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. 1994 Jul 1;13(13):3065–3076. doi: 10.1002/j.1460-2075.1994.tb06605.x

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.

P Leboulch 1, G M Huang 1, R K Humphries 1, Y H Oh 1, C J Eaves 1, D Y Tuan 1, I M London 1
PMCID: PMC395197  PMID: 8039501

Abstract

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.

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Selected References

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

  1. Behringer R. R., Hammer R. E., Brinster R. L., Palmiter R. D., Townes T. M. Two 3' sequences direct adult erythroid-specific expression of human beta-globin genes in transgenic mice. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7056–7060. doi: 10.1073/pnas.84.20.7056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender M. A., Gelinas R. E., Miller A. D. A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells. Mol Cell Biol. 1989 Apr;9(4):1426–1434. doi: 10.1128/mcb.9.4.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang J. C., Liu D., Kan Y. W. A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3107–3110. doi: 10.1073/pnas.89.7.3107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chao M. V., Mellon P., Charnay P., Maniatis T., Axel R. The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells. Cell. 1983 Feb;32(2):483–493. doi: 10.1016/0092-8674(83)90468-3. [DOI] [PubMed] [Google Scholar]
  5. Collis P., Antoniou M., Grosveld F. Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression. EMBO J. 1990 Jan;9(1):233–240. doi: 10.1002/j.1460-2075.1990.tb08100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cone R. D., Weber-Benarous A., Baorto D., Mulligan R. C. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. Mol Cell Biol. 1987 Feb;7(2):887–897. doi: 10.1128/mcb.7.2.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Curtin P. T., Liu D. P., Liu W., Chang J. C., Kan Y. W. Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7082–7086. doi: 10.1073/pnas.86.18.7082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Danos O., Mulligan R. C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6460–6464. doi: 10.1073/pnas.85.17.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deshler J. O., Rossi J. J. Unexpected point mutations activate cryptic 3' splice sites by perturbing a natural secondary structure within a yeast intron. Genes Dev. 1991 Jul;5(7):1252–1263. doi: 10.1101/gad.5.7.1252. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dzierzak E. A., Papayannopoulou T., Mulligan R. C. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature. 1988 Jan 7;331(6151):35–41. doi: 10.1038/331035a0. [DOI] [PubMed] [Google Scholar]
  12. Forrester W. C., Novak U., Gelinas R., Groudine M. Molecular analysis of the human beta-globin locus activation region. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5439–5443. doi: 10.1073/pnas.86.14.5439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Forrester W. C., Thompson C., Elder J. T., Groudine M. A developