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Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation

Gene Therapy volume 9, pages 1351–1358 (2002)Cite this article

Abstract

Intramuscular (i.m.) injection of plasmids followed by electropermeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels. To further improve skeletal muscle as a bioreactor, we identified a formulation that elevates transgene expression in myofibers after i.m. injection and electroporation. With secreted placental alkaline phosphate (SEAP) as reporter gene, plasmid formulated with poly-L-glutamate produced two- to eight-fold higher levels of SEAP in mouse serum than plasmid in saline. Various concentrations and molecular weights of poly-L-glutamate were similarly effective, but 6 mg/ml of 15–50 kDa poly-L-glutamate consistently yielded the highest expression levels. The poly-L-glutamate formulation was effective in two different muscle groups in mice at various plasmid doses for several transgenes, including an erythropoietin (EPO) gene, for which expression was elevated four- to 12-fold in comparison to animals that received EPO plasmid in saline. Transgene expression was localized to myofibers. Poly-L-glutamate may improve transgene expression in part by increasing plasmid retention in skeletal muscle. Poly-L-glutamate did not enhance gene transfer in the absence of electroporation. Therefore, the polymer is a novel formulation that specifically enhances the transfer and expression of genes delivered with electroporation.

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References

  1. MacColl GS, Goldspink G, Bouloux PM . Using skeletal muscle as an artificial endocrine tissue J Endocrinol 1999 162: 1–9

    Article  CAS  PubMed  Google Scholar 

  2. Wolff JA et al. Direct gene transfer into mouse muscle in vivo Science 1990 247: 1465–1468

    Article  CAS  PubMed  Google Scholar 

  3. Manthorpe M et al. Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice Hum Gene Ther 1993 4: 419–431

    Article  CAS  PubMed  Google Scholar 

  4. Danko I et al. High expression of naked plasmid DNA in muscles of young rodents Hum Mol Genet 1997 6: 1435–1443

    Article  CAS  PubMed  Google Scholar 

  5. Zhang G et al. Efficient expression of naked DNA delivered intra-arterially to limb muscles of nonhuman primates Hum Gene Ther 2001 12: 427–438

    Article  CAS  PubMed  Google Scholar 

  6. Mir LM et al. High-efficiency gene transfer into skeletal muscle mediated by electric pulses Proc Natl Acad Sci USA 1999 96: 4262–4267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mumper RJ et al. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle J Controlled Release 1998 52: 191–203

    Article  CAS  Google Scholar 

  8. Lemieux P et al. A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle Gene Ther 2000 7: 986–991

    Article  CAS  PubMed  Google Scholar 

  9. Rols MP et al. In vivo electrically mediated protein and gene transfer in murine melanoma Nature Biotechnol 1998 16: 168–171

    Article  CAS  Google Scholar 

  10. Yoshizato K et al. Gene delivery with optimized electroporation parameters shows potential for treatment of gliomas Int J Oncol 2000 16: 899–905

    CAS  PubMed  Google Scholar 

  11. Goto T et al. Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene Proc Natl Acad Sci USA 2000 97: 354–359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Heller L et al. Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo Gene Ther 2000 7: 826–829

    Article  CAS  PubMed  Google Scholar 

  13. Titomirov AV, Sukharev S, Kistanova E . In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA Biochim Biophys Acta 1991 1088: 131–134

    Article  CAS  PubMed  Google Scholar 

  14. Banga AK, Prausnitz MR . Assessing the potential of skin electroporation for the delivery of protein- and gene-based drugs Trends Biotechnol 1998 16: 408–412

    Article  CAS  PubMed  Google Scholar 

  15. Heller R et al. In vivo gene electroinjection and expression in rat liver FEBS Lett 1996 389: 225–228

    Article  CAS  PubMed  Google Scholar 

  16. Suzuki T et al. Direct gene transfer into rat liver cells by in vivo electroporation FEBS Lett 1998 425: 436–440

    Article  CAS  PubMed  Google Scholar 

  17. Aihara H, Miyazaki J . Gene transfer into muscle by electroporation in vivo Nature Biotechnol 1998 16: 867–870

    Article  CAS  Google Scholar 

  18. Mathiesen I . Electropermeabilization of skeletal muscle enhances gene transfer in vivo Gene Ther 1999 6: 508–514

    Article  CAS  PubMed  Google Scholar 

  19. Vicat JM et al. Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression Hum Gene Ther 2000 11: 909–916

    Article  CAS  PubMed  Google Scholar 

  20. Kriess P et al. Erythropoietin secretion and physiological effect in mouse after intramuscular plasmid DNA electrotransfer J Gene Med 1999 1: 245–250

    Article  Google Scholar 

  21. Rizzuto G et al. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation Proc Natl Acad Sci USA 1999 96: 6417–6422

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Maruyama H et al. Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation Hum Gene Ther 2000 11: 429–437

    Article  CAS  PubMed  Google Scholar 

  23. Bettan M et al. High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle Mol Ther 2000 2: 204–210

    Article  CAS  PubMed  Google Scholar 

  24. Fewell JG et al. Gene therapy for the treatment of hemophilia B using PINC-formulated plasmid delivered to muscle with electroporation Mol Ther 2001 3: 574–583

    Article  CAS  PubMed  Google Scholar 

  25. Krieg AM . Direct Immunologic activities of CpG DNA and implications for gene therapy J Gene Med 1999 1: 56–63

    Article  CAS  PubMed  Google Scholar 

  26. Barry ME et al. Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection Hum Gene Ther 1999 10: 2461–2480

    Article  CAS  PubMed  Google Scholar 

  27. Mahato RI et al. Biodistribution and gene expression of lipid/plasmid complexes after systemic administration Hum Gene Ther 1998 9: 2083–2099

    Article  CAS  PubMed  Google Scholar 

  28. Mumper RJ et al. Polyvinyl derivatives as novel interactive polymers for controlled gene delivery to muscle Pharm Res 1996 13: 701–709

    Article  CAS  PubMed  Google Scholar 

  29. Levy MY et al. Mechanism of gene uptake and expression in adult mouse skeletal muscle Pharm Res 1994 11: 317

    Article  Google Scholar 

  30. Neumann E, Kakorin S, Toensing K . Fundamentals of electroporative delivery of drugs and genes Bioelectrochem Bioenerg 1999 48: 3–16

    Article  CAS  PubMed  Google Scholar 

  31. Sukharev SI et al. Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores Biophys J 1992 63: 1320–1327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wolf H et al. Control by pulse parameters of electric field-mediated gene transfer in mammalian cells Biophys J 1994 66: 524–531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fujii T et al. Inhibition of microtubule assembly by poly(L-glutamic acid) and the site of its action Biochem Cell Biol 1986 64: 615–621

    Article  CAS  PubMed  Google Scholar 

  34. Matteoni R, Kreis TE . Translocation and clustering of endosomes and lysosomes depends on microtubules J Cell Biol 1987 105: 1253–1265

    Article  CAS  PubMed  Google Scholar 

  35. Courvalin JC, Domontier M, Bornens M . Solubilization of nuclear structures by the polyanion heparin J Biol Chem 1982 257: 456–463

    CAS  PubMed  Google Scholar 

  36. Stein A, Mitchell M . Generation of different nucleosome spacing periodicities in vitro. Possible origin of cell type specificity J Mol Biol 1998 203: 1029–1043

    Article  Google Scholar 

  37. Moran RG . Roles of Folylpoly-γ-glutamate synthetase in therapeutics with tetrahydrofolate antimetabolites: an overview Semin Oncol 1999 26: 4–32

    Google Scholar 

  38. Wilcheck M, Frensdorff A, Sela M . New synthesis of poly-L-glutamic acid and poly-L-glutamyl proteins Arch Biochem Biophys 1966 113: 742–749

    Article  Google Scholar 

  39. Li C et al. Complete regression of well-established tumors using a novel water-soluble poly(L-glutamic acid)-paclitaxel conjugate Cancer Res 1998 58: 2404–2409

    CAS  PubMed  Google Scholar 

  40. Li C et al. Antitumor activity of poly(L-glutamic acid)-paclitaxel on syneneic and xenografted tumors Clin Cancer Res 1999 5: 891–897

    CAS  PubMed  Google Scholar 

  41. Singer JW et al. Water-soluble poly-(L-glutamic acid)-Gly-camptothecin conjugates enhance camptothecin stability and efficacy in vivo J Controlled Release 2001 74: 243–247

    Article  CAS  Google Scholar 

  42. Otani Y, Tabata Y, Ikada Y . Rapidly curable biological glue composed of gelatin and poly(L-glutamic acid) Biomaterials 1996 17: 1367–1391

    Article  Google Scholar 

  43. Otani Y, Tabata Y, Ikada Y . Hemostatic capability of rapidly curable glues from gelatin, poly(L-glutamic acid), and carbodiimide Biomaterials 1998 19: 2091–2098

    Article  CAS  PubMed  Google Scholar 

  44. Maurer PH . Antigenicity of polypeptides (poly-α-amino acids) J Immunol 1965 95: 1095–1099

    CAS  PubMed  Google Scholar 

  45. Abruzzese RV et al. Ligand-dependent regulation of plasmid-based transgene expression in vivo Hum Gene Ther 1999 10: 1499–1507

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank Margaret Gondo, Vidya Mehta, Debra Bruce, Ingrid Anscombe, Valarie Florack and Jason Fewell for technical assistance and useful comments.

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  1. F Nicol

    Present address: ALZA Corporation, 1900 Charleston Road, PO Box 7210, Mountain View, 94039–7210, CA, USA

Authors and Affiliations

  1. Valentis, Inc, The Woodlands, TX, USA

    F Nicol, M Wong, F C MacLaughlin, J Perrard, E Wilson, J L Nordstrom & L C Smith

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Nicol, F., Wong, M., MacLaughlin, F. et al. Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation. Gene Ther 9, 1351–1358 (2002). https://doi.org/10.1038/sj.gt.3301806

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  • DOI: https://doi.org/10.1038/sj.gt.3301806

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