Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Sep;86(17):6748–6752. doi: 10.1073/pnas.86.17.6748

Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells.

C Bordignon 1, S F Yu 1, C A Smith 1, P Hantzopoulos 1, G E Ungers 1, C A Keever 1, R J O'Reilly 1, E Gilboa 1
PMCID: PMC297923  PMID: 2549545

Abstract

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.

Full text

PDF
6748

Images in this article

6750Image
on p.6750
6751Image
on p.6751
6751Image
on p.6751
6751Image
on p.6751

Selected References

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

  1. Anderson W. F. Prospects for human gene therapy. Science. 1984 Oct 26;226(4673):401–409. doi: 10.1126/science.6093246. [DOI] [PubMed] [Google Scholar]
  2. Armentano D., Yu S. F., Kantoff P. W., von Ruden T., Anderson W. F., Gilboa E. Effect of internal viral sequences on the utility of retroviral vectors. J Virol. 1987 May;61(5):1647–1650. doi: 10.1128/jvi.61.5.1647-1650.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belmont J. W., Henkel-Tigges J., Chang S. M., Wager-Smith K., Kellems R. E., Dick J. E., Magli M. C., Phillips R. A., Bernstein A., Caskey C. T. Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer. Nature. 1986 Jul 24;322(6077):385–387. doi: 10.1038/322385a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Dick J. E., Magli M. C., Huszar D., Phillips R. A., Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell. 1985 Aug;42(1):71–79. doi: 10.1016/s0092-8674(85)80102-1. [DOI] [PubMed] [Google Scholar]
  6. Eglitis M. A., Kantoff P., Gilboa E., Anderson W. F. Gene expression in mice after high efficiency retroviral-mediated gene transfer. Science. 1985 Dec 20;230(4732):1395–1398. doi: 10.1126/science.2999985. [DOI] [PubMed] [Google Scholar]
  7. Gartner S., Kaplan H. S. Long-term culture of human bone marrow cells. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4756–4759. doi: 10.1073/pnas.77.8.4756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gilboa E. Retrovirus vectors and their uses in molecular biology. Bioessays. 1986 Dec;5(6):252–257. doi: 10.1002/bies.950050605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gordon M. Y., Riley G. P., Watt S. M., Greaves M. F. Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment. 1987 Mar 26-Apr 1Nature. 326(6111):403–405. doi: 10.1038/326403a0. [DOI] [PubMed] [Google Scholar]
  10. Hock R. A., Miller A. D. Retrovirus-mediated transfer and expression of drug resistance genes in human haematopoietic progenitor cells. Nature. 1986 Mar 20;320(6059):275–277. doi: 10.1038/320275a0. [DOI] [PubMed] [Google Scholar]
  11. Kantoff P. W., Gillio A. P., McLachlin J. R., Bordignon C., Eglitis M. A., Kernan N. A., Moen R. C., Kohn D. B., Yu S. F., Karson E. Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer. J Exp Med. 1987 Jul 1;166(1):219–234. doi: 10.1084/jem.166.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kantoff P. W., Kohn D. B., Mitsuya H., Armentano D., Sieberg M., Zwiebel J. A., Eglitis M. A., McLachlin J. R., Wiginton D. A., Hutton J. J. Correction of adenosine deaminase deficiency in cultured human T and B cells by retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6563–6567. doi: 10.1073/pnas.83.17.6563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keller G., Paige C., Gilboa E., Wagner E. F. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature. 1985 Nov 14;318(6042):149–154. doi: 10.1038/318149a0. [DOI] [PubMed] [Google Scholar]
  14. Kwok W. W., Schuening F., Stead R. B., Miller A. D. Retroviral transfer of genes into canine hemopoietic progenitor cells in culture: a model for human gene therapy. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4552–4555. doi: 10.1073/pnas.83.12.4552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lemischka I. R., Raulet D. H., Mulligan R. C. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell. 1986 Jun 20;45(6):917–927. doi: 10.1016/0092-8674(86)90566-0. [DOI] [PubMed] [Google Scholar]
  16. Meera Khan P. Enzyme electrophoresis on cellulose acetate gel: zymogram patterns in mgh-mouse and man--Chinese hamster somatic cell hybrids. Arch Biochem Biophys. 1971 Aug;145(2):470–483. doi: 10.1016/s0003-9861(71)80007-3. [DOI] [PubMed] [Google Scholar]
  17. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pike B. L., Robinson W. A. Human bone marrow colony growth in agar-gel. J Cell Physiol. 1970 Aug;76(1):77–84. doi: 10.1002/jcp.1040760111. [DOI] [PubMed] [Google Scholar]
  19. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  20. Spangrude G. J., Heimfeld S., Weissman I. L. Purification and characterization of mouse hematopoietic stem cells. Science. 1988 Jul 1;241(4861):58–62. doi: 10.1126/science.2898810. [DOI] [PubMed] [Google Scholar]
  21. Welte K., Platzer E., Lu L., Gabrilove J. L., Levi E., Mertelsmann R., Moore M. A. Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1526–1530. doi: 10.1073/pnas.82.5.1526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wiginton D. A., Adrian G. S., Friedman R. L., Suttle D. P., Hutton J. J. Cloning of cDNA sequences of human adenosine deaminase. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7481–7485. doi: 10.1073/pnas.80.24.7481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wiginton D. A., Kaplan D. J., States J. C., Akeson A. L., Perme C. M., Bilyk I. J., Vaughn A. J., Lattier D. L., Hutton J. J. Complete sequence and structure of the gene for human adenosine deaminase. Biochemistry. 1986 Dec 16;25(25):8234–8244. doi: 10.1021/bi00373a017. [DOI] [PubMed] [Google Scholar]
  24. Williams D. A., Orkin S. H., Mulligan R. C. Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2566–2570. doi: 10.1073/pnas.83.8.2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. van der Weyden M. B., Bailey L. A micromethod for determining adenosine deaminase and purine nucleoside phosphorylase activity in cells from human peripheral blood. Clin Chim Acta. 1978 Jan 2;82(1-2):179–184. doi: 10.1016/0009-8981(78)90041-4. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES