Generation of non-immunogenic islet cells using genetic engineering

  • Michael Brownlee


Can non-immunogenic insulin-producing pancreatic cells be created by genetic engineering? Brownlee relates the rationale for and substantive progress to date in this’ star Wars’ level research. By inserting an immortalized cell line with no potential for malignant mutagenesis, murine β-cells from transgenic mice expressing a rat insulin promoter-driven immortalizing gene plus an internal suicide gene were synthesized. Brownlee envisions combination of several genetic engineering strategies to produce a non-immunogenic β-cell line that will be clinically effective. Protection against autoimmune destruction would be included by transducing these cells with an anti-apoptotic gene. Alternatively, the insulin-producing cells would be encapsulated as a means of avoiding rejection. How long will it be before genetic engineering makes obsolete all previous treatment regimes for type 1 diabetes? As in so many fields of medicine, traditional physicians observe the rapidly arriving future in a state of wonderment. Clearly, 21st Century diabetes care is likely to be unrecognizable in the protocols now becoming obsolete.


Islet Cell Internal Ribosome Entry Site Islet Transplantation Autoimmune Destruction Islet Allograft 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Barker CF, Naji A. Perspectives in pancreatic and islet transplantation. N Engl J Med 327: 271–273.Google Scholar
  2. 2.
    Sarvetnick N, Shizuru J, Liggitt D, Martin L, Mclntyre B, Gregory A, Parslow T, Stewart T. Loss of pancreatic islet tolerance induced by beta-cell expression of interferon gamma. Nature 1990; 346: 844–847.PubMedCrossRefGoogle Scholar
  3. 3.
    Gray DW. The development and current status of pancreas and islet transplantation. Transplant Immunol 1994; 2: 127–129.CrossRefGoogle Scholar
  4. 4.
    Zeng YJ, Ricordi C, Tzakis A, Rilo HL, Carroll PB, Starzl TE, Ildstad ST. Long-term survival of donor-specific pancreatic islet xenografts in fully xenogeneic chimeras (WF rat-B10 mouse). Transplantation 1992; 53: 277–283.PubMedCrossRefGoogle Scholar
  5. 5.
    Li H, Colson YL, Ildstad ST. Mixed allogeneic chimerism achieved by lethal and nonlethal conditioning approaches induces donor-specific tolerance to simultaneous islet allografts. Transplantation 1996; 60: 523–529.CrossRefGoogle Scholar
  6. 6.
    Li H, Kaufman CL, Boggs SS, Johnson PC, Patrene KD, Ildstad ST. Mixed allogeneic chimerism induced by a sublethal approach prevents autoimmune diabetes and reverses insulitis in nonobese diabetic (NOD) mice. J Immunol 1996;156: 380–388.PubMedGoogle Scholar
  7. 7.
    Posselt AM, Barker CF, Tomaszewski JE, Markmann JF, Choti MA, Naji A. Induction of donor-specific unresponsiveness by intrathymic islet transplantation. Science 1990; 249: 1293–1295.PubMedCrossRefGoogle Scholar
  8. 8.
    Posselt AM, Barker CF, Friedman AL, Naji A. Prevention of autoimmune diabetes in the BB rat by intrathymic islet transplantation at birth. Science 1992; 256: 1321–1324.PubMedCrossRefGoogle Scholar
  9. 9.
    Lenschow DJ, Zeng Y, Hathcock KS, Zuckerman LA, Freeman G, Thistlethwaite JR, Gray GS. Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-l antibodies. Transplantation 1995; 60: 1171–1178.PubMedCrossRefGoogle Scholar
  10. 10.
    Larsen CP, Elwood ET, Alexander DZ, Ritchie SC, Hendrix R, Tucker-Burden C, Cho HR, Aruffo A, Hollenbaugh D, Linsley PS, Winn KJ, Pearson TC. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 1996; 381: 434–438.PubMedCrossRefGoogle Scholar
  11. 11.
    Lacy PE, Hegre OD, Gerasimidi-Vazeou A, Gentile FT, Dionne KE. Maintenance of normogly-cemia in diabetic mice by subcutaneous xenografts of encapsulated islets. Science 1991; 254: 1782–1784.PubMedCrossRefGoogle Scholar
  12. 12.
    Cole DR, Waterfall M, Mclntyre M, Baird JD. Microencapsulated islet grafts in the BB/E rat: a possible role for cytokines in graft failure. Diabetologia 1992; 35: 231–237.PubMedCrossRefGoogle Scholar
  13. 13.
    Devos P, Wolters GH, Fritschy WM, Van Schilfgaarde R. Obstacles in the application of microencapsulation in islet transplantation. Int J Artif Organs 1993; 16: 205–212.Google Scholar
  14. 14.
    Schrezenmeir J, Kirchgessner J, Gero L, Kunz LA, Beyer J, Mueller-Klieser W. Effect of microencapsulation on oxygen distribution in islets organs. Transplantation 1994; 57: 1308–1314.PubMedCrossRefGoogle Scholar
  15. 15.
    Sun Y, Ma X, Zhou D, Vacek I, Sun AM. Normalization of Diabetes in Spontaneously Diabetic Cynomologus monkeys by xenografts of microencapsulated porcine islets without immunosup-pression. J Clin Invest 1996: 98: 1417–1422.PubMedCrossRefGoogle Scholar
  16. 16.
    Efrat S, Fejer G, Brownlee M, Horwitz M. Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes. Proc Natl Acad Sci USA 1995; 92: 6947–6951.PubMedCrossRefGoogle Scholar
  17. 17.
    Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A, Duke RC. A role for CD95 ligand in preventing graft rejection. Nature 1995; 377: 630–632.PubMedCrossRefGoogle Scholar
  18. 18.
    Lau HT, Yu M, Fontana A, Stoeckert CJ Jr. Prevention of islet allograft rejection with engineered myoblasts expressing Fas L in mice. Science 1996; 273: 109–112.PubMedCrossRefGoogle Scholar
  19. 19.
    Soon-Shiong P, Heintz RE, Merideth N, Yao QX, Yao Z, Zheng T, Murphy M, Moloney MK, Schmehl M, Harris M, Mendez Robert, Mendez Raphael, Sandford P. Insulin independence in a type 1 diabetic patient after encapsulated islet transplantation. Lancet. 1994; 353: 950–951.CrossRefGoogle Scholar
  20. 20.
    Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley K A, Goodwin RG, Smith CA, Ramsdell F, Lynch DH. Fas ligand mediates activation-induced cell death in human T lymphocytes. J Exp Med 1995; 181: 71–77.PubMedCrossRefGoogle Scholar
  21. 21.
    Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Itoh N, Suda T Nagata S. Lethal effect of the anti-Fas antibody in mice. Nature 1993; 364: 806–809.PubMedCrossRefGoogle Scholar
  22. 22.
    Galle PR, Hofmann WJ, Walczak H, Schaller H, Otto G, Stremmel W, Krammer PH, Runkel L. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 1995; 182: 1223–1230.PubMedCrossRefGoogle Scholar
  23. 23.
    Rodriguez I, Matsuura K, Khatib K, Reed JC, Nagata S, Vassalli P. A bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection. J Exp Med 1996; 183: 1031–1036.PubMedCrossRefGoogle Scholar
  24. 24.
    Markmann JF, Bassiri H, Desai NM, Odorico JS, Kim JI, Koller BH, Smithies O, Barker CF. Indefinite survival of MHC Class I-Deficient murine pancreatic islet allocrafts. Transplantation 1992; 54: 1085–1089.PubMedCrossRefGoogle Scholar
  25. 25.
    Paabo S, Bhat BM, Wold WS, Peterson PA. A short sequence in the COOH-Terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum. Cell 1987; 50:311–317.PubMedCrossRefGoogle Scholar
  26. 26.
    Beier DC, Cox JH, Vining DR, Cresswell P, Engelhard VH. Association of human Class I MHC alleles with the adenovirus E3/19K protein1. J Immunol 1994; 152: 3862–3872.PubMedGoogle Scholar
  27. 27.
    Tanaka Y, Tevethia S. Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb-and H-2Db Class I antigens and H-2Kb-and H-2Db-restricted SV40-specific CTL-mediated lysis. Virology 1988; 165: 357–366.PubMedCrossRefGoogle Scholar
  28. 28.
    Oldstone MBA, Herrath M von, Evans CF, Horwitz MS. Virus-Induced autoimmune disease: Transgenic approach to mimic insulin-dependent diabetes mellitus and multiple sclerosis. Curr Top Microbiol Immunol 1995; 206: 67–83.CrossRefGoogle Scholar
  29. 29.
    Cole D, Waterfall M, McIntyre M, Baird J. Microencapsulated islet grafts in the BB/E rat: a possible role for cytokines in graft failure. Diabetologia 1992; 35: 231–237.PubMedCrossRefGoogle Scholar
  30. 30.
    Rabinovitch A, Suarez-Pinzon W, Shi Y, Morgan R, Bleackley R. DNA fragmentation is an early event in cytokine-induced islet beta cell destruction. Diabetologia 1994a; 37: 733–738.PubMedCrossRefGoogle Scholar
  31. 31.
    Almemri E, Robertson N, Fernandez T, Croce C, Litwack G. Overexpressed full-length human Bcl-2 extends survival of baculovirus-infected SF9 insect cells. Proc Natl Acad Sci USA 1992; 89: 7295–7299.CrossRefGoogle Scholar
  32. 32.
    Allsopp T, Wyatt S, Patterson H, Davies A. The proto-oncogene bcl-2 can selectively rescue neurotrophic factor dependent neurons from apoptosis. Cell 1993; 73: 295–307.PubMedCrossRefGoogle Scholar
  33. 33.
    Behl C, Hovey LI, Krajewski S, Schubert D, Reed J. Bcl-2 prevents killing of neuronal cells by glutamate but not by amyloid beta protein. Biochem. Biophys Res Commun 1993; 197: 949–956.PubMedCrossRefGoogle Scholar
  34. 34.
    Zhong L-T, Sarafian T, Kane D, Charles A, Mah S, Edwards R, Bredesen D. Bcl-2 inhibits death of central neural cells induced by multiple agents. Proc Natl Acad Sci USA 1993; 90: 4533–4537.PubMedCrossRefGoogle Scholar
  35. 35.
    Liu Y, Rabinovitch A, Suarez-Pinzon W, Muhkerjee B, Brownlee M, Edelstein D, Federoff HJ. Expression of the bcl-2 Gene from a Defective HSV-1 amplicon vector protects pancreatic b-cells from apoptosis. Human Gene Therapy 1996; 7: 1719–1726.PubMedCrossRefGoogle Scholar
  36. 36.
    Lacy PE, Davie JM. Transplantation of pancreatic islets. Annu Rev Immunol 1984; 2: 183–198.PubMedCrossRefGoogle Scholar
  37. 37.
    Efrat S, Linde S, Kofod H, Spector D, Delammoy M, Grant S, Hanahan D, Baekkeskov S. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci USA 1988; 85: 9037–9041.PubMedCrossRefGoogle Scholar
  38. 38.
    Hamaguch K, Gaskins HR, Leiter EH. NIT-1, a pancreatic β-cell line established from a transgenic NOD/Lt mouse. Diabetes 1991; 4: 842–849.CrossRefGoogle Scholar
  39. 39.
    Hicks BA, Stein R, Efrat S, Grant S, Hanahan D, Demetrion AA. Transplantation of beta cells from transgenic mice into nude athymic, diabetic rats restores glucose regulation. Diabetes Res Clin Pract 1991; 14: 157–164.PubMedCrossRefGoogle Scholar
  40. 40.
    Tal M, Thorens B, Surana M, Fleischer N, Lodish H, Hanahan D, Efrat S. Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice. Mol Cell Biol 1990; 12: 422–432.Google Scholar
  41. 41.
    Newgard, CB. Cellular engineering and gene therapy strategies for insulin replacement in diabetes. Diabetes 1994; 43: 341–350.PubMedCrossRefGoogle Scholar
  42. 42.
    Efrat S, Fusco-Demane D, Lemberg H, Emran OA, Wang X. Conditional transformation of a pancreatic B-cell line derives from transgenic mice expressing a tetracycline-regulated oncogene. Proc Natl Acad Sci USA 1995; 92: 3576–3580.PubMedCrossRefGoogle Scholar
  43. 43.
    Christopherson KS, Mark MR, Bajaj V, Godowski PJ. Ecdysteroid-dependent regulation of genes in mammalian cells by a Drosphila ecdysone receptor and chimeric transactivators. Proc Natl Acad Sci USA 1992; 89: 6314–6318.PubMedCrossRefGoogle Scholar
  44. 44.
    Wang Y, O’Malley JR, Tsai SY, O’Malley BW. A regulatory systen for use in gene transfer. Proc Natl Acad Sci 1994; 91: 8180–8184.PubMedCrossRefGoogle Scholar
  45. 45.
    Hanahan D. Heritable formation of pancreatic β-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 1985; 315: 115–122.PubMedCrossRefGoogle Scholar
  46. 46.
    Miyazaki JI, Araki K, Yamato E, Ikegami H, Asano T, Shibasaki Y, Oka Y, Yamamura KI. Establishment of a pancreatic β-cell line that retains glucose-inducible insulin secreation: special reference to expression of glucose transporter isoforms. Endocrinology 1990; 127: 126–132.PubMedCrossRefGoogle Scholar
  47. 47.
    Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination. 1.Recombination between loxp sites. J Mol Biol 1981; 150: 467–486.PubMedCrossRefGoogle Scholar
