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Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme

An Erratum to this article was published on 08 November 2002

Abstract

Down-regulation of retroviral vector expression occurs in a number of cell types after transplantation. Although a number of vector elements have been shown to affect expression in specific experimental situations, the results can vary depending on the specific cDNA being expressed, the individual retroviral elements included in vectors, the promoter, or the inclusion of selectable markers. In previous experiments with the lysosomal enzyme β-glucuronidase, silencing has occurred in more than 95% of transduced cells regardless of the position of the expression unit within the vector, whether a eukaryotic or viral promoter was used, whether a bacterial selectable marker gene was present or not, the target cell type, or the species of the host. It has been a consistent finding that a small number of continuously expressing cells persist for long periods after transplantation. In this study we found that deletion of all the transcriptional regulatory elements from the vector LTR, inclusion of a permissive primer binding site sequence, and use of a eukaryotic housekeeping promoter could greatly increase the number of expressing cells in fibroblast grafts in subcutaneous neo-organs and in the brain. Furthermore, the level of enzyme expression was increased five-fold on a per positive cell basis, indicating that the deleted regulatory elements were exerting a negative effect on expression in the few cells that were positive before modification of the vector. This resulted in more than a 50-fold increase in total activity compared with the previous highest expressing vector.

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References

  1. Svoboda J et al. Retroviruses in foreign species and the problem of provirus silencing Gene 2000 261: 181–188

    Article  CAS  PubMed  Google Scholar 

  2. Halene S, Kohn DB . Gene therapy using hematopoietic stem cells: Sisyphus approaches the crest Hum Gene Ther 2000 11: 1259–1267

    Article  CAS  PubMed  Google Scholar 

  3. Palmer TD, Rosman GJ, Osborne WR, Miller AD . Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes Proc Natl Acad Sci USA 1991 88: 1330–1334

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Liu C et al. Long-term expression, systemic delivery, and macrophage uptake of recombinant human glucocerebrosidase in mice transplanted with genetically modified primary myoblasts Hum Gene Ther 1998 9: 2375–2384

    Article  CAS  PubMed  Google Scholar 

  5. Lynch CM et al. Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy Proc Natl Acad Sci USA 1992 89: 1138–1142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Barker JE, Wolfe JH, Rowe LB, Birkenmeier EH . Advantages of gradient vs 5-fluorouracil enrichment of stem cells for retroviral-mediated gene transfer Exp Hematol 1993 21: 47–54

    CAS  PubMed  Google Scholar 

  7. Casal ML, Wolfe JH . In utero transplantation of fetal liver cells in the mucopolysaccharidosis type VII mouse results in low-level chimerism, but overexpression of beta-glucuronidase can delay onset of clinical signs Blood 2001 97: 1625–1634

    Article  CAS  PubMed  Google Scholar 

  8. Lorincz MC et al. Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity J Biol Chem 1999 274: 657–665

    Article  CAS  PubMed  Google Scholar 

  9. Moullier P, Bohl D, Heard JM, Danos O . Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts Nat Genet 1993 4: 154–159

    Article  CAS  PubMed  Google Scholar 

  10. Taylor RM, Wolfe JH . Decreased lysosomal storage in the adult MPS VII mouse brain in the vicinity of grafts of retroviral vector-corrected fibroblasts secreting high levels of β-glucuronidase Nat Med 1997 3: 771–774

    Article  CAS  PubMed  Google Scholar 

  11. Wolfe JH et al. Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer Nature 1992 360: 749–753

    Article  CAS  PubMed  Google Scholar 

  12. Wolfe JH et al. Gene transfer of low levels of beta-glucuronidase corrects hepatic lysosomal storage in a large animal model of mucopolysaccharidosis VII Mol Ther 2000 2: 552–561

    Article  CAS  PubMed  Google Scholar 

  13. Bowtell DD, Cory S, Johnson GR, Gonda TJ . Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes J Virol 1988 62: 2464–2473

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Correll PH, Colilla S, Dave HP, Karlsson S . High levels of human glucocerebrosidase activity in macrophages of long-term reconstituted mice after retroviral infection of hematopoietic stem cells Blood 1992 80: 331–336

