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. 1992 Oct 1;89(19):8981–8985. doi: 10.1073/pnas.89.19.8981

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

R M Adams 1, H E Soriano 1, M Wang 1, G Darlington 1, D Steffen 1, F D Ledley 1
PMCID: PMC50048  PMID: 1329086

Abstract

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

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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. 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. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chowdhury J. R., Grossman M., Gupta S., Chowdhury N. R., Baker J. R., Jr, Wilson J. M. Long-term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR-deficient rabbits. Science. 1991 Dec 20;254(5039):1802–1805. doi: 10.1126/science.1722351. [DOI] [PubMed] [Google Scholar]
  6. Cornetta K., Morgan R. A., Anderson W. F. Safety issues related to retroviral-mediated gene transfer in humans. Hum Gene Ther. 1991 Spring;2(1):5–14. doi: 10.1089/hum.1991.2.1-5. [DOI] [PubMed] [Google Scholar]
  7. Davis G. R., Blumeyer K., DiMichele L. J., Whitfield K. M., Chappelle H., Riggs N., Ghosh S. S., Kao P. M., Fahy E., Kwoh D. Y. Detection of human immunodeficiency virus type 1 in AIDS patients using amplification-mediated hybridization analyses: reproducibility and quantitative limitations. J Infect Dis. 1990 Jul;162(1):13–20. doi: 10.1093/infdis/162.1.13. [DOI] [PubMed] [Google Scholar]
  8. Enat R., Jefferson D. M., Ruiz-Opazo N., Gatmaitan Z., Leinwand L. A., Reid L. M. Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1411–1415. doi: 10.1073/pnas.81.5.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fritsch E. F., Temin H. M. Inhibition of viral DNA synthesis in stationary chicken embryo fibroblasts infected with avian retroviruses. J Virol. 1977 Nov;24(2):461–469. doi: 10.1128/jvi.24.2.461-469.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  11. Gupta S., Aragona E., Vemuru R. P., Bhargava K. K., Burk R. D., Chowdhury J. R. Permanent engraftment and function of hepatocytes delivered to the liver: implications for gene therapy and liver repopulation. Hepatology. 1991 Jul;14(1):144–149. doi: 10.1002/hep.1840140124. [DOI] [PubMed] [Google Scholar]
  12. Gupta S., Chowdhury N. R., Jagtiani R., Gustin K., Aragona E., Shafritz D. A., Chowdhury J. R., Burk R. D. A novel system for transplantation of isolated hepatocytes utilizing HBsAg-producing transgenic donor cells. Transplantation. 1990 Sep;50(3):472–475. [PubMed] [Google Scholar]
  13. Jaenisch R., Hoffmann E. Transcription of endogenous C-type viruses in resting and proliferating tissues of BALB/Mo mice. Virology. 1979 Oct 30;98(2):289–297. doi: 10.1016/0042-6822(79)90552-x. [DOI] [PubMed] [Google Scholar]
  14. Kalser S. C., Hoofnagle J. H. Reports from the NIH DDDN. Human liver tissue for research. Gastroenterology. 1990 Apr;98(4):1086–1087. doi: 10.1016/0016-5085(90)90038-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lanford R. E., Carey K. D., Estlack L. E., Smith G. C., Hay R. V. Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium. In Vitro Cell Dev Biol. 1989 Feb;25(2):174–182. doi: 10.1007/BF02626175. [DOI] [PubMed] [Google Scholar]
  17. Ledley F. D. Clinical application of somatic gene therapy in inborn errors of metabolism. J Inherit Metab Dis. 1990;13(4):597–616. doi: 10.1007/BF01799515. [DOI] [PubMed] [Google Scholar]
  18. Ledley F. D., Darlington G. J., Hahn T., Woo S. L. Retroviral gene transfer into primary hepatocytes: implications for genetic therapy of liver-specific functions. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5335–5339. doi: 10.1073/pnas.84.15.5335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ledley F. D., Grenett H. E., McGinnis-Shelnutt M., Woo S. L. Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells. Proc Natl Acad Sci U S A. 1986 Jan;83(2):409–413. doi: 10.1073/pnas.83.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ledley F. D., Woo S. L., Ferry G. D., Whisennand H. H., Brandt M. L., Darlington G. J., Demmler G. J., Finegold M. J., Pokorny W. J., Rosenblatt H. Hepatocellular transplantation in acute hepatic failure and targeting genetic markers to hepatic cells. Hum Gene Ther. 1991 Winter;2(4):331–358. doi: 10.1089/hum.1991.2.4-331. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Miller A. D., Garcia J. V., von Suhr N., Lynch C. M., Wilson C., Eiden M. V. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol. 1991 May;65(5):2220–2224. doi: 10.1128/jvi.65.5.2220-2224.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  26. Miller D. G., Adam M. A., Miller A. D. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol. 1990 Aug;10(8):4239–4242. doi: 10.1128/mcb.10.8.4239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morgan R. A., Cornetta K., Anderson W. F. Applications of the polymerase chain reaction in retroviral-mediated gene transfer and the analysis of gene-marked human TIL cells. Hum Gene Ther. 1990 Summer;1(2):135–149. doi: 10.1089/hum.1990.1.2-135. [DOI] [PubMed] [Google Scholar]
  28. O'Neill R. R., Buckler C. E., Theodore T. S., Martin M. A., Repaske R. Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus. J Virol. 1985 Jan;53(1):100–106. doi: 10.1128/jvi.53.1.100-106.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ou C. Y., Moore J. L., Schochetman G. Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques. 1991 Apr;10(4):442–446. [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Rosenberg S. A., Aebersold P., Cornetta K., Kasid A., Morgan R. A., Moen R., Karson E. M., Lotze M. T., Yang J. C., Topalian S. L. Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med. 1990 Aug 30;323(9):570–578. doi: 10.1056/NEJM199008303230904. [DOI] [PubMed] [Google Scholar]
  33. Smith D. D., Jr, Campbell J. W. Distribution of glutamine synthetase and carbamoyl-phosphate synthetase I in vertebrate liver. Proc Natl Acad Sci U S A. 1988 Jan;85(1):160–164. doi: 10.1073/pnas.85.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Southard J. H., van Gulik T. M., Ametani M. S., Vreugdenhil P. K., Lindell S. L., Pienaar B. L., Belzer F. O. Important components of the UW solution. Transplantation. 1990 Feb;49(2):251–257. doi: 10.1097/00007890-199002000-00004. [DOI] [PubMed] [Google Scholar]
  35. Springett G. M., Moen R. C., Anderson S., Blaese R. M., Anderson W. F. Infection efficiency of T lymphocytes with amphotropic retroviral vectors is cell cycle dependent. J Virol. 1989 Sep;63(9):3865–3869. doi: 10.1128/jvi.63.9.3865-3869.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Threadgill D. W., Wilkmeyer M., Womack J. E., Ledley F. D. Localization of the murine methylmalonyl CoA mutase (Mut) locus on chromosome 17 by in situ hybridization. Cytogenet Cell Genet. 1990;53(2-3):112–114. doi: 10.1159/000132907. [DOI] [PubMed] [Google Scholar]
  37. Varmus H. E., Padgett T., Heasley S., Simon G., Bishop J. M. Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell. 1977 Jun;11(2):307–319. doi: 10.1016/0092-8674(77)90047-2. [DOI] [PubMed] [Google Scholar]
  38. Wilkemeyer M. F., Crane A. M., Ledley F. D. Primary structure and activity of mouse methylmalonyl-CoA mutase. Biochem J. 1990 Oct 15;271(2):449–455. doi: 10.1042/bj2710449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wilson J. M., Chowdhury J. R. Prospects for gene therapy of familial hypercholesterolemia. Mol Biol Med. 1990 Jun;7(3):223–232. [PubMed] [Google Scholar]
  40. Wilson J. M., Chowdhury N. R., Grossman M., Wajsman R., Epstein A., Mulligan R. C., Chowdhury J. R. Temporary amelioration of hyperlipidemia in low density lipoprotein receptor-deficient rabbits transplanted with genetically modified hepatocytes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8437–8441. doi: 10.1073/pnas.87.21.8437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wilson J. M., Johnston D. E., Jefferson D. M., Mulligan R. C. Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4421–4425. doi: 10.1073/pnas.85.12.4421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wolff J. A., Yee J. K., Skelly H. F., Moores J. C., Respess J. G., Friedmann T., Leffert H. Expression of retrovirally transduced genes in primary cultures of adult rat hepatocytes. Proc Natl Acad Sci U S A. 1987 May;84(10):3344–3348. doi: 10.1073/pnas.84.10.3344. [DOI] [PMC free article] [PubMed] [Google Scholar]

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