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Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer

Gene Therapy volume 9, pages 963–971 (2002)Cite this article

Abstract

To test whether hepatocytes engineered in vivo can serve as surrogate β cells by similarly secreting mature insulin in a glucose-sensitive manner, we prepared adenoviral vectors encoding wild-type proinsulin (hIns-wt), a modified proinsulin cleavable by the ubiquitously expressed protease furin (hIns-M3), or each of the two β cell specific pro-insulin convertases PC2 and PC3. Following a detailed in vitro characterization of the proteins produced by our vectors, we infected the liver and, for comparison, the muscle of a chemically induced murine model of type I diabetes. Insulin expression from the transduced tissues was extensively characterized and showed to be constitutive rather than regulated. To obtain regulated expression, we placed expression of hIns-M3 under the control of the dimerizer-inducible transcription system. Hormone secretion from mouse liver was negligible in the absence of the dimerizer drug rapamycin, was inducible in a dose-dependent manner upon its administration, and reversible following drug withdrawal. These data confirm liver as a promising target for in vivo expression of processed insulin. While suggesting that hepatocytes cannot provide authentic glucose-responsive regulation, these results demonstrate that pharmacological regulation is a promising alternative route to the controlled delivery of insulin following hepatic gene transfer.

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References

  1. Taylor SI . Diabetes mellitus Scriver CM, Beaudet AL, Sly WS (eds); The Metabolic and Molecular Bases of Inherited Disease McGraw-Hill 1995 pp 843–896

  2. Rosenzweig JL . Principles of insulin therapy Kahn RC, Weir GC (eds); Joslin's Diabetes Mellitus Williams & Wilkins 1994 pp 460–488

  3. Halban PA, Kahn SE, Lernmark A, Rhodes CJ . Gene and cell-replacement therapy in the treatment of type 1 diabetes: how high must the standards be set? Diabetes 2001 50: 2181–2191

    Article  CAS  PubMed  Google Scholar 

  4. Shoelson SE, Halban PA . Insulin biosynthesis and chemistry Kahn RC, Weir GC (eds); Joslin's Diabetes Mellitus Williams & Wilkins 1994 pp 29–55

  5. Halban PA . Proinsulin processing in the regulated and the constitutive secretory pathway Diabetologia 1994 37: S65–S72

    Article  CAS  PubMed  Google Scholar 

  6. Halban PA . Proinsulin processing in the regulated and the constitutive secretory pathway Johnson JH, Newgard CB. The Role of Glucose Transport and Phosphorylation in Glucose-Stimulated Insulin Secretion. Lippincott-Raven: Philadelphia, 1996.

  7. Dong H, Woo SL . Hepatic insulin production for type 1 diabetes Trends Endocrinol Metab 2001 12: 441–446

    Article  CAS  PubMed  Google Scholar 

  8. Kafri T et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors Nat Genet 1997 17: 314–317

    Article  CAS  PubMed  Google Scholar 

  9. Fisher KJ et al. Recombinant adeno-associated virus for muscle directed gene therapy Nat Med 1997 3: 306–312

    Article  CAS  PubMed  Google Scholar 

  10. Snyder RO et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors Nat Genet 1997 16: 270–276

    Article  CAS  PubMed  Google Scholar 

  11. Zaret KS . Liver specification and early morphogenesis Mech Dev 2000 92: 83–88

    Article  CAS  PubMed  Google Scholar 

  12. Granner D, Pilkis S . The genes of hepatic glucose metabolism J Biol Chem 1990 265: 10173–10176

    CAS  PubMed  Google Scholar 

  13. Malhi H et al. KATP channels regulate mitogenically induced proliferation in primary rat hepatocytes and human liver cell lines. Implications for liver growth control and potential therapeutic targeting J Biol Chem 2000 275: 26050–26057

    Article  CAS  PubMed  Google Scholar 

  14. Simpson AM et al. Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g) Gene Therapy 1997 4: 1202–1215

    Article  CAS  PubMed  Google Scholar 

  15. Hayashi M, Taguchi M, Fujita T . The IRI release from insulin gene-transduced hepatic cells responds to ambient glucose concentration Biochem Biophys Res Commun 1997 233: 470–475

    Article  CAS  PubMed  Google Scholar 

  16. Rivera VM et al. A humanized system for pharmacologic control of gene expression Nat Med 1996 2: 1028–1032

    Article  CAS  PubMed  Google Scholar 

  17. Kaufmann JE, Irminger JC, Mungall J, Halban PA . Proinsulin conversion in GH3 cells after coexpression of human proinsulin with the endoproteases PC2 and/or PC3 Diabetes 1997 46: 978–982

    Article  CAS  PubMed  Google Scholar 

  18. Groskreutz DJ, Sliwkowski MX, Gorman CM . Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin J Biol Chem 1994 269: 6241–6245

    CAS  PubMed  Google Scholar 

  19. Rivera VM et al. Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum Science 2000 287: 826–830

    Article  CAS  PubMed  Google Scholar 

  20. Tiedge M et al. Engineering of a glucose-responsive surrogate cell for insulin replacement therapy of experimental insulin-dependent diabetes Hum Gene Ther 2000 11: 403–414

    Article  CAS  PubMed  Google Scholar 

  21. MacFarlane WM et al. Engineering a glucose-responsive human insulin-secreting cell line from islets of Langerhans isolated from a patient with persistent hyperinsulinemic hypoglycemia of infancy J Biol Chem 1999 274: 34059–34066

    Article  CAS  PubMed  Google Scholar 

  22. Rhoads DB et al. Evidence for expression of the facilitated glucose transporter in rat hepatocytes Proc Natl Acad Sci USA 1988 85: 9042–9046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kolodka TM, Finegold M, Moss L, Woo SL . Gene therapy for diabetes mellitus in rats by hepatic expression of insulin Proc Natl Acad Sci USA 1995 92: 3293–3297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Short DK, Okada S, Yamauchi K, Pessin JE . Adenovirus-mediated transfer of a modified human proinsulin gene reverses hyperglycemia in diabetic mice Am J Physiol 1998 275: E748–E756

    Article  CAS  PubMed  Google Scholar 

  25. Lee HC et al. Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue Nature 2000 408: 483–488

    Article  CAS  PubMed  Google Scholar 

  26. Chen R, Meseck ML, Woo SL . Auto-regulated hepatic insulin gene expression in type 1 diabetic rats Mol Ther 2001 3: 584–590

    Article  CAS  PubMed  Google Scholar 

  27. Dong H et al. Hepatic insulin expression improves glycemic control in type 1 diabetic rats Diabetes Res Clin Pract 2001 52: 153–163

    Article  CAS  PubMed  Google Scholar 

  28. Thule PM, Liu JM . Regulated hepatic insulin gene therapy of STZ-diabetic rats Gene Therapy 2000 7: 1744–1752

    Article  CAS  PubMed  Google Scholar 

  29. Irminger JC, Meyer K, Halban P . Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus Biochem J 1996 320: 11–15

    Article  PubMed  PubMed Central  Google Scholar 

  30. Smeekens SS et al. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2 and PC3 Proc Natl Acad Sci USA 1992 89: 8822–8826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Vollenweider F, Kaufmann J, Irminger JC, Halban PA . Processing of proinsulin by furin, PC2 and PC3 in (co) transfected COS (monkey kidney) cells Diabetes 1995 44: 1075–1080

    Article  CAS  PubMed  Google Scholar 

  32. Like AA, Rossini AA . Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus Science 1976 193: 415–417

    Article  CAS  PubMed  Google Scholar 

  33. Steiner DF et al. The role of prohormone convertases in insulin biosynthesis: evidence for inherited defects in their action in man and experimental animals Diabetes Metab 1996 22: 94–104

    CAS  PubMed  Google Scholar 

  34. Cheung AT et al. Glucose-dependent insulin release from genetically engineered K cells Science 2000 290: 1959–1962

    Article  CAS  PubMed  Google Scholar 

  35. Olefsky JM . Diabetes. Gene therapy for rats and mice (news) Nature 2000 408: 420–421

    Article  CAS  PubMed  Google Scholar 

  36. Ferber S et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia Nat Med 2000 6: 568–572

    Article  CAS  PubMed  Google Scholar 

  37. Rossi FM, Blau HM . Recent advances in inducible gene expression systems Curr Opin Biotechnol 1998 9: 451–456

    Article  CAS  PubMed  Google Scholar 

  38. Clackson T . Regulated gene expression systems Gene Therapy 2000 7: 120–125

    Article  CAS  PubMed  Google Scholar 

  39. Rivera VM et al. Long-term regulated expression of growth hormone in mice after intramuscular gene transfer Proc Natl Acad Sci USA 1999 96: 8657–8662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ye X et al. Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer Science 1999 283: 88–91

    Article  CAS  PubMed  Google Scholar 

  41. Davis AR, Meyers K, Wilson JM . High throughput method for creating and screening recombinant adenoviruses Gene Therapy 1998 5: 1148–1152

    Article  CAS  PubMed  Google Scholar 

  42. Gao GP, Yang Y, Wilson JM . Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy J Virol 1996 70: 8934–8943

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Gao GP, Yang Y, Wilson JM . Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy Harlow E, Lane D (eds). Immuno-affinity purification. In: Antibodies. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1988, pp 511–552.

  44. Auricchio A et al. Epidermal growth factor induces protein tyrosine phosphorylation and association of p190 with ras-GTP-ase activating protein in Caco-2 cells FEBS Lett 1994 353: 16–20

    Article  CAS  PubMed  Google Scholar 

  45. Allen E, Yu QC, Fuchs E . Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation J Cell Biol 1996 133: 1367–1382

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The technical assistance of the Animal Models group, Vector and Cell Morphology Cores of the Institute for Human Gene Therapy and of Xiurong Wang is gratefully acknowledged. We thank Alex Shifrin for helping with the animal studies. This work was supported by the NIH (P30 DK47757-07) and the Juvenile Diabetes Foundation. JMW holds equity in Targeted Genetics. AA is recipient of a fellowship from Telethon-Italia (371/B).

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Authors and Affiliations

  1. Department of Medicine, Institute for Human Gene Therapy, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA

    A Auricchio, G-P Gao, QC Yu & JM Wilson

  2. Department of Surgery, University of Pennsylvania, Philadelphia, PA, USA

    S Raper

  3. ARIAD Gene Therapeutics, Cambridge, MA, USA

    VM Rivera & T Clackson

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  1. A Auricchio
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  2. G-P Gao
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Auricchio, A., Gao, GP., Yu, Q. et al. Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer. Gene Ther 9, 963–971 (2002). https://doi.org/10.1038/sj.gt.3301746

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