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Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume

Gene Therapy volume 13, pages 1696–1702 (2006)Cite this article

Abstract

The naked plasmid DNA transfer method of rapid injection with large volume has been useful for gene therapy in experimental study. However, only small animals like rodents have usually been reported on. In this study, the authors attempted to transfect naked plasmid DNA to the porcine liver by modified hydrodynamic method. We decided to transfer plasmid DNA to a part of the liver using the angio-catheter to reduce the liver damage. To discern the condition of injection, naked plasmid DNA-encoding green fluorescent protein (GFP) was transferred for use as a marker gene. The GFP gene expression was markedly observed in gene-transferred pig livers. In large animals, not only the naked gene quantity, the solution volume containing the plasmid DNA and the injection speed, but also the additional treatments of the portal vein and the hepatic artery preparation were crucial. We found that the following injection condition were needed: plasmid DNA, 3 mg; the solution volume, 150 ml and the injection speed, 5 ml/s. The portal vein and the hepatic artery were clamped during gene delivery and the blood flow of the portal vein was flushed out using normal saline. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig) gene was used to test for secretory protein. CTLA4-Ig gene was injected with a large volume of solution via the hepatic vein to the left outer lobe of the liver selectively. CTLA4-Ig was detected in the pig blood at a maximum serum level of 161.7 ng/ml 1 day after gene transfer, and the CTLA4-Ig was detected for several weeks. Our new technique of inserting a catheter into only a selected portion of the liver reduced liver toxicity and increased gene transfer efficiency. This is the first report of successful gene transfer, using a hydrodynamic method, to the segmental liver in pigs, and achieved more than enough secretory protein for the clinically therapeutic level in pigs.

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References

  1. Niidome T, Huang L . Gene therapy progress and prospects: nonviral vectors. Gene Therapy 2002; 9: 1647–1652.

    Article  CAS  Google Scholar 

  2. Liu F, Song Y, Liu D . Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy 1999; 6: 1258–1266.

    Article  CAS  Google Scholar 

  3. Zhang G, Budker V, Wolff JA . High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 1999; 10: 1735–1737.

    Article  CAS  Google Scholar 

  4. Maruyama H, Higuchi N, Nishikawa Y, Kameda S, Iino N, Kazama JJ et al. High-level expression of naked DNA delivered to rat liver via tail vein injection. J Gene Med 2002; 4: 333–341.

    Article  CAS  Google Scholar 

  5. Liang KW, Nishikawa M, Liu F, Sun B, Ye Q, Huang L . Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA. Gene Therapy 2004; 11: 901–908.

    Article  CAS  Google Scholar 

  6. Danialou G, Comtois AS, Matecki S, Nalbantoglu J, Karpati G, Gilbert R et al. Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model. Mol Ther 2005; 11: 257–266.

    Article  CAS  Google Scholar 

  7. Zhang G, Budker V, Williams P, Subbotin V, Wolff JA . Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther 2001; 12: 427–438.

    Article  CAS  Google Scholar 

  8. Crespo A, Peydro A, Dasi F, Benet M, Calvete JJ, Revert F et al. Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles. Gene Therapy 2005; 12: 927–935.

    Article  CAS  Google Scholar 

  9. Zhang G, Gao X, Song YK, Vollmer R, Stolz DB, Gasiorowski JZ et al. Hydroporation as the mechanism of hydrodynamic delivery. Gene Therapy 2004; 11: 675–682.

    Article  CAS  Google Scholar 

  10. Eastman SJ, Baskin KM, Hodges BL, Chu Q, Gates A, Dreusicke R et al. Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA. Hum Gene Ther 2002; 13: 2065–2077.

    Article  CAS  Google Scholar 

  11. Zhang G, Vargo D, Budker V, Armstrong N, Knechtle S, Wolff JA . Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers. Hum Gene Ther 1997; 8: 1763–1772.

    Article  CAS  Google Scholar 

  12. Herrero MJ, Dasi F, Noguera I, Sanchez M, Moret I, Sanmartin I et al. Mouse and pig nonviral liver gene therapy: success and trials. Gene Ther Mol Biol 2005; 9: 169–180.

    Google Scholar 

  13. Inoue S, Hakamata Y, Kaneko M, Kobayashi E . Gene therapy for organ grafts using rapid injection of naked DNA: application to the rat liver. Transplantation 2004; 77: 997–1003.

    Article  CAS  Google Scholar 

  14. Sato Y, Ajiki T, Inoue S, Hakamata Y, Murakami T, Kaneko T et al. A novel gene therapy to the graft organ by a rapid injection of naked DNA I: long-lasting gene expression in a rat model of limb transplantation. Transplantation 2003; 76: 1294–1298.

    Article  CAS  Google Scholar 

  15. Sato Y, Ajiki T, Inoue S, Fujishiro J, Yoshino H, Igarashi Y et al. Gene silencing in rat-liver and limb grafts by rapid injection of small interference RNA. Transplantation 2005; 79: 240–243.

    Article  CAS  Google Scholar 

  16. Herweijer H, Wolff JA . Progress and prospects: naked DNA gene transfer and therapy. Gene Therapy 2003; 10: 453–458.

    Article  CAS  Google Scholar 

  17. Kawabata K, Takakura Y, Hashida M . The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharm Res 1995; 12: 825–830.

    Article  CAS  Google Scholar 

  18. Yang Z, Wu X, Tsui TY, Hou Y, Luk JM, Fan ST . Long-term liver allograft survival induced by combined treatment with rAAV-hCTLA4Ig gene transfer and low-dose FK506. Transplantation 2003; 75: 303–308.

    Article  CAS  Google Scholar 

  19. Nakamura M, Wang J, Murakami T, Ajiki T, Hakamata Y, Kaneko T et al. DNA immunization of the grafted liver by particle-mediated gene gun. Transplantation 2003; 76: 1369–1375.

    Article  CAS  Google Scholar 

  20. Komiya K, Sato Y, Wainai T, Murayama T, Yamada M, Hiruta A et al. Evaluation of intraoperative infusion solution using a complete anhepatic model in baby pigs. Transplant Proc 2005; 37: 2341–2346.

    Article  CAS  Google Scholar 

  21. Ajiki T, Murakami T, Kobayashi Y, Hakamata Y, Wang J, Inoue S et al. Long-lasting gene expression by particle-mediated intramuscular transfection modified with bupivacaine: combinatorial gene therapy with IL-12 and IL-18 cDNA against rat sarcoma at a distant site. Cancer Gene Ther 2003; 10: 318–329.

    Article  CAS  Google Scholar 

  22. Hakamata Y, Tahara K, Uchida H, Sakuma Y, Nakamura M, Kume A et al. Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophys Res Commun 2001; 286: 779–785.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan

    H Yoshino & E Kobayashi

  2. Department of Pediatric Surgery, University of Tokyo, Tokyo, Japan

    H Yoshino & K Hashizume

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  1. H Yoshino
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  2. K Hashizume
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  3. E Kobayashi
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Correspondence to H Yoshino or E Kobayashi.

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Yoshino, H., Hashizume, K. & Kobayashi, E. Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume. Gene Ther 13, 1696–1702 (2006). https://doi.org/10.1038/sj.gt.3302833

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  • DOI: https://doi.org/10.1038/sj.gt.3302833

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