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. 1993 Dec 15;90(24):11548–11552. doi: 10.1073/pnas.90.24.11548

Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

R J Cristiano 1, L C Smith 1, M A Kay 1, B R Brinkley 1, S L Woo 1
PMCID: PMC48021  PMID: 8265587

Abstract

Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies.

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Selected References

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

  1. Armentano D., Thompson A. R., Darlington G., Woo S. L. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: potential for gene therapy of hemophilia B. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6141–6145. doi: 10.1073/pnas.87.16.6141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Belmont J. W., MacGregor G. R., Wager-Smith K., Fletcher F. A., Moore K. A., Hawkins D., Villalon D., Chang S. M., Caskey C. T. Expression of human adenosine deaminase in murine hematopoietic cells. Mol Cell Biol. 1988 Dec;8(12):5116–5125. doi: 10.1128/mcb.8.12.5116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Cristiano R. J., Smith L. C., Woo S. L. Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2122–2126. doi: 10.1073/pnas.90.6.2122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Foisner R., Wiche G. Promotion of MAP/MAP interaction by taxol. J Ultrastruct Res. 1985 Oct-Nov;93(1-2):33–41. doi: 10.1016/0889-1605(85)90083-7. [DOI] [PubMed] [Google Scholar]
  10. Gao L., Wagner E., Cotten M., Agarwal S., Harris C., Rømer M., Miller L., Hu P. C., Curiel D. Direct in vivo gene transfer to airway epithelium employing adenovirus-polylysine-DNA complexes. Hum Gene Ther. 1993 Feb;4(1):17–24. doi: 10.1089/hum.1993.4.1-17. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Kay M. A., Li Q., Liu T. J., Leland F., Toman C., Finegold M., Woo S. L. Hepatic gene therapy: persistent expression of human alpha 1-antitrypsin in mice after direct gene delivery in vivo. Hum Gene Ther. 1992 Dec;3(6):641–647. doi: 10.1089/hum.1992.3.6-641. [DOI] [PubMed] [Google Scholar]
  13. Kay M. A., Rothenberg S., Landen C. N., Bellinger D. A., Leland F., Toman C., Finegold M., Thompson A. R., Read M. S., Brinkhous K. M. In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs. Science. 1993 Oct 1;262(5130):117–119. doi: 10.1126/science.8211118. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Michael S. I., Huang C. H., Rømer M. U., Wagner E., Hu P. C., Curiel D. T. Binding-incompetent adenovirus facilitates molecular conjugate-mediated gene transfer by the receptor-mediated endocytosis pathway. J Biol Chem. 1993 Apr 5;268(10):6866–6869. [PubMed] [Google Scholar]
  17. Nabel E. G., Gordon D., Yang Z. Y., Xu L., San H., Plautz G. E., Wu B. Y., Gao X., Huang L., Nabel G. J. Gene transfer in vivo with DNA-liposome complexes: lack of autoimmunity and gonadal localization. Hum Gene Ther. 1992 Dec;3(6):649–656. doi: 10.1089/hum.1992.3.6-649. [DOI] [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. Peng H., Armentano D., MacKenzie-Graham L., Shen R. F., Darlington G., Ledley F. D., Woo S. L. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8146–8150. doi: 10.1073/pnas.85.21.8146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ponder K. P., Gupta S., Leland F., Darlington G., Finegold M., DeMayo J., Ledley F. D., Chowdhury J. R., Woo S. L. Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1217–1221. doi: 10.1073/pnas.88.4.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wagner E., Plank C., Zatloukal K., Cotten M., Birnstiel M. L. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7934–7938. doi: 10.1073/pnas.89.17.7934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wagner E., Zatloukal K., Cotten M., Kirlappos H., Mechtler K., Curiel D. T., Birnstiel M. L. Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6099–6103. doi: 10.1073/pnas.89.13.6099. [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. Wickham T. J., Mathias P., Cheresh D. A., Nemerow G. R. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell. 1993 Apr 23;73(2):309–319. doi: 10.1016/0092-8674(93)90231-e. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. Yang Y., Raper S. E., Cohn J. A., Engelhardt J. F., Wilson J. M. An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4601–4605. doi: 10.1073/pnas.90.10.4601. [DOI] [PMC free article] [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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