    CAS  PubMed  Google Scholar 

  15. Heim DA et al. Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model Mol Ther 2000 1: 533–544

    Article  CAS  PubMed  Google Scholar 

  16. Linney E et al. Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells Nature 1984 308: 470–472

    Article  CAS  PubMed  Google Scholar 

  17. Tsukiyama T, Niwa O, Yokoro K . Mechanism of suppression of the long terminal repeat of Moloney leukemia virus in mouse embryonal carcinoma cells Mol Cell Biol 1989 9: 4670–4676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Flanagan J, Krieg A, Max E, Khan A . Negative control region at the 5’ end of murine leukemia virus long terminal repeats Mol Cell Biol 1989 9: 739–746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Flanagan JR et al. Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus Mol Cell Biol 1992 12: 38–44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Feuer G, Taketo M, Hanecak RC, Fan H . Two blocks in Moloney murine leukemia virus expression in undifferentiated F9 embryonal carcinoma cells as determined by transient expression assays J Virol 1989 63: 2317–2324

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Loh TP, Sievert LL, Scott RW . Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells Mol Cell Biol 1990 10: 4045–4057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Boyes J, Bird A . DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein Cell 1991 64: 1123–1134

    Article  CAS  PubMed  Google Scholar 

  23. Cedar H . DNA methylation and gene activity Cell 1988 53: 3–4

    Article  CAS  PubMed  Google Scholar 

  24. Challita PM et al. Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells J Virol 1995 69: 748–755

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang L, Robbins PB, Carbonaro DA, Kohn DB . High-resolution analysis of cytosine methylation in the long terminal repeat of retroviral vectors Hum Gene Ther 1998 9: 2321–2330

    Article  CAS  PubMed  Google Scholar 

  26. Birkenmeier EH . Murine mucopolysaccharidosis type VII. Characterization of a mouse with beta-glucuronidase deficiency J Clin Invest 1989 83: 1258–1266

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Miller AD, Rosman GJ . Improved retroviral vectors for gene transfer and expression Biotechniques 1989 7: 980–982

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Park K, Atchison ML . Isolation of a candidate repressor/activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 3’ enhancer and the immunoglobulin heavy-chain mu E1 site Proc Natl Acad Sci USA 1991 88: 9804–9808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Petersen R, Kempler G, Barklis E . A stem cell-specific silencer in the primer-binding site of a retrovirus Mol Cell Biol 1991 11: 1214–1221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Halene S et al. Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector Blood 1999 94: 3349–3357

    CAS  PubMed  Google Scholar 

  31. Hawley RG, Lieu FH, Fong AZ, Hawley TS . Versatile retroviral vectors for potential use in gene therapy Gene Therapy 1994 1: 136–138

    CAS  PubMed  Google Scholar 

  32. Robbins PB et al. Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells Proc Natl Acad Sci USA 1998 95: 10182–10187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wolfe JH et al. High level expression and export of beta-glucuronidase from murine mucopolysaccharidosis VII cells corrected by a double-copy retrovirus vector Gene Therapy 1995 2: 70–78

    CAS  PubMed  Google Scholar 

  34. Hantzopoulos PA, Sullenger BA, Ungers G, Gilboa E . Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector Proc Natl Acad Sci USA 1989 86: 3519–3523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Grez M, Akgun E, Hilberg F, Ostertag W . Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells Proc Natl Acad Sci USA 1990 87: 9202–9206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shipley JM et al. Analysis of the 5’ flanking region of the human beta-glucuronidase gene Genomics 1991 10: 1009–1018

    Article  CAS  PubMed  Google Scholar 

  37. Southern PJ, Berg P . Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter J Mol Appl Genet 1982 1: 327–341

    CAS  PubMed  Google Scholar 

  38. Markowitz D, Goff S, Bank A . Construction and use of a safe and efficient amphotropic packaging cell line Virology 1988 167: 400–406

    Article  CAS  PubMed  Google Scholar 

  39. Wolfe JH, Sands MS . Murine mucopolysaccharidosis type VII: a model system for somatic gene therapy of the central nervous system Lowenstein PR, Enquist LW (eds); Protocols for Gene Transfer in Neuroscience: Towards Gene Therapy of Neurological Disorders John Wiley 1996 pp 263–274

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Acknowledgements

We thank A Polesky for expert technical assistance, and Drs C Kang for help with real-time PCR and M Atchison for reading the manuscript and helpful discussion. This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney diseases (DK42707, DK46637) to JHW. DKT was supported by the Veterinary Medical Scientist Training Grant (GM07170); GGH was supported by a fellowship from the National Institute of Mental Health (MH12285) and an NIH training grant to the Institute for Human Gene Therapy (DK07748).

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Prasad Alur, R., Foley, B., Parente, M. et al. Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme. Gene Ther 9, 1146–1154 (2002). https://doi.org/10.1038/sj.gt.3301777

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