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Nuclear entry of nonviral vectors

Gene Therapy volume 12, pages 881–890 (2005)Cite this article

Abstract

Nonviral gene delivery is limited to a large extent by multiple extracellular and intracellular barriers. One of the major barriers, especially in nondividing cells, is the nuclear envelope. Once in the cytoplasm, plasmids must make their way into the nucleus in order to be expressed. Numerous studies have demonstrated that transfections work best in dividing populations of cells in which the nuclear envelope disassembles during mitosis, thus largely eliminating the barrier. However, since many of the cells that are targets for gene therapy do not actively undergo cell division during the gene transfer process, the mechanisms of nuclear transport of plasmids in nondividing cells are of critical importance. In this review, we summarize recent studies designed to elucidate the mechanisms of plasmid nuclear import in nondividing cells and discuss approaches to either exploit or circumvent these processes to increase the efficiency of gene transfer and therapy.

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References

  1. Capecchi MR . High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 1980; 22: 479–488.

    CAS  PubMed  Google Scholar 

  2. Graessman M et al. Helper activity for gene expression, a novel function of the SV40 enhancer. Nucleic Acids Res 1989; 17: 6603–6612.

    Google Scholar 

  3. Zabner J et al. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 1995; 270: 18997–19007.

    CAS  PubMed  Google Scholar 

  4. Mirzayans R, Remy AA, Malcom PC . Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection. Mutat Res 1992; 281: 115–122.

    CAS  PubMed  Google Scholar 

  5. Thornburn AM, Alberts AS . Efficient expression of miniprep plasmid DNA after needle micro-injection into somatic cells. Biotechniques 1993; 14: 356–358.

    Google Scholar 

  6. Labat-Moleur F et al. An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Therapy 1996; 3: 1010–1017.

    CAS  PubMed  Google Scholar 

  7. Dean DA, Dean BS, Muller S, Smith LC . Sequence requirements for plasmid nuclear entry. Exp Cell Res 1999; 253: 713–722.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Lewis PF, Emerman M . Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994; 68: 510–516.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Miller DG, Adam MA, Miller AD . Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990; 10: 4239–4242.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dvorin JD, Malim MH . Intracellular trafficking of HIV-1 cores: journey to the center of the cell. Curr Top Microbiol Immunol 2003; 281: 179–208.

    CAS  PubMed  Google Scholar 

  11. Fasbender A, Zabner J, Zeiher BG, Welsh MJ . A low rate of cell proliferation and reduced DNA uptake limit cationic lipid-mediated gene transfer to primary cultures of ciliated human airway epithelia. Gene Therapy 1997; 4: 1173–1180.

    CAS  PubMed  Google Scholar 

  12. Brunner S et al. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Therapy 2000; 7: 401–407.

    CAS  PubMed  Google Scholar 

  13. Escriou V et al. Critical assessment of the nuclear import of plasmid during cationic lipid-mediated gene transfer. J Gene Med 2001; 3: 179–187.

    CAS  PubMed  Google Scholar 

  14. Coonrod A, Li FQ, Horwitz M . On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Therapy 1997; 4: 1313–1321.

    CAS  PubMed  Google Scholar 

  15. Tseng W, Haselton F, Giorgio T . Transfection by cationic liposomes using simultaneous single cell measurements of plasmid delivery and transgene expression. J Biol Chem 1997; 272: 25641–25647.

    CAS  PubMed  Google Scholar 

  16. James MB, Giorgio TD . Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells. Mol Ther 2000; 1: 339–346.

    CAS  PubMed  Google Scholar 

  17. Tachibana R, Harashima H, Shinohara Y, Kiwada H . Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviral vectors. Adv Drug Deliv Rev 2001; 52: 219–226.

    CAS  PubMed  Google Scholar 

  18. Sebestyén MG et al. DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA. Nat Biotechnol 1998; 16: 80–85.

    PubMed  Google Scholar 

  19. Ludtke JJ, Sebestyen MG, Wolff JA . The effect of cell division on the cellular dynamics of microinjected DNA and dextran. Mol Ther 2002; 5: 579–588.

    CAS  PubMed  Google Scholar 

  20. Lechardeur D et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Therapy 1999; 6: 482–497.

    Article  CAS  PubMed  Google Scholar 

  21. Pollard H et al. Ca2+-senssitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids. J Gene Med 2001; 3: 153–164.

    CAS  PubMed  Google Scholar 

  22. Escriou V et al. Cationic lipid-mediated gene transfer: analysis of cellular uptake and nuclear import of plasmid DNA. Cell Biol Toxicol 1998; 14: 95–104.

