Molecular Biotechnology

, Volume 38, Issue 2, pp 109–119 | Cite as

Naked Plasmid DNA-Based α-Galactosidase A Gene Transfer Partially Reduces Systemic Accumulation of Globotriaosylceramide in Fabry Mice

  • Gen Nakamura
  • Hiroki MaruyamaEmail author
  • Satoshi Ishii
  • Masaaki Shimotori
  • Shigemi Kameda
  • Toru Kono
  • Jun-ichi Miyazaki
  • Ashok B. Kulkarni
  • Fumitake Gejyo


Fabry disease is an X-linked recessive inborn metabolic disorder in which a deficiency in lysosomal enzyme α-galactosidase A (Gal A) causes the systemic accumulation of globotriaosylceramide (Gb3). Although many investigators have attempted to treat α-Gal A knock-out mice (Fabry mice) with gene therapy, no report has demonstrated therapeutic effects by the retrograde renal vein injection of naked DNA. We recently developed a naked plasmid vector-mediated kidney-targeted gene transfer technique. A solution containing naked plasmid DNA encoding human α-Gal A (pKSCX-α-Gal A) was rapidly injected into the left kidney of Fabry mice (pKSCX-α-Gal A mice). pKSCX was used for mock transfections (pKSCX mice). We confirmed that vector-derived human α-Gal A mRNA was present in the left kidney but not in other tissues, by reverse transcriptase polymerase chain reaction. Compared with the pKSCX mice, the pKSCX-α-Gal A mice showed partial therapeutic effects: increased α-Gal A activity in the injected kidney and in the liver, heart, and plasma, and decreased Gb3 in the injected kidney, contralateral kidney, liver, heart, and spleen. Our results demonstrated that, although further studies are needed to improve the outcome, this method has promise as a potential treatment option for Fabry disease.


Naked plasmid DNA Fabry disease Catheter-based gene transfer Renal vein injection CAG promoter Hydrodynamics-based transfection 



This work was partly supported by a Grant-in-aid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture and a Yujin Memorial Grant to Hiroki Maruyama.


  1. 1.
    Desnick R. J., Ionnou Y. A., & Eng C. M. (2001). α-Galactosidase A deficiency: Fabry disease, In Scriver C. R., Beaudet A. L., Sly W. S., & Valle D., Eds., The metabolic and molecular bases of inherited disease, 8th ed. (pp. 3733–3774). New York: McGraw-Hill.Google Scholar
  2. 2.
    Brady, R. O., Gal, A. E., Bradley, R. M., Martensson, E., Warshaw, A. L., & Laster, L. (1967). Enzymatic defect in Fabry’s disease. Ceramidetrihexosidase deficiency. New England Journal of Medicine, 276, 1163–1167.PubMedCrossRefGoogle Scholar
  3. 3.
    Marguery, M. C., Giordano, F., Parant, M., Samalens, G., Levade, T., Salvayre, R., Maret, A., Calvas, P., Bourrouillou, G., & Cantala, P. (1993). Fabry’s disease: Heterozygous form of different expression in two monozygous twin sisters. Dermatology, 187, 9–15.PubMedGoogle Scholar
  4. 4.
    Whybra, C., Kampmann, C., Willers, I., Davies, J., Winchester, B., Kriegsmann, J., Bruhl, K., Gal, A., Bunge, S., & Beck, M. (2001). Anderson-Fabry disease: clinical manifestations of disease in female heterozygotes. Journal of Inherited Metabolic Disease, 24, 715–724.PubMedCrossRefGoogle Scholar
  5. 5.
    Takahashi, H., Hirai, Y., Migita, M., Seino, Y., Fukuda, Y., Sakuraba, H., Kase, R., Kobayashi, T., & Hashimoto, Y. (2002). Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer. Proceedings of the National Academy of Sciences of the United States of America, 99, 13777–13782.PubMedCrossRefGoogle Scholar
  6. 6.
    Wolfe, J. H., Sands, M. S., Barker, J. E., Gwynn, B., Rowe, L. B., Vogler, C. A., & Birkenmeier, E. H. (1992). Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer. Nature, 360, 749–753.PubMedCrossRefGoogle Scholar
  7. 7.
    Ishii, S., Kase, R., & Sakuraba, H. (1995). The functional role of glutamine-280 and threonine-282 in human alpha-galactosidase. Biochimica et Biophysica Acta, 1270, 163–167.PubMedGoogle Scholar
  8. 8.
    Ziegler, R. J., Li, C., Cherry, M., Zhu, Y., Hempel, D., van Rooijen, N., Ioannou, Y. A., Desnick, R. J., Goldberg, M. A., Yew, N. S., & Cheng, S. H. (2002). Correction of the nonlinear dose response improves the viability of adenoviral vectors for gene therapy of Fabry disease. Human Gene Therapy, 13, 935–945.PubMedCrossRefGoogle Scholar
  9. 9.
    Jung, S. C., Han, I. P., Limaye, A., Xu, R., Gelderman, M. P., Zerfas, P., Tirumalai, K., Murray, G. J., During, M. J., Brady, R. O., & Qasba, P. (2001). Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice. Proceedings of the National Academy of Sciences of the United States of America, 98, 2676–2681.PubMedCrossRefGoogle Scholar
  10. 10.
    Ziegler, R. J., Lonning, S. M., Armentano, D., Li, C., Souza, D. W., Cherry, M., Ford, C., Barbon, C. M., Desnick, R. J., Gao, G., Wilson, J. M., Peluso, R., Godwin, S., Carter, B. J., Gregory, R. J., Wadworth, S. C., & Cheng, S. H. (2004). AAV2 vector harboring a liver-restricted promoter facilitates sustained expression of therapeutic levels of alpha-galactosidase A and the induction of immune tolerance in Fabry mice. Molecular Therapy, 9, 231–240.PubMedCrossRefGoogle Scholar
  11. 11.
    Li, C., Ziegler, R. J., Cherry, M., Desnick, R. J., Yew, N. S., & Cheng, S. H. (2002). Adenovirus-transduced lung as a portal for delivering alpha-galactosidase A into systemic circulation for Fabry disease. Molecular Therapy, 5, 745–754.PubMedCrossRefGoogle Scholar
  12. 12.
    Przybylska, M., Wu, I. H., Zhao, H., Ziegler, R. J., Tousignant, J. D., Desnick, R. J., Scheule, R. K., Cheng, S. H., & Yew, N. S. (2004). Partial correction of the alpha-galactosidase A deficiency and reduction of glycolipid storage in Fabry mice using synthetic vectors. Journal of Gene Medicine, 6, 85–92.PubMedCrossRefGoogle Scholar
  13. 13.
    Yoshimitsu, M., Sato, T., Tao, K., Walia, J. S., Rasaiah, V. I., Sleep, G. T., Murray, G. J., Poeppl, A. G., Underwood, J., West, L., Brady, R. O., & Medin, J. A. (2004). Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors. Proceedings of the National Academy of Sciences of the United States of America, 101, 16909–16914.PubMedCrossRefGoogle Scholar
  14. 14.
    Park, J., Murray, G. J., Limaye, A., Quirk, J. M., Gelderman, M. P., Brady, R. O., & Qasba, P. (2003). Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer. Proceedings of the National Academy of Sciences of the United States of America, 100, 3450–3454.PubMedCrossRefGoogle Scholar
  15. 15.
    Ziegler, R. J., Yew, N. S., Li, C., Cherry, M., Berthelette, P., Romanczuk, H., Ioannou, Y. A., Zeidner, K. M., Desnick, R. J., & Cheng, S. H. (1999). Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer. Human Gene Therapy, 10, 1667–1682.PubMedCrossRefGoogle Scholar
  16. 16.
    Lavigne, M. D., Pohlschmidt, M., Novo, J. F., Higgins, B., Alakhov, V., Lochmuller, H., Sakuraba, H., Goldspink, G., MacDermot, K., & Gorecki, D. C. (2005). Promoter dependence of plasmid-pluronics targeted alpha galactosidase A expression in skeletal muscle of Fabry mice. Molecular Therapy, 12, 985–990.PubMedCrossRefGoogle Scholar
  17. 17.
    Branton, M., Schiffmann, R., & Kopp, J. B. (2002). Natural history and treatment of renal involvement in Fabry disease. Journal of the American Society of Nephrology, 13(Suppl 2), 139–143.Google Scholar
  18. 18.
    Branton, M. H., Schiffmann, R., Sabnis, S. G., Murray, G. J., Quirk, J. M., Altarescu, G., Goldfarb, L., Brady, R. O., Balow, J. E., Austin, H. A. III, & Kopp, J. B. (2002). Natural history of Fabry renal disease: influence of alpha-galactosidase A activity and genetic mutations on clinical course. Medicine, 81, 122–138.PubMedCrossRefGoogle Scholar
  19. 19.
    Maruyama, H., Higuchi, N., Nishikawa, Y., Hirahara, H., Iino, N., Kameda, S., Kawachi, H., Yaoita, E., Gejyo, F., & Miyazaki, J. (2002). Kidney-targeted naked DNA transfer by retrograde renal vein injection in rats. Human Gene Therapy, 13, 455–468.PubMedCrossRefGoogle Scholar
  20. 20.
    Kameda, S., Maruyama, H., Higuchi, N., Iino, N., Nakamura, G., Miyazaki, J., & Gejyo, F. (2004). Kidney-targeted naked DNA transfer by retrograde injection into the renal vein in mice. Biochemical and Biophysical Research Communications, 314, 390–395.PubMedCrossRefGoogle Scholar
  21. 21.
    Niwa, H., Yamamura, K., & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108, 193–199.PubMedCrossRefGoogle Scholar
  22. 22.
    Maruyama, H., Sugawa, M., Moriguchi, Y., Imazeki, I., Ishikawa, Y., Ataka, K., Hasegawa, S., Ito, Y., Higuchi, N., Kazama, J. J., Gejyo, F., & Miyazaki, J. (2000). Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation. Human Gene Therapy, 11, 429–437.PubMedCrossRefGoogle Scholar
  23. 23.
    Ishii, S., Kase, R., Sakuraba, H., Fujita, S., Sugimoto, M., Tomita, K., Semba, T., & Suzuki, Y. (1994). Human alpha-galactosidase gene expression: significance of two peptide regions encoded by exons 1–2 and 6. Biochimica et Biophysica Acta, 1204, 265–270.PubMedGoogle Scholar
  24. 24.
    Ohshima, T., Murray, G. J., Swaim, W. D., Longenecker, G., Quirk, J. M., Cardarelli, C. O., Sugimoto, Y., Pastan, I., Gottesman, M. M., Brady, R. O., & Kulkarni, A. B. (1997). Alpha-galactosidase A deficient mice: A model of Fabry disease. Proceedings of the National Academy of Sciences of the United States of America, 94, 2540–2544.PubMedCrossRefGoogle Scholar
  25. 25.
    Ishii, S., Yoshioka, H., Mannen, K., Kulkarni, A. B., & Fan, J. Q. (2004). Transgenic mouse expressing human mutant alpha-galactosidase A in an endogenous enzyme deficient background: A biochemical animal model for studying active-site specific chaperone therapy for Fabry disease. Biochimica et Biophysica Acta, 1690, 250–257.PubMedGoogle Scholar
  26. 26.
    Fan, J. Q., Ishii, S., Asano, N., & Suzuki, Y. (1999). Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nature Medicine, 5, 112–115.PubMedCrossRefGoogle Scholar
  27. 27.
    Mayes, J. S., Scheerer, J. B., Sifers, R. N., & Donaldson, M.L. (1981). Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry’s disease. Clinica Chimica Acta., 112, 245–251.CrossRefGoogle Scholar
  28. 28.
    McCluer, R. H., Williams, M. A., Gross S. K., & Meisler, M. H., (1981). Testosterone effects on the induction and urinary excretion of mouse kidney glycosphingolipids associated with lysosomes. Journal of Biological Chemistry, 256, 13112–13120.PubMedGoogle Scholar
  29. 29.
    Pharmaceuticals and Medical Devices Agency for Evaluation of Medicinal Products. January 2004,
  30. 30.
    Ioannou, Y. A., Zeidner, K. M., Gordon, R. E., & Desnick, R. J. (2001). Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice. American Journal of Human Genetics, 68, 14–25.PubMedCrossRefGoogle Scholar
  31. 31.
    Brady, R. O., Murray, G. J., Moore, D. F., & Schiffmann, R. (2001). Enzyme replacement therapy in Fabry disease. Journal of Inherited Metabolic Disease, 24(Suppl 2), 18–24.PubMedCrossRefGoogle Scholar
  32. 32.
    Lee, K., Jin, X., Zhang, K., Copertino, L., Andrews, L., Baker-Malcolm, J., Geagan, L., Qiu, H., Seiger, K., Barngrover, D., McPherson, J. M., & Edmunds, T. (2003). A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease. Glycobiology, 13, 305–313.PubMedCrossRefGoogle Scholar
  33. 33.
    Monahan, P. E. & Samulski, R. J. (2000). Adeno-associated virus vectors for gene therapy: More pros than cons?. Molecular Medicine Today, 6, 433–440.CrossRefGoogle Scholar
  34. 34.
    Chirmule, N., Propert, K., Magosin, S., Qian, Y., Qian, R., & Wilson, J. (1999). Immune responses to adenovirus and adeno-associated virus in humans. Gene Therapy, 6, 1574–1583.PubMedCrossRefGoogle Scholar
  35. 35.
    Garman, S. C. & Garboczi, D. N. (2004). The molecular defect leading to Fabry disease: structure of human alpha-galactosidase. Journal of Molecular Biology, 337, 319–335.CrossRefGoogle Scholar
  36. 36.
    Schiffmann, R., Kopp, J. B., Austin, H. A., Sabnis, S., Moore, D. F., Weibel, T., Balow, J. E., & Brady, R. O. (2001). Enzyme replacement therapy in Fabry disease: a randomized controlled trial. JAMA—Journal of the American Medical Association, 285, 2743–2749.CrossRefGoogle Scholar
  37. 37.
    Stern, A. S., Klotman, M. E., Ioannou, Y. A., Burrow, C. R., Wilson, P. D., Klotman, P. E., & Lipkowitz, M. S. (2002). Polarity of alpha-galactosidase A uptake by renal tubule cells. Kidney International, 61(Suppl 1), 52–55.PubMedCrossRefGoogle Scholar
  38. 38.
    Thurberg, B. L., Rennke, H., Colvin, R. B., Dikman, S., Gordon, R. E., Collins, A. B., Desnick, R. J., & O’callaghan, M. (2002). Globotriaosylceramide accumulation in the Fabry kidney is cleared from multiple cell types after enzyme replacement therapy. Kidney International, 62, 1933–1946.PubMedCrossRefGoogle Scholar
  39. 39.
    Maddox D. A. & Brenner, B. M. (2000). Glomerular ultrafiltration, in Elements of normal renal structure and function, vol. 1. In Brenner B. M. (Ed.), Brenner & Rector’s The kidney 7th ed. (pp. 353–412). Philadelphia: WB Saunders.Google Scholar
  40. 40.
    Ye, P., Thompson, A. R., Sarkar, R., Shen, Z., Lillicrap, D. P., Kaufman, R. J., Ochs, H. D., Rawlings, D. J., & Miao, C. H. (2004). Naked DNA transfer of Factor VIII induced transgene-specific, species-independent immune response in hemophilia A mice. Molecular Therapy, 10, 117–126.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Gen Nakamura
    • 1
  • Hiroki Maruyama
    • 2
    Email author
  • Satoshi Ishii
    • 3
  • Masaaki Shimotori
    • 1
  • Shigemi Kameda
    • 1
  • Toru Kono
    • 4
  • Jun-ichi Miyazaki
    • 5
  • Ashok B. Kulkarni
    • 6
  • Fumitake Gejyo
    • 1
  1. 1.Division of Clinical Nephrology and RheumatologyNiigata University Graduate School of Medical and Dental SciencesNiigataJapan
  2. 2.Department of Clinical NephroscienceNiigata University Graduate School of Medical and Dental SciencesNiigataJapan
  3. 3.Department of Agricultural and Life SciencesObihiro University of Agriculture and Veterinary MedicineObihiroJapan
  4. 4.Division of Gastroenterology, Department of Surgery IIAsahikawa Medical CollegeAsahikawaJapan
  5. 5.Division of Stem Cell Regulation Research, G6Osaka University Medical SchoolSuitaJapan
  6. 6.Functional Genomics Section CDBRB NIDCR, NIHBethesdaUSA

Personalised recommendations