  48. 48.
    Sauer B. Manipulation of transgenes by sitespecific recombination: use of cre recombination. Meth Enzymol 1993; 225: 890–900.PubMedCrossRefGoogle Scholar
  49. 49.
    Gu H, Zou Y, Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch region evidenced through cre-loxp-mediated gene targeting. Cell 1993; 73: 1155.PubMedCrossRefGoogle Scholar
  50. 50.
    Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. Deletion of a DNA polymerase B gene segment in T cells using cell type-specific gene targeting. Science 1994; 265: 103–106.PubMedCrossRefGoogle Scholar
  51. 51.
    Borrelli E, Heyman R, Hsi M, Evans RM. Targeting of an inducible toxic phenotype in animal cells. Proc Natl Acad Sci USA 1988; 85: 7572–7576.PubMedCrossRefGoogle Scholar
  52. 52.
    Vile RG, Hart IR. Use of tissue-specific expression of the Herpes Simplex Virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA. Cancer Res 1993; 53: 3860–3864.PubMedGoogle Scholar
  53. 53.
    Freeman SM, Whartenby KA, Freeman JL, Abboud CN, Marrogi AJ. In situ use of suicide genes for cancer therapy. Semin Oncol 1996; 23: 31–45.PubMedGoogle Scholar
  54. 54.
    Schmid M, Wimmer E. IRES-controlled protein synthesis and genome replication of poliovirus. Arch Virol 9: 279–289.Google Scholar
  55. 55.
    Lexander L, Lu HH, Gromeler M, Wimmer E. Dicistronic poliovirus as expression vectors for foreign genes. AIDS Res Human Retrovir 1994; 10(Suppl 2): S57–S60.Google Scholar
  56. 56.
    Mountford P, Zevnik B, Duwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A. Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci USA 1994; 91: 4303–4307.PubMedCrossRefGoogle Scholar
  57. 57.
    Mountford P, Smith AG. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis. TIG 1995; 11: 179–184.PubMedCrossRefGoogle Scholar
  58. 58.
    Frenkel N, Singer O, Kwong AD. Minireview: the herpes simplex virus amplicon — a versatile defective virus vector. Gene Therapy 1994; 1(Suppl 1): S40–S46.PubMedGoogle Scholar
  59. 59.
    Glorioso JC, Deluca NA, Goins WF, Fink DJ. Development of herpes simplex virus vectors for gene transfer to the central nervour system. In: Wolff JA (ed.), Gene Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser, Cambridge, MA; 1994: 281–302.Google Scholar
  60. 60.
    Kaplitt MG, Kwong AD, Kleopoulos SP, Mobbs CV, Rabkin SD, Pfaff DW. Preproenkephalin promoter yields region-specific and long-term expression in adult brain after direct in vivo gene transfer via a defective herpes simplex viral vector. Proc Natl Acad Sci USA 1994; 91: 8979–8983.PubMedCrossRefGoogle Scholar
  61. 61.
    Mesri BA, Federoff HJ, Brownlee M. Expression of vascular endothelial growth factor from a defective herpes simplex virue type 1 amplicon vector induces angiogenesis in mice. Circ Res 1995; 76: 161–167.PubMedCrossRefGoogle Scholar
  62. 62.
    Geschwind MD, Hartnick CK, Liu W, Amat J, Van de Water TR, Federoff HJ. Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration. Human Gene Ther 1996; 7: 173–182.CrossRefGoogle Scholar
  63. 63.
    Riddell SR, Elliott M, Lewinsohn DA, Gilbert MJ, Wilson L, Manley SA, Lupton SD, Overell RW, Reynolds TC, Corey L, Greenbery PD. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocyes in HIV-infected patients. Nature Med 1996; 2: 216–223.PubMedCrossRefGoogle Scholar
  64. 64.
    Yang Y, Li Q, Ert H, Wilson J. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 1995; 69: 2004–2015.PubMedGoogle Scholar
  65. 65.
    Koenig S. A lesson from the HIV patient: the immune response is still the bane (or promise) of gene therapy. Nature Med 1996; 2: 165–167.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 1998

Authors and Affiliations

  • Michael Brownlee

There are no affiliations available

Personalised recommendations