    CAS  PubMed  Google Scholar 

  23. Zelphati O, Liang X, Hobart P, Felgner PL . Gene chemistry: functionally and conformationally intact fluorescent plasmid DNA. Hum Gene Ther 1999; 10: 15–24.

    CAS  PubMed  Google Scholar 

  24. Banks GA, Roselli RJ, Chen R, Giorgio TD . A model for the analysis of nonviral gene therapy. Gene Therapy 2003; 10: 1766–1775.

    CAS  PubMed  Google Scholar 

  25. Pollard H et al. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J Biol Chem 1998; 273: 7507–7511.

    Article  CAS  PubMed  Google Scholar 

  26. Ogris M et al. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery [In Process Citation]. Gene Therapy 1999; 6: 595–605.

    CAS  PubMed  Google Scholar 

  27. Carlisle RC et al. Adenovirus hexon protein enhances nuclear delivery and increases transgene expression of polyethylenimine/plasmid DNA vectors. Mol Ther 2001; 4: 473–483.

    CAS  PubMed  Google Scholar 

  28. Dowty ME et al. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes. Proc Natl Acad Sci USA 1995; 92: 4572–4576.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Gasiorowski JZ, Dean DA . Mechanisms of nuclear transport and interventions. Adv Drug Deliv Rev 2003; 55: 703–716.

    CAS  PubMed  Google Scholar 

  30. Dean DA . Import of plasmid DNA into the nucleus is sequence specific. Exp Cell Res 1997; 230: 293–302.

    CAS  PubMed  Google Scholar 

  31. Dean BS, Byrd Jr JN, Dean DA . Nuclear targeting of plasmid DNA in human corneal cells. Cur Eye Res 1999; 19: 66–75.

    CAS  Google Scholar 

  32. Vacik J, Dean BS, Zimmer WE, Dean DA . Cell-specific nuclear import of plasmid DNA. Gene Therapy 1999; 6: 1006–1014.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Young JL, Benoit JN, Dean DA . Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature. Gene Therapy 2003; 10: 1465–1470.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Mir B, Piedrahita JA . Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts. Nucleic Acids Res 2004; 32: e25.

    PubMed  PubMed Central  Google Scholar 

  35. Blomberg P et al. Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle. Biochem Biophys Res Commun 2002; 298: 505–510.

    CAS  PubMed  Google Scholar 

  36. Wildeman AG . Regulation of SV40 early gene expression. Biochem Cell Biol 1988; 66: 567–577.

    CAS  PubMed  Google Scholar 

  37. Whittaker GR . Virus nuclear import. Adv Drug Deliv Rev 2003; 55: 733–747.

    CAS  PubMed  Google Scholar 

  38. Kuwahara J, Azumano M, Takeda T . Nuclear localization of transcription factor Sp1. Nucleic Acids Symp Ser 1999; 42: 293–294.

    CAS  Google Scholar 

  39. Chan CK, Jans DA . Synergy of importin alpha recognition and DNA binding by the yeast transcriptional activator GAL4. FEBS Lett 1999; 462: 221–224.

    CAS  PubMed  Google Scholar 

  40. Chan CK, Hubner S, Hu W, Jans DA . Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery. Gene Therapy 1998; 5: 1204–1212.

    CAS  PubMed  Google Scholar 

  41. Müller CW et al. Structure of the NF-κB p50 homodimer bound to DNA. Nature 1995; 373: 311–317.

    PubMed  Google Scholar 

  42. Mesika A, Grigoreva I, Zohar M, Reich Z . A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei. Mol Ther 2001; 3: 653–657.

    CAS  PubMed  Google Scholar 

  43. Mesika A et al. Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein. Hum Gene Ther 16: 200–208.

    CAS  PubMed  Google Scholar 

  44. Wilson GL, Dean BS, Wang G, Dean DA . Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J Biol Chem 1999; 274: 22025–22032.

    CAS  PubMed  Google Scholar 

  45. Langle-Rouault F et al. Up to 100-fold increase of apparent gene expression in the presence of Epstein–Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids. J Virol 1998; 72: 6181–6185.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Vaysse L et al. Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein. J Biol Chem 2004; 279: 5555–5564.

