performed the experiments, analyzed data; WSQ designed the experiments; ZZ drafted the manuscript; YJ, LDD and LQJ performed the experiments; WSQ designed and supervised the study. REFERENCES (1) Braasch D. A., Corey D. R. (2002) Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry 41, 4503-4510. (2) Stephenson M. L., Zamecnik P. C. (1978) Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A 75, 285-288. (3) Ackermann D., Famulok M. (2013) Pseudo-complementary PNA actuators as reversible switches in dynamic DNA nanotechnology. Nucleic Acids Res 41, 4729-4739. (4) Lehto T., Castillo Alvarez A., Gauck S., Gait M. J., Coursindel T., Wood M. J., Lebleu B., Boisguerin P. (2014) Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells. Nucleic Acids Res 42, 3207-3217. (5) Shabanpoor F., McClorey G., Saleh A. F., Jarver P., Wood M. J., Gait M. J. (2015) Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy. Nucleic Acids Res 43, 29-39. (6) Sato Y., Sato T., Nishizawa S., Teramae N. (2014) The effect of LNA nucleobases as 12


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Figure 1. Identification of the binding selectivity of the phage clones. A. Phage clones binding to HA, NA, IAV or wild-type phages were detected using an HRP-conjugated anti-M13 phage antibody. PBS and URps (unrelated phage, an amplified phage randomly selected from the original phage peptide library) were used as negative controls. Triplicate determinations were performed at each data point, and the average OD450 nm values of the two types of cells are shown. *, n=3, p < 0.05 (vs. URps). B. Intracellular distribution of FITC-labelled HABP (a) and Cy3 (b)-labelled IAV after co-incubation for 6 h with A549 cells. HABP and IAV can be co-stained in the cells (c and d). Bar: 20 µM.

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Figure 2. Characterization of FT-HABP. A. Schematic description of the synthesis of FT-HABP. B. Mass spectrum of the FT-HABP conjugate. C. Agarose gel analysis of proteinase K digests of FT-HABP (n=3). D. Agarose gel analysis of FT-HABP after incubation in 10% FBS at 37 °C for up to 48 h (n=3).

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Figure 3. HABP enhanced the delivery of the ODNs in the IAV-infected cultured cells and mice. A. BALB/c mice from seven groups (n=3) were intranasally administered with FITC-labelled FT-HABP (FT-HABP) and other control chemicals. Then, the mice were inoculated with IAV. Individual organs were isolated from animals 4 h after IAV inoculation. The FITC signal was evaluated quantitatively using an in vivo imaging system. Data represent the means and standard deviations, n = 3, *, P < 0.05, versus control groups. B. In vivo FT- HABP bio-distribution. FITC-labelled FT-HABP (FT-HABP), FITC-labelled FT (FT) and FITC-labelled FT-control peptide (CP, FT-CP) were intranasally administered to mice infected (V) or uninfected (C) with 17



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IAV. Arrow, co-staining of the IAV and FT-HABP. Bar: 20 µM.

Figure 4. Effect of FT-HABP on IAV replication in vitro and in vivo. A. IAV-infected A549 cells were incubated with FT-HABP for 12 h. Then, the supernatant was collected for plaque assay in the MDCK cells (n=3). B. Effects of FT-HABP on survival rate and body weight loss in infected mice. BALB/c mice were pretreated with normal saline, FT-HABP (14.4 mg/kg/day), and molar matchable FT, FT-random, FT-CP, and HABP dissolved in normal saline at 30 min and 24 h before infection. After drug pretreatment, each mouse was infected intranasally with 400 pfu of A/FM/1/47 (H1N1) in sterile normal saline or normal saline only (uninfected group) at a total volume of 20 µL. Mice (n = 10 in each group) were monitored for 14 days starting from virus infection. (b, left panel) Effects of FT-HABP on the survival of the infected mice; (b, right panel) effects of FT-HABP on the body weight loss of the infected mice. C. Expression of viral M1 vRNA was inhibited by FT-HABP in mock-infected and IAV-infected mice lung tissue. At 6 days post inoculation, mice lung tissue samples were prepared for detection of M1 vRNA by real-time 18


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RT-PCR. GAPDH served as the internal control. Data represent the means and standard deviations, n = 3, *, P < 0.05, versus FT-HABP group.

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Delivery System Targeting Hemagglutinin of ... - ACS Publications

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