Bioconjugate Chem. 1996, 7, 3−6
3
COMMUNICATIONS Block Polycationic Oligonucleotide Derivative: Synthesis and Inhibition of Herpes Virus Reproduction Sergey V. Vinogradov,† Yulia G. Suzdaltseva, and Alexander V. Kabanov*,‡ Moscow Institute of Biotechnology, Inc., and Department of Polymer Sciences, Faculty of Chemistry, Moscow State University, Vorobievy Gory, Moscow 119899, Russia. Received August 7, 1995X
The block polycationic oligonucleotide (oligo) consisting of a phosphodiester 12-mer linked to the polycation chain at the 3′-end and cholesteryl group at the 5′-end was synthesized. The polycation chain was grown on the solid support using the monomer, H-phosphonate of 1-O-(4,4′-dimethoxytrityl)1,3-butanediol. Amino groups were introduced in the polymer backbone using 1,4-diaminobutane, and then the oligo chain was formed at the free end of the polymer. The last stage of the synthesis was the attachment of the cholesteryl group to the 5′-end of the oligo prior to cleavage and deprotection of the copolymer. The nucleotide sequence of this copolymer, CGTTCCTCCTGC, was complementary to the splicing site of immediate early (IE) mRNA 4 and 5 of herpes simplex virus type 1 (HSV-1). The stability of the duplexes formed between the copolymer and the complementary 12-mer was similar to that of unmodified oligo. The stability of the block polycationic oligo against phosphodiesterase digestion was significantly increased compared to that of the unmodified oligo. The block polycationic oligo inhibited the reproduction of HSV-1 in Vero cells; however, the effect was significantly less than the effect of 12-mer oligo modified with cholesterol at the 5′-end. The decreased antiviral activity of the copolymer is explained by the polycation-induced stimulation of the virus infection.
Antisense oligonucleotides (oligos) are selective inhibitors of gene expression and viral reproduction (1-3). Two significant obstacles currently limiting the therapeutical application of oligos are poor uptake into cells and rapid degradation by nucleases. To resolve these problems, a variety of structural modifications to oligo have recently been advanced. Specifically, we and other investigators have demonstrated that the addition of hydrophobic substituents to the oligo chain can significantly increase their antisense activity (4-6). Reduction of a net negative charge of oligo backbone also enhances oligo uptake and stability, as has been shown for methyl phosphonates (7) and other derivatives (2, 3), including novel cationic backbone substituents, aminoethyl phosphonates (8). One way to reduce the net negative charge of the DNA molecule is to link it with a polycation. Leonetti et al., (9) have described oligo conjugated with poly(L-lysine) that exhibited greater specific activity on cells compared to unmodified oligos. Other authors are designing various conjugated structures incorporating cationic peptide and oligo blocks (10). The electrostatic binding of polycations to negatively charged polynucleotide molecules has also been shown to increase the (1) stability against nuclease digestion, (2) uptake into the cells, and (3) biological activity of these polynucleotides (cell transfec* Corresponding author. † Present address: Centre National de la Recherche ´ Scientifique, Centre de Biophysique Moleculaire, rue Charles Sadron, 45071, Orleans, Cedex 2, France. ‡ Present address: Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6025. Telephone: (402) 559-9364. Fax (402) 559-5060. X Abstract published in Advance ACS Abstracts, December 15, 1995.
