Sequence-Specific Activity of Antisense Oligonucleotides Conjugated to Poly (L-Lysine) Carriersa GENEVIEVE DEGOLS, JEAN-PAUL L E O N E ~ I AND , BERNARD LEBLEU University of Montpellier II Sciences et Techniques du Languedoc UA CNRS 11 91 Gknktique Molkculaire Place E. Bataillon 34095 Montpellier C k d a 5, France Antisense oligonucleotides (ODN) offer interesting prospects for the specific control of viral gene expression, as demonstrated for HIV-1 and other virus-infected cells in vitro. Their efficacy is limited by several factors including metabolic stability, cellular uptake, and intracellular bioavailability (reviewed in ref. 1). The chemical conjugation of poly(L-lysine) (PLL) to the 3’ end of antisense ODNs considerably increases their antiviral activity against the vesicular stomatitis virus without loss of sequence specificity2; increased cellular uptake and protection against phosphodiesterase degradation presumably contribute to the increased efficacy of these ODN-PLL
conjugate^.^ Synthetic oligonucleotides have been used as well for the control of HIV-1 infection in several T-cell lines in vitro (as reviewed in ref. 4), but little or no sequence specificity has been documented. The most active compound is a (dC)28 phosphorothioate derivative (EC50 = 0.5 pM).Nonconjugated and PLL-conjugated 15-mer ODNs complementary to the translation initiation region of tat mRNA have been compared in acutely infected (1,000 TCID 50/ml) MT4 lymphocytes (TABLE1). A strictly sequence-specific and dose-dependent inhibition (EC50 = 0.15 p M ) has been obtained with PLL conjugates; little sequence recognition and a lower efficiency (EC50 = 20 pM) are seen with free ODN, in agreement with published data.4 The lack of sequence recognition probably results from nonspecific inhibition of HIV reverse transcriptase and/or virus adsorption. Whether and how PLL conjugation prevents it cannot be ascertained; however, PCR analysis of viral DNA synthesis has confirmed differences in the intracellular targets of free and PLL-conjugate ODNs (not shown). The antiviral activity of ODN-PLL conjugates is only transient and is limited by the toxicity of PLL for T lymphocytes. The association of these ODN-PLL conjugates with sulfated polyanions, such as heparin, is beneficial in several aspects, as sulfated polyanions (1) interfere with early steps in the HIV multiplication cycle, thereby diminishing initial virus load; (2) strongly reduce PLL cytotoxicity as documented by trypan blue exclusion; and (3) modify the release of the conjugated material. Such ternary complexes between ODN-PLL conjugates at doses as small as 75 nM and heparin (50 pg/ml) completely suppress HIV production when given in a single addition immediately prior to virus inoculation. As a matter of comparison, AZT (0.5 FM), heparin (50 pg/ml), or their combination only transiently suppresses nThis work has been supported by CNRS and the Agence Nationale pour la Recherche sur le SIDA. 33 1
ANNALS NEW YORK ACADEMY OF SCIENCES
332
TABLE 1. Antiviral Activity of Free and PLL-Conjugated Antisense Oligonucleotides"
ODN tat BRU-PLL tat ELI-PLL tat BRU tat ELI
Percentaee RT Inhibition 40 pM ODN 0.5 FM ODN HIV-1 BRU HIV-1 ELI HIV-1 BRU HIV-1 ELI 93 21 ND ND 31 89 ND ND 5 ND 95 78 ND 0 92 90
'MT4 cells (3 x 106 cells/ml) were infected with the HIV-1 BRU, or HIV-1 ELI isolate (1,OOO TCIDSO/ml) and incubated 30 minutes at 4°C. Cells were washed, diluted to 3 x 105 cells/ml, and incubated at 37°C in the presence of PLL conjugated or unconjugated oligomers. tat BRU(s'CTAGGATCTACTGGCTrA3'), tat ELI(5'TTAGGATCTACTGGATrA3') and VSV(s'CATI"ITGATTACTGTrA3') oligomers were synthesized on a riboadenosinederivatized support using a Biosearch Cyclone automatic DNA synthesizer and purified by reverse phase chromatography. Covalent linkage to PLL through an N-morpholine ring was achieved by periodic acid oxidation and borocyanohydride reduction of the 3' end ribose, as previously de~cribed.~ tat BRU and tat ELI have been designed to hybridize fully to the translation initiation region of tat mRNAs in the corresponding isolates. Culture samples were removed 4 days after virus inoculation for HIV-1 BRU and 5 days after infection for HIV-1 ELI to determine cell count, viability, syncytia formation (not shown), and reverse transcriptase activity, according to established procedures. 50000
P
0 0
10
20
30
I
Time post infection FIGURE 1. Potentiation of antisense-PLL conjugate activity by sulfated polyanions. MT4 cells were left untreated or were treated with various combinations of antiviral agents at the indicated concentrations. The cells were then infected with HIV-1 BRU, and viral production 1. Time post infection refers to days. (0--O), untreated was monitored as described in TABLE cells; 0-----a, cells treated with heparin (50 kg/ml) and a control (complementary to VSV N cells treated protein mRNA initiation site) oligonucleotide-PLL conjugate (0.5 pM); A----A, with heparin (50 pg/ml) and AZT (0.5 pM): O----O,cells treated with heparin (50 p.g/ml) and tat BRU-PLL conjugate (0.5 pM).
