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. 1996 Jun 1;24(11):2176–2182. doi: 10.1093/nar/24.11.2176

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

A Bielinska 1, J F Kukowska-Latallo 1, J Johnson 1, D A Tomalia 1, J R Baker Jr 1
PMCID: PMC145901  PMID: 8668551

Abstract

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.

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Selected References

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  1. Barth R. F., Adams D. M., Soloway A. H., Alam F., Darby M. V. Boronated starburst dendrimer-monoclonal antibody immunoconjugates: evaluation as a potential delivery system for neutron capture therapy. Bioconjug Chem. 1994 Jan-Feb;5(1):58–66. doi: 10.1021/bc00025a008. [DOI] [PubMed] [Google Scholar]
  2. Bennett C. F., Chiang M. Y., Chan H., Shoemaker J. E., Mirabelli C. K. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol. 1992 Jun;41(6):1023–1033. [PubMed] [Google Scholar]
  3. Bielinska A., Shivdasani R. A., Zhang L. Q., Nabel G. J. Regulation of gene expression with double-stranded phosphorothioate oligonucleotides. Science. 1990 Nov 16;250(4983):997–1000. doi: 10.1126/science.2237444. [DOI] [PubMed] [Google Scholar]
  4. Boutorin A. S., Gus'kova L. V., Ivanova E. M., Kobetz N. D., Zarytova V. F., Ryte A. S., Yurchenko L. V., Vlassov V. V. Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3'-terminus and their interaction with DNA within mammalian cells. FEBS Lett. 1989 Aug 28;254(1-2):129–132. doi: 10.1016/0014-5793(89)81023-3. [DOI] [PubMed] [Google Scholar]
  5. Brossalina E., Pascolo E., Toulmé J. J. The binding of an antisense oligonucleotide to a hairpin structure via triplex formation inhibits chemical and biological reactions. Nucleic Acids Res. 1993 Dec 11;21(24):5616–5622. doi: 10.1093/nar/21.24.5616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ch'ng J. L., Mulligan R. C., Schimmel P., Holmes E. W. Antisense RNA complementary to 3' coding and noncoding sequences of creatine kinase is a potent inhibitor of translation in vivo. Proc Natl Acad Sci U S A. 1989 Dec;86(24):10006–10010. doi: 10.1073/pnas.86.24.10006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colige A., Sokolov B. P., Nugent P., Baserga R., Prockop D. J. Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells. Biochemistry. 1993 Jan 12;32(1):7–11. doi: 10.1021/bi00052a002. [DOI] [PubMed] [Google Scholar]
  8. Denhardt D. T. Mechanism of action of antisense RNA. Sometime inhibition of transcription, processing, transport, or translation. Ann N Y Acad Sci. 1992 Oct 28;660:70–76. doi: 10.1111/j.1749-6632.1992.tb21059.x. [DOI] [PubMed] [Google Scholar]
  9. Fisher T. L., Terhorst T., Cao X., Wagner R. W. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res. 1993 Aug 11;21(16):3857–3865. doi: 10.1093/nar/21.16.3857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fréchet J. M. Functional polymers and dendrimers: reactivity, molecular architecture, and interfacial energy. Science. 1994 Mar 25;263(5154):1710–1715. doi: 10.1126/science.8134834. [DOI] [PubMed] [Google Scholar]
  11. Gareis M., Harrer P., Bertling W. M. Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice. Cell Mol Biol. 1991;37(2):191–203. [PubMed] [Google Scholar]
  12. Goodarzi G., Gross S. C., Tewari A., Watabe K. Antisense oligodeoxyribonucleotides inhibit the expression of the gene for hepatitis B virus surface antigen. J Gen Virol. 1990 Dec;71(Pt 12):3021–3025. doi: 10.1099/0022-1317-71-12-3021. [DOI] [PubMed] [Google Scholar]
  13. Johnson L. G. Gene therapy for cystic fibrosis. Chest. 1995 Feb;107(2 Suppl):77S–83S. doi: 10.1378/chest.107.2_supplement.77s. [DOI] [PubMed] [Google Scholar]
  14. Krieg A. M., Yi A. K., Matson S., Waldschmidt T. J., Bishop G. A., Teasdale R., Koretzky G. A., Klinman D. M. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 1995 Apr 6;374(6522):546–549. doi: 10.1038/374546a0. [DOI] [PubMed] [Google Scholar]
  15. Krystal G. W., Armstrong B. C., Battey J. F. N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts. Mol Cell Biol. 1990 Aug;10(8):4180–4191. doi: 10.1128/mcb.10.8.4180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. MacGregor G. R., Caskey C. T. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 1989 Mar 25;17(6):2365–2365. doi: 10.1093/nar/17.6.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miller P. S. Oligonucleoside methylphosphonates as antisense reagents. Biotechnology (N Y) 1991 Apr;9(4):358–362. doi: 10.1038/nbt0491-358. [DOI] [PubMed] [Google Scholar]
  18. Milligan J. F., Matteucci M. D., Martin J. C. Current concepts in antisense drug design. J Med Chem. 1993 Jul 9;36(14):1923–1937. doi: 10.1021/jm00066a001. [DOI] [PubMed] [Google Scholar]
  19. Perez J. R., Li Y., Stein C. A., Majumder S., van Oorschot A., Narayanan R. Sequence-independent induction of Sp1 transcription factor activity by phosphorothioate oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5957–5961. doi: 10.1073/pnas.91.13.5957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ranade K., Poteete A. R. A switch in translation mediated by an antisense RNA. Genes Dev. 1993 Aug;7(8):1498–1507. doi: 10.1101/gad.7.8.1498. [DOI] [PubMed] [Google Scholar]
  21. Roessler B. J., Hartman J. W., Vallance D. K., Latta J. M., Janich S. L., Davidson B. L. Inhibition of interleukin-1-induced effects in synoviocytes transduced with the human IL-1 receptor antagonist cDNA using an adenoviral vector. Hum Gene Ther. 1995 Mar;6(3):307–316. doi: 10.1089/hum.1995.6.3-307. [DOI] [PubMed] [Google Scholar]
  22. Singh P., Moll F., 3rd, Lin S. H., Ferzli C., Yu K. S., Koski R. K., Saul R. G., Cronin P. Starburst dendrimers: enhanced performance and flexibility for immunoassays. Clin Chem. 1994 Sep;40(9):1845–1849. [PubMed] [Google Scholar]
  23. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  24. Stein C. A., Tonkinson J. L., Yakubov L. Phosphorothioate oligodeoxynucleotides--anti-sense inhibitors of gene expression? Pharmacol Ther. 1991 Dec;52(3):365–384. doi: 10.1016/0163-7258(91)90032-h. [DOI] [PubMed] [Google Scholar]
  25. Wagner R. W. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994 Nov 24;372(6504):333–335. doi: 10.1038/372333a0. [DOI] [PubMed] [Google Scholar]
  26. Wagner R. W., Matteucci M. D., Lewis J. G., Gutierrez A. J., Moulds C., Froehler B. C. Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science. 1993 Jun 4;260(5113):1510–1513. doi: 10.1126/science.7684856. [DOI] [PubMed] [Google Scholar]
  27. Yakubov L., Khaled Z., Zhang L. M., Truneh A., Vlassov V., Stein C. A. Oligodeoxynucleotides interact with recombinant CD4 at multiple sites. J Biol Chem. 1993 Sep 5;268(25):18818–18823. [PubMed] [Google Scholar]
  28. Zamecnik P. C., Stephenson M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):280–284. doi: 10.1073/pnas.75.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]

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