1043-1802/96/2907-0003$12.00/0
tion and antisense inhibition of gene expression) (11). It has been suggested (12, 13) that a polycation molecule is able to electrostatically bind with both the negatively charged deoxyribophosphate backbone of the oligo and the acidic phospholipids of the plasma membrane, thus increasing the stability of the nucleic acid and its uptake into target cells. This paper describes the synthesis, stability, and specific activity of a block polycationic derivative of oligo, consisting of a 12-mer phosphodiester oligo linked to a cholesteryl group at the 5′-end and a polycationic chain at the 3′-end. The oligo sequence, CGTTCCTCCTGC, was complementary to the splicing site of immediate early (IE) mRNA 4 and 5 of herpes simplex virus type 1 (HSV-1). This target site was first identified by Kulka et al. (14, 15). The sequence-specific inhibition of HSV-1 reproduction with the hydrophobically modified oligo CGTTCCTCCTGC has previously been reported (16, 17). This paper evaluates the effects of the polycationic derivative of this oligo on reproduction of HSV-1 in Vero cells. The block polycationic oligo (Figure 1) was produced by automated DNA synthesis. During the first stage of this synthesis (Scheme 1), the polycation chains were grown on a solid support modified with 1-O-DMT-1,3butanediol1 using a 380B-02 DNA synthesizer (Applied Biosystems, U.S.A.) at a 10 µmol scale. The monomer 2, H-phosphonate of 1-O-DMT-1,3-butanediol, was prepared as previously reported (18). Briefly, 3.6 g (40 mmol) of butane-1,3-diol (Merck) dissolved in 50 mL of anhydrous pyridine (Aldrich) was reacted with 6.8 g (20 1 Abbreviations: Chol, cholesteryl group; DMT, 4,4′-dimethoxytrityl group; EDTA, ethylenediaminetetraacetic acid; PAGE, polyacrylamide gel electrophoresis; PFU, patch-forming unit; 2,4,6-TNBS, 2,4,6-trinitrobenzenesulfonic acid.
© 1996 American Chemical Society
4 Bioconjugate Chem., Vol. 7, No. 1, 1996
Vinogradov et al.
Figure 1. Block polycationic oligo structure. Scheme 1. Growth of the Polycation Chain
Scheme 2. Propagation of the Oligo Chain
Scheme 3. Introduction of the Cholesteryl Group
mmol) of 4,4′-dimethoxytrityl chloride (Sigma) for 1.5 h at 20 °C. The completion of the reaction was determined using thin layer chromatography on the silica gel plates developed with chloroform/methanol (95:5). The Rf of the product was 0.6. The reaction mixture was added to 200 mL of an 8% aqueous solution of the sodium bicarbonate, the product was extracted with chloroform, and chloroform was evaporated. The resulting oily intermediate (4.7 g, 12 mmol) was dissolved in 30 mL of anhydrous 1,4-dioxane containing 2.32 g (18 mmol) of diisopropylethylamine (Aldrich), and the reaction system was then supplemented with 3.64 g (18 mmol) of salicyl chlorophosphite (Aldrich) which was added using a syringe in an argon atmosphere. The reaction mixture was incubated for 1 h at 20 °C, and formation of the product was monitored using thin layer chromatography as described above (Rf of the product was 0.05). After the completion of the reaction, 10 mL of water was added to the reaction mixture for 30 min, and then the solvents evaporated. The product was dissolved in 100 mL of chloroform, and the solution obtained was extracted with 100 mL of 8% aqueous solution of the sodium bicarbonate, then 100 mL of 0.2 M triethylammonium acetate solution, and finally 100 mL of water. The chloroform was evaporated, and the oily residue containing the monomer 2 was purified on a silica gel chromatographic column, using a stepwise gradient of (1) chloroform, (2) 3% methanol in chloroform, and (3) 6% methanol in chloroform. The yield of the 2 was 4.1 g (7.3 mmol, 63%). During the automated synthesis, a 6-fold excess of 2 (with respect to the 1,3butanediol linked to the support) was used as a building
block at each condensation cycle, and a 3-fold excess of pivaloyl chloride in pyridine/acetonitrile (1:1) was used as an activating agent. The yield of the polymer 3 after nine condensation cycles was about 96%, as determined by DMT cation release test (498 nm; extinction coefficient, 14 300) (19). Polymer 3 was then oxidized using the Atterton-Todd reaction (19) for 30 min at room temperature by 10-fold excess (with respect to the total concentration of phosphonate groups) of N-(trifluoroacetyl)-1,4-diaminobutane in chloroform/pyridine (1:1). During the second stage of the synthesis, standard phosphoramidite chemistry was used to form the oligo chain at the free end of the polymer attached to the solid support (Scheme 2). At the third stage of the synthesis (Scheme 3), the cholesteryl group was introduced at the 5′-end of the oligo using the N-[6-[[(cholesteryloxy)carbonyl]amino]hexyl]-O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite 6 as previously described (16). The copolymeric product was cleaved from the support and deprotected by treatment with concentrated aqueous ammonia for 17 h at 50 °C. The copolymer was purified by reverse phase high-performance liquid chromatography (HPLC) on a Silasorb C16 column (9 × 240 mm, 10 µm, NPO “Chromatographia”, Moscow, Russia) using a linear gradient of acetonitrile (5 to 40%) in 20 mM triethylammonium acetate buffer (pH 7.5). The presence of free amino groups in the copolymer was determined using a ninhydrine test. The net yield of the desired copolymer was 27%; the remaining amount accounted for shorter oligomers and oligomers that did not contain amino groups. The copolymer obtained revealed one major band in PAGE (20% gel, 7 M urea, tris-borate buffer, pH 8.3) and migrated substantially more slowly than a 12-mer oligo, modified at the 5′-end with cholesteryl groups as previously described (16). Titration with 2,4,6-TNBS (20) indicated that the concentration of the free amino groups was 11.2 µmol per µmol of the oligo. In this experiment, the oligo concentration was determined spectrophotometrically (18), and it is probable that
Bioconjugate Chem., Vol. 7, No. 1, 1996 5
Communications
Figure 2. Effects of various concentrations of block polycationic oligo 1, cholesteryl-modified oligo, and polycation on the HSV-1 reproduction in Vero cells.
the difference between experimental ()11.2) and theoretical ()9) concentration of the amino groups in the copolymer 1 accounts for the change of molar extinction of oligo after modification with the polycation. The straight polycation, which was used in the control experiment on virus reproduction, was obtained via solid support synthesis without growing the oligo chain (Scheme 1), purified as a DMT derivative by reverse phase HPLC, and analyzed using the DMT release test and 2,4,6-TNBS titration as described above. The copolymer 1 formed duplexes with the complementary 14-mer oligo, which contained one desoxythymidine unit at both 3′- and 5′-ends [0.05 M phosphate buffer (pH 7.0) containing 0.01 M EDTA and 0.15 M NaCl; oligo and copolymer concentrations were 1 µM]. The stability of these duplexes was not statistically different from the stability of the duplexes formed by the unmodified 12-mer with the same nucleotide structure (Tm equaled 44 and 45 °C, respectively). Furthermore, the stability of the block polycationic oligo against nuclease digestion was significantly increased compared to that of the unmodified oligo. No digestion products were detected when the copolymer was exposed to Crotalus durissus phosphodiesterase (Boehringer Manheim) for 2 h at 37 °C [0.1 M sodium acetate buffer (pH 8.0)], while PAGE demonstrated that this treatment resulted in complete digestion of the unmodified oligomer. The effects of the block polycationic oligo on the reproduction of HSV-1 in the Vero cells were investigated and compared to the effects of (1) the 12-mer oligo with the same polynucleotide sequence, modified at the 5′-end with cholesteryl groups as previously described (19), and (2) the straight polycation synthesized as described above (Scheme 1). The experiment on HSV-1 reproduction was performed as previously described (16). Briefly, monolayers of Vero cells were infected at 0.1 PFU/cell multiplicity with the virus. The oligos or polycation was added to the cells at various concentrations 1 h prior to infection. After 8 h of incubation of the infected cells in the presence of oligonucleotides, the medium was replaced with fresh media containing fetal calf serum. The virus infectious titer (PFU/mL) was determined 24 h postinfection on monolayers of Vero cells (21). All experiments were performed in triplicate. The variations in the infectious titers determined were less than 25%. The results of the experiment are presented in Figure 2. The block polycationic oligo decreased the virus titer in the narrow range of oligo concentration of 1-4 µM, this decrease approximating 30-fold at 2 µM. At higher concentrations of oligo, the increase in the virus titer was observed. Conversely, the cholesteryl-modified oligo that
did not contain the polycationic block significantly inhibited the infection in the same concentration range, decreasing the virus titer by more than 10000-fold at 4 µM. The treatment with the straight polycation unlinked to an oligo chain resulted in an increase in the titer, this increase approximating 100-fold at 8 µM. Therefore, decreased antiviral activity of the copolymer compared to the activity of the cholesteryl-modified oligo is probably explained by the polycation-induced stimulation of the virus infection. In this case, the net effect of the copolymer was a sum of the effects of the oligo chain that inhibited the virus reproduction and the polycation chain that enhanced the infection. The promotion of the infection by polycations has previously been described for various polycations and viruses (22-24). Although the mechanism of this phenomena is not completely understood, it was commonly observed when the polycations were added prior to or simultaneously with the infection, suggesting the polycation effects on the virus adsorption and uptake. We also observed the promotion of HSV-1 infection in Vero cells by short-chain polyspermines added prior to infection (data not presented). One previous observation suggests that polylysine and polyarginine inhibit infection of the cells with HSV-1, by interfering with the virus interactions with cell receptors (25). However, according to Langeland et al. (25), the inhibitory effect decreased with the decrease of the polycation length, and it was observed with polycations of much higher molecular mass than the molecular mass of polycations studied in our work. Because of the polycation effects on infection, the HSV-1 system described in this work may not be suitable as a virus model for investigation of the biological activity of the block polycationic oligos. However, these oligos may become useful for inhibition of reproduction of other viruses and expression of genes in the target cells. The automated synthesis is convenient and allows blocks of varying lengths and molecular masses to be synthesized precisely. Furthermore, it can also be used for introduction of various functional moieties in the block polycationic oligo such as, for example, other hydrophobes (5, 16), polyoxyethylene chains (26, 27), and polypeptides (28, 29). LITERATURE CITED (1) Helene, C. (1991) Rational design of sequence-specific oncogene inhibitors based on antisense and antigene oligonucleotides. Eur. J. Cancer 27, 1466-1471. (2) Crooke, R. M. (1991) In vitro toxicology and pharmacokinetics of antisense oligonucleotides. Anti-Cancer Drug Des. 6, 609-646. (3) Stein, C. A., and Cheng, Y.-C. (1993) Antisense oligonucleotides as therapeutic agents - is the bullet really magical? Science 261, 1004-1012. (4) Letsinger, R. L., Zhang, X., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989) Cholesteryl-conjugated oligonucleotides: synthesis, properties and activity as inhibitors of replication of human immunodeficiencey virus in cell culture. Proc. Natl. Acad. Sci. U.S.A. 86, 6553-6556. (5) Kabanov, A. V., Vinogradov, S. V., Ovcharenko, A. V., Krivonos, A. V., Melik-Nubarov, N. S., Kiselev, V. I., and Severin, E. S. (1990) A new class of antivirals: antisense oligonucleotides combined with a hydrophobic substituent effectively inhibit influenza virus reproduction and synthesis of virus-specific proteins in MDCK cells. FEBS Lett. 259, 327-330. (6) Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotide conjugates. Nucleic Acids Res. 18, 3777-3783. (7) Miller, P. S. (1989) Non-ionic antisense oligonucleotides. In Antisense Inhibitors of Gene Expression (J. Cohen, Ed.) CRC Press, Boca Raton, FL.
6 Bioconjugate Chem., Vol. 7, No. 1, 1996 (8) Fathi, R., Huang, Q., Coppola, G., Delaney, W., Teasdale, R., Krieg, A. M., and Cook, A. F. (1994) Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates. Nucleic Acids Res. 22, 5416-5424. (9) Leonetti, J.-P., Degols, G., and Lebleu, B. (1990) Biological Activity of Oligonucleotide-Poly(L-Lysine) Conjugates: Mechanism of Cell Uptake. Bioconjugate Chem. 1, 149-153. (10) Wei, Z., Tung, C. H., Zhu, T., Stein, S. (1994) Synthesis of Oligoarginine-Oligonucleotide Conjugates and OligoarginineBridged Oligonucleotide Pairs. Bioconjugate Chem. 5, 468474. (11) Kabanov, A. V., and Kabanov, V. A. (1995) DNA Complexes with Polycations for Delivery of Genetic Material into Cells. Bioconjugate Chem. 6, 7-20. (12) Kabanov, A. V., Astafieva, I. V., Maksimova, I. V., Lukanidin, E. M., Georgiev, G. P., and Kabanov, V. A. (1993) Efficient Transformation of Mammalian Cells Using DNA Interpolyelectrolyte Complexes with Carbon Chain Polycations. Bioconjugate Chem. 4, 448-454. (13) Kabanov, A. V., Astafieva, I. V., Chikindas, M. L., Rosenblat, G. F., Kiselev, V. I., Severin, E. S., and Kabanov, V. A. (1991) DNA interpolyelectrolyte complexes as a tool for efficient cell transformation. Biopolymers 31, 1437-1443. (14) Kulka, M., Smith, C. C., Aurelian, L., Fishelevich, R., Meade, K., Miller, P., and Ts’o, P. O. P. (1989) Site specificity of the inhibitory effects of oligo(nucleoside methylphosphonate)s complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4. Proc. Natl. Acad. Sci. U.S.A. 86, 6868-6872. (15) Kulka, M., Smith, C. C., Levis, J., Fishelevich, R., Hunter, J. C. R., Cushman, C. D., Miller, P. S., Ts’o, P. O. P., and Aurelian, L. (1994) Synergistic antiviral activities of oligonucleotide methylphosphonates complementary to herpes simplex virus type 1 immediate-early mRNAs 4,5 and 1. Antimicrob. Agents Chemother. 38, 675-680. (16) Vinogradov, S. V., Suzdaltseva, Yu., Alakhov, V. Yu., and Kabanov, A. V. (1994) Inhibition of Herpes simplex virus 1 reproduction with hydrophobized antisense oligonucleotides. Biochem. Biophys. Res. Commun. 203, 959-966. (17) Kabanov, A. V., Vinogradov, S. V., Suzdaltseva, Yu. G., and Alakhov, V. Yu. (1995) Water-Soluble Block Polycations as Carriers for Oligonucleotide Delivery, Bioconjugate Chem. 6, 639-643. (18) Agrawal, S. (1994) Functionalization of oligonucleotides with amino groups and attachment of amino specific reporter
Vinogradov et al. groups. In Protocols for Oligonucleotide Conjugates (S. Agrawal, Ed.) pp 93-120, Humana Press, Totowa, NJ. (19) Atkinson, T., and Smith, M. (1984) Solid-phase synthesis of oligodeoxyribonucleotides by the phosphite-triester method. In Oligonucleotide Synthesis. A Practical Approach (M. J. Gait, Ed.) pp 35-82, IRL Press, Oxford, Washington, DC. (20) Friser, H. (1975) An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal. Biochem. 64, 284-288. (21) Freshney, R. I. (1994) Culture of Animal Cells, 3rd ed., John Willey & Sons, Inc., New York. (22) Bailey, C. A., Miller, D. K., and Lenard, J. (1984) Effects of DEAE-dextran on infection and homolysis by VSV. Evidence that nonspecific electrostatic interactions mediate effective binding of VSV to cells. Virology 133, 111-118. (23) Prakash, J., and Foxe, M. J. (1985) Optimization of conditions for infection of isolated potato protoplasts with potato virus X. Arch. Virol. 85, 269-279. (24) Rousculp, M. D., Goldsmith, K. T., and Garver, R. I. (1992) Quantative evaluation of retroviral gene transduction efficiency in human lung cancer cells. Hum. Gene Ther. 3, 471477. (25) Langeland, N., Moore, L. J., Holmsen, H., and Haarr, L. (1988) Interaction of polylysine with the cellular receptor for herpes simplex virus type 1. J. Gen. Virol. 69, 1137-1145. (26) Jasschke, A., Furste, J. P., Nordorf, E., Hillenkapm, F., Cech, D., and Erdmann, V. A. (1994) Synthesis and properties of oligodeoxyribonucleotide-polyethylene glycol conjugates. Nucleic Acids Res. 22, 4810-4817. (27) Jones, D. S., Hachmann, J. P., Osgood, S. A., Hayag, M. S., Barstad, P. A., Iversen, G. M., and Couttus, S. M. (1994) Conjugates of Double-Stranded Oligonucleotides with Poly(ethylene glycol) and Keyhole Limpet Hemocyanin: A Model for Treating Systemic Lupus Erythematosus. Bioconjugate Chem. 5, 390-399. (28) Soukchareun, S., Tregear, G. W., and Haralambidis, J. (1995) Preparation and Characterization of Antisense Oligonucleotide-Peptide Hybrids Containing Viral Fusion Peptides. Bioconjugate Chem. 6, 43-53. (29) Arar, K., Aubertin, A.-M., Roche, A.-C., Monsigny, M., and Mayer, R. (1995) Synthesis and Antiviral Activity of PeptideOligonucleotide Conjugates Prepared by Using NR-(Bromoacetyl)peptides. Bioconjugate Chem. 6, 573-577.
BC9500913