    CAS  PubMed  Google Scholar 

  47. Dean DA . Nucleocytoplasmic trafficking. In: Mahato RI (ed). Pharmaceutical Perspectives of Nucleic Acid-based Therapeutics. Harwood Academic Publishers: London, 2002, pp 229–260.

    Google Scholar 

  48. Carson JA, Fillmore RA, Schwartz RJ, Zimmer WE . The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor. J Biol Chem 2000; 275: 39061–39072.

    CAS  PubMed  Google Scholar 

  49. Browning CL et al. The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes. Dev Biol 1998; 194: 18–37.

    CAS  PubMed  Google Scholar 

  50. Kovacs AM, Zimmer WE . Cell specific transcription of the smooth muscle γ-actin gene requires both positive and negative acting cis-elements. Gene Exp 1998; 7: 115–129.

    CAS  Google Scholar 

  51. Patterson C et al. Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. J Biol Chem 1995; 270: 23111–23118.

    CAS  PubMed  Google Scholar 

  52. Young JL, Byrd JN, Wyatt CR, Dean DA . Endothelial cell-specific plasmid nuclear import. Mol Biol Cell 1999; 10S: 443a.

    Google Scholar 

  53. Adam SA, Marr RS, Gerace L . Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 1990; 111: 807–816.

    CAS  PubMed  Google Scholar 

  54. Colin M et al. The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptide containing nuclear localisation properties. Gene Therapy 2001; 8: 1643–1653.

    CAS  PubMed  Google Scholar 

  55. Bustamante JO et al. Dendrimer-assisted patch-clamp sizing of nuclear pores. Pflugers Arch 2000; 439: 829–837.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Bustamante JO et al. Calcium, ATP and nuclear pore channel gating. Pflugers Arch 2000; 439: 433–444.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Bustamante JO . Nuclear pore ion channel behavior in live syncytial nuclei. Pflugers Arch 2002; 444: 286–290.

    CAS  PubMed  Google Scholar 

  58. Collas P, Alestrom P . Rapid targeting of plasmid DNA to zebrafish embryo nuclei by the nuclear localization signal of SV40 T antigen. Mol Mar Biol Biotechnol 1997; 6: 48–58.

    CAS  PubMed  Google Scholar 

  59. Collas P, Husebye H, Alestrom P . The nuclear localization sequence of the SV40T antigen promotes transgene uptake and expression in zebrafish embryo nuclei. Transgenic Res 1996; 5: 451–458.

    CAS  PubMed  Google Scholar 

  60. Collas P, Alestrom P . Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro. Mol Reprod Dev 1996; 45: 431–438.

    CAS  PubMed  Google Scholar 

  61. Aronsohn AI, Hughes JA . Nuclear localization signal peptides enhance cationic liposome-mediated gene therapy. J Drug Target 1998; 5: 163–169.

    CAS  PubMed  Google Scholar 

  62. Chan CK, Jans DA . Enhancement of polylysine-mediated transferrinfection by nuclear localization sequences: polylysine does not function as a nuclear localization sequence. Hum Gene Ther 1999; 10: 1695–1702.

    CAS  PubMed  Google Scholar 

  63. Subramanian A, Ranganathan P, Diamond SL . Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells. Nat Biotechnol 1999; 17: 873–877.

    CAS  PubMed  Google Scholar 

  64. Kaneda Y, Iwai K, Uchida T . Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science 1989; 243: 375–378.

    CAS  PubMed  Google Scholar 

  65. Fritz JD, Herweijer H, Zhang G, Wolff JA . Gene transfer into mammalian cells using histone-condensed plasmid DNA. Hum Gene Ther 1996; 7: 1395–1404.

    CAS  PubMed  Google Scholar 

  66. Bottger M et al. Acid nuclear extracts as mediators of gene transfer and expression. Biochim Biophys Acta 1998; 1395: 78–87.

    CAS  PubMed  Google Scholar 

  67. Hagstrom JE et al. Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation. Biochim Biophys Acta 1996; 1284: 47–55.

    PubMed  Google Scholar 

  68. Chan CK, Jans DA . Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway. Gene Therapy 2001; 8: 166–171.

    CAS  PubMed  Google Scholar 

  69. Hagstrom JE et al. Nuclear import of DNA in digitonin-permeabilized cells. J Cell Sci 1997; 110: 2323–2331.

    CAS  PubMed  Google Scholar 

  70. Ludtke JJ, Zhang G, Sebestyen MG, Wolff JA . A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA. J Cell Sci 1999; 112: 2033–2041.