DEGOLS et al.: SEQUENCE-SPECIFIC ACTMTY
333
HIV-1 production in this model system. Interestingly, sequence specificity is maintained in this formulation (FIG.1 and data not shown). Additional studies are required to understand the underlying mechanisms, exploring the relative efficacy in this model of various ODN chemistries and targets in the infected cells. Whether comparable inhibition can be attained in chronically infected T lymphocytes or in macrophage reservoirs will also be of interest.
ACKNOWLEDGMENTS The authors are indebted to Dr. C. Devaux for helpful discussion and access to appropriate containment facilities. REFERENCES B., G. DEGOLS & J. P. LEONETTI. 1991.Antisense oligonucleotides as pharmacolog1. LEBLEU, ical modulators of genes expression. In Targeted Drug Delivery. R. L. Juliano, ed.: 329-354. Springer-Verlag. Heidelberg. M., B. BAYARD & B. LEBLEU. 1987. Specific antiviral activity of a poly (L-lysine) 2. LEMAITRE, conjugated oligodeoxyribonucleotidecomplementary to vesicular stomatitis virus Nprotein mRNA initiation site. Proc. Natl. Acad. Sci. USA 84: 648-652. J. P., G. DEGOLS& B. LEBLEU.1990. Biological activity of oligonucleotide-poly 3. LEONETTI, (L-lysine) conjugates: Mechanism of cell uptake. Bioconjugate Chem. 1: 149-153. M., H. MITSUYA & S. BRODER.1991. A new concepts in AIDS treatment: An 4. MATSUKURA, antisense approach and its current status towards clinical application. In Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. E. Wickstrom, ed.: 159-178. Wiley-Liss, Inc. New York.
conjugate^.^ Synthetic oligonucleotides have been used as well for the control of HIV-1 infection in several T-cell lines in vitro (as reviewed in ref. 4), but little or no sequence specificity has been documented. The most active compound is a (dC)28 phosphorothioate derivative (EC50 = 0.5 pM).Nonconjugated and PLL-conjugated 15-mer ODNs complementary to the translation initiation region of tat mRNA have been compared in acutely infected (1,000 TCID 50/ml) MT4 lymphocytes (TABLE1). A strictly sequence-specific and dose-dependent inhibition (EC50 = 0.15 p M ) has been obtained with PLL conjugates; little sequence recognition and a lower efficiency (EC50 = 20 pM) are seen with free ODN, in agreement with published data.4 The lack of sequence recognition probably results from nonspecific inhibition of HIV reverse transcriptase and/or virus adsorption. Whether and how PLL conjugation prevents it cannot be ascertained; however, PCR analysis of viral DNA synthesis has confirmed differences in the intracellular targets of free and PLL-conjugate ODNs (not shown). The antiviral activity of ODN-PLL conjugates is only transient and is limited by the toxicity of PLL for T lymphocytes. The association of these ODN-PLL conjugates with sulfated polyanions, such as heparin, is beneficial in several aspects, as sulfated polyanions (1) interfere with early steps in the HIV multiplication cycle, thereby diminishing initial virus load; (2) strongly reduce PLL cytotoxicity as documented by trypan blue exclusion; and (3) modify the release of the conjugated material. Such ternary complexes between ODN-PLL conjugates at doses as small as 75 nM and heparin (50 pg/ml) completely suppress HIV production when given in a single addition immediately prior to virus inoculation. As a matter of comparison, AZT (0.5 FM), heparin (50 pg/ml), or their combination only transiently suppresses nThis work has been supported by CNRS and the Agence Nationale pour la Recherche sur le SIDA. 33 1
ANNALS NEW YORK ACADEMY OF SCIENCES
332
TABLE 1. Antiviral Activity of Free and PLL-Conjugated Antisense Oligonucleotides"
ODN tat BRU-PLL tat ELI-PLL tat BRU tat ELI
Percentaee RT Inhibition 40 pM ODN 0.5 FM ODN HIV-1 BRU HIV-1 ELI HIV-1 BRU HIV-1 ELI 93 21 ND ND 31 89 ND ND 5 ND 95 78 ND 0 92 90
'MT4 cells (3 x 106 cells/ml) were infected with the HIV-1 BRU, or HIV-1 ELI isolate (1,OOO TCIDSO/ml) and incubated 30 minutes at 4°C. Cells were washed, diluted to 3 x 105 cells/ml, and incubated at 37°C in the presence of PLL conjugated or unconjugated oligomers. tat BRU(s'CTAGGATCTACTGGCTrA3'), tat ELI(5'TTAGGATCTACTGGATrA3') and VSV(s'CATI"ITGATTACTGTrA3') oligomers were synthesized on a riboadenosinederivatized support using a Biosearch Cyclone automatic DNA synthesizer and purified by reverse phase chromatography. Covalent linkage to PLL through an N-morpholine ring was achieved by periodic acid oxidation and borocyanohydride reduction of the 3' end ribose, as previously de~cribed.~ tat BRU and tat ELI have been designed to hybridize fully to the translation initiation region of tat mRNAs in the corresponding isolates. Culture samples were removed 4 days after virus inoculation for HIV-1 BRU and 5 days after infection for HIV-1 ELI to determine cell count, viability, syncytia formation (not shown), and reverse transcriptase activity, according to established procedures. 50000
P
0 0
10
20
30
I
Time post infection FIGURE 1. Potentiation of antisense-PLL conjugate activity by sulfated polyanions. MT4 cells were left untreated or were treated with various combinations of antiviral agents at the indicated concentrations. The cells were then infected with HIV-1 BRU, and viral production 1. Time post infection refers to days. (0--O), untreated was monitored as described in TABLE cells; 0-----a, cells treated with heparin (50 kg/ml) and a control (complementary to VSV N cells treated protein mRNA initiation site) oligonucleotide-PLL conjugate (0.5 pM); A----A, with heparin (50 pg/ml) and AZT (0.5 pM): O----O,cells treated with heparin (50 p.g/ml) and tat BRU-PLL conjugate (0.5 pM).
DEGOLS et al.: SEQUENCE-SPECIFIC ACTMTY
333
HIV-1 production in this model system. Interestingly, sequence specificity is maintained in this formulation (FIG.1 and data not shown). Additional studies are required to understand the underlying mechanisms, exploring the relative efficacy in this model of various ODN chemistries and targets in the infected cells. Whether comparable inhibition can be attained in chronically infected T lymphocytes or in macrophage reservoirs will also be of interest.
ACKNOWLEDGMENTS The authors are indebted to Dr. C. Devaux for helpful discussion and access to appropriate containment facilities. REFERENCES B., G. DEGOLS & J. P. LEONETTI. 1991.Antisense oligonucleotides as pharmacolog1. LEBLEU, ical modulators of genes expression. In Targeted Drug Delivery. R. L. Juliano, ed.: 329-354. Springer-Verlag. Heidelberg. M., B. BAYARD & B. LEBLEU. 1987. Specific antiviral activity of a poly (L-lysine) 2. LEMAITRE, conjugated oligodeoxyribonucleotidecomplementary to vesicular stomatitis virus Nprotein mRNA initiation site. Proc. Natl. Acad. Sci. USA 84: 648-652. J. P., G. DEGOLS& B. LEBLEU.1990. Biological activity of oligonucleotide-poly 3. LEONETTI, (L-lysine) conjugates: Mechanism of cell uptake. Bioconjugate Chem. 1: 149-153. M., H. MITSUYA & S. BRODER.1991. A new concepts in AIDS treatment: An 4. MATSUKURA, antisense approach and its current status towards clinical application. In Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. E. Wickstrom, ed.: 159-178. Wiley-Liss, Inc. New York.
