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. 1993 Mar 15;90(6):2122–2126. doi: 10.1073/pnas.90.6.2122

Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.

R J Cristiano 1, L C Smith 1, S L Woo 1
PMCID: PMC46037  PMID: 8384712

Abstract

We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bender M. A., Gelinas R. E., Miller A. D. A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells. Mol Cell Biol. 1989 Apr;9(4):1426–1434. doi: 10.1128/mcb.9.4.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cotten M., Längle-Rouault F., Kirlappos H., Wagner E., Mechtler K., Zenke M., Beug H., Birnstiel M. L. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4033–4037. doi: 10.1073/pnas.87.11.4033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crystal R. G. Alpha 1-antitrypsin deficiency, emphysema, and liver disease. Genetic basis and strategies for therapy. J Clin Invest. 1990 May;85(5):1343–1352. doi: 10.1172/JCI114578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Curiel D. T., Agarwal S., Wagner E., Cotten M. Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8850–8854. doi: 10.1073/pnas.88.19.8850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Curiel D. T., Wagner E., Cotten M., Birnstiel M. L., Agarwal S., Li C. M., Loechel S., Hu P. C. High-efficiency gene transfer mediated by adenovirus coupled to DNA-polylysine complexes. Hum Gene Ther. 1992 Apr;3(2):147–154. doi: 10.1089/hum.1992.3.2-147. [DOI] [PubMed] [Google Scholar]
  7. Dzierzak E. A., Papayannopoulou T., Mulligan R. C. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature. 1988 Jan 7;331(6151):35–41. doi: 10.1038/331035a0. [DOI] [PubMed] [Google Scholar]
  8. Jiao S., Williams P., Berg R. K., Hodgeman B. A., Liu L., Repetto G., Wolff J. A. Direct gene transfer into nonhuman primate myofibers in vivo. Hum Gene Ther. 1992 Feb;3(1):21–33. doi: 10.1089/hum.1992.3.1-21. [DOI] [PubMed] [Google Scholar]
  9. Kay M. A., Baley P., Rothenberg S., Leland F., Fleming L., Ponder K. P., Liu T., Finegold M., Darlington G., Pokorny W. Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):89–93. doi: 10.1073/pnas.89.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lim B., Apperley J. F., Orkin S. H., Williams D. A. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8892–8896. doi: 10.1073/pnas.86.22.8892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Liu T. J., Kay M. A., Darlington G. J., Woo S. L. Reconstitution of enzymatic activity in hepatocytes of phenylalanine hydroxylase-deficient mice. Somat Cell Mol Genet. 1992 Jan;18(1):89–96. doi: 10.1007/BF01233451. [DOI] [PubMed] [Google Scholar]
  12. MacGregor G. R., Mogg A. E., Burke J. F., Caskey C. T. Histochemical staining of clonal mammalian cell lines expressing E. coli beta galactosidase indicates heterogeneous expression of the bacterial gene. Somat Cell Mol Genet. 1987 May;13(3):253–265. doi: 10.1007/BF01535207. [DOI] [PubMed] [Google Scholar]
  13. Mannino R. J., Gould-Fogerite S. Liposome mediated gene transfer. Biotechniques. 1988 Jul-Aug;6(7):682–690. [PubMed] [Google Scholar]
  14. Massie B., Gluzman Y., Hassell J. A. Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen. Mol Cell Biol. 1986 Aug;6(8):2872–2883. doi: 10.1128/mcb.6.8.2872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McDonald J. D., Bode V. C. Hyperphenylalaninemia in the hph-1 mouse mutant. Pediatr Res. 1988 Jan;23(1):63–67. doi: 10.1203/00006450-198801000-00014. [DOI] [PubMed] [Google Scholar]
  16. Means G. E. Reductive alkylation of amino groups. Methods Enzymol. 1977;47:469–478. doi: 10.1016/0076-6879(77)47047-2. [DOI] [PubMed] [Google Scholar]
  17. Nielsen D. A., Chou J., MacKrell A. J., Casadaban M. J., Steiner D. F. Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5198–5202. doi: 10.1073/pnas.80.17.5198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Palmer T. D., Hock R. A., Osborne W. R., Miller A. D. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1055–1059. doi: 10.1073/pnas.84.4.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schwartz A. L., Rup D., Lodish H. F. Difficulties in the quantification of asialoglycoprotein receptors on the rat hepatocyte. J Biol Chem. 1980 Oct 10;255(19):9033–9036. [PubMed] [Google Scholar]
  20. Van Lenten L., Ashwell G. Studies on the chemical and enzymatic modification of glycoproteins. A general method for the tritiation of sialic acid-containing glycoproteins. J Biol Chem. 1971 Mar 25;246(6):1889–1894. [PubMed] [Google Scholar]
  21. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  22. Wagner E., Cotten M., Foisner R., Birnstiel M. L. Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4255–4259. doi: 10.1073/pnas.88.10.4255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wagner E., Zenke M., Cotten M., Beug H., Birnstiel M. L. Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc Natl Acad Sci U S A. 1990 May;87(9):3410–3414. doi: 10.1073/pnas.87.9.3410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Williams R. S., Johnston S. A., Riedy M., DeVit M. J., McElligott S. G., Sanford J. C. Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2726–2730. doi: 10.1073/pnas.88.7.2726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wilson J. M., Grossman M., Wu C. H., Chowdhury N. R., Wu G. Y., Chowdhury J. R. Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits. J Biol Chem. 1992 Jan 15;267(2):963–967. [PubMed] [Google Scholar]
  26. Wu C. H., Wilson J. M., Wu G. Y. Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J Biol Chem. 1989 Oct 15;264(29):16985–16987. [PubMed] [Google Scholar]
  27. Wu G. Y., Wilson J. M., Shalaby F., Grossman M., Shafritz D. A., Wu C. H. Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats. J Biol Chem. 1991 Aug 5;266(22):14338–14342. [PubMed] [Google Scholar]
  28. Wu G. Y., Wu C. H. Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro. Biochemistry. 1988 Feb 9;27(3):887–892. doi: 10.1021/bi00403a008. [DOI] [PubMed] [Google Scholar]
  29. Wu G. Y., Wu C. H. Receptor-mediated gene delivery and expression in vivo. J Biol Chem. 1988 Oct 15;263(29):14621–14624. [PubMed] [Google Scholar]
  30. Wu G. Y., Wu C. H. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem. 1987 Apr 5;262(10):4429–4432. [PubMed] [Google Scholar]
  31. Zenke M., Steinlein P., Wagner E., Cotten M., Beug H., Birnstiel M. L. Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells. Proc Natl Acad Sci U S A. 1990 May;87(10):3655–3659. doi: 10.1073/pnas.87.10.3655. [DOI] [PMC free article] [PubMed] [Google Scholar]

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