    CAS  PubMed  Google Scholar 

  71. Ciolina C et al. Coupling of nuclear localization signals to plasmid DNA and specific interaction of the conjugates with importin alpha. Bioconjug Chem 1999; 10: 49–55.

    CAS  PubMed  Google Scholar 

  72. van der Aa MA et al. An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems. J Gene Med 2005; 7: 208–217.

    CAS  PubMed  Google Scholar 

  73. Zanta MA, Belguise-Valladier P, Behr JP . Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc Natl Acad Sci USA 1999; 96: 91–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Branden LJ, Mohamed AJ, Smith CI . A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA. Nat Biotechnol 1999; 17: 784–787.

    CAS  PubMed  Google Scholar 

  75. Neves C, Byk G, Scherman D, Wils P . Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation. FEBS Lett 1999; 453: 41–45.

    CAS  PubMed  Google Scholar 

  76. Zelphati O et al. PNA-dependent gene chemistry: stable coupling of peptides and oligonucleotides to plasmid DNA. Biotechniques 2000; 28: 304–316.

    CAS  PubMed  Google Scholar 

  77. Branden LJ, Christensson B, Smith CI . In vivo nuclear delivery of oligonucleotides via hybridizing bifunctional peptides. Gene Therapy 2001; 8: 84–87.

    CAS  PubMed  Google Scholar 

  78. Liang KW, Hoffman EP, Huang L . Targeted delivery of plasmid DNA to myogenic cells via transferrin-conjugated peptide nucleic acid. Mol Ther 2000; 1: 236–243.

    CAS  PubMed  Google Scholar 

  79. Morris MC et al. Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression. Gene Therapy 2004; 11: 757–764.

    CAS  PubMed  Google Scholar 

  80. Cartier R, Reszka R . Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems. Gene Therapy 2002; 9: 157–167.

    CAS  PubMed  Google Scholar 

  81. Carriere M, Escriou V, Savarin A, Scherman D . Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties. BMC Biotechnol 2003; 3: 14.

    PubMed  PubMed Central  Google Scholar 

  82. Rebuffat A et al. Selective enhancement of gene transfer by steroid-mediated gene delivery. Nat Biotechnol 2001; 19: 1155–1161.

    CAS  PubMed  Google Scholar 

  83. Rebuffat AG et al. Gene delivery by a steroid-peptide nucleic acid conjugate. FASEB J 2002; 16: 1426–1428.

    CAS  PubMed  Google Scholar 

  84. Arenal A et al. The SV40T antigen nuclear localization sequence enhances nuclear import of vector DNA in embryos of a crustacean (Litopenaeus schmitti). Gene 2004; 337: 71–77.

    CAS  PubMed  Google Scholar 

  85. Li S et al. Muscle-specific enhancement of gene expression by incorporation of the SV40 enhancer in the expression plasmid. Gene Therapy 2001; 8: 494–497.

    CAS  PubMed  Google Scholar 

  86. Salman H et al. Kinetics and mechanism of DNA uptake into the cell nucleus. Proc Natl Acad Sci USA 2001; 98: 7247–7252.

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Utvik JK, Nja A, Gundersen K . DNA injection into single cells of intact mice. Hum Gene Ther 1999; 10: 291–300.

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Rui Zhou and Joshua Z Gasiorowski for providing figures and all the members of our labs for intriguing discussions and critical reading of the manuscript. Work in the authors labs was supported in parts by grants HL59956 (DAD), HL71643 (DAD), and CA95608 (WEZ) from the NIH, and by The National Medical Test Bed and the US Department of the Army (Cooperative Agreement Number DAMD17-97-2-7016). The content of the information does not necessarily reflect the position or policy of the government or the NMTB. DDS is supported by the Office of Research and Development, Medical Research Service, Department of Veteran Affairs.

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  1. Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    D A Dean

  2. VA Loma Linda Health Care System, Loma Linda, CA, USA

    D D Strong

  3. Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University, College Station, TX, USA

    W E Zimmer

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Dean, D., Strong, D. & Zimmer, W. Nuclear entry of nonviral vectors. Gene Ther 12, 881–890 (2005). https://doi.org/10.1038/sj.gt.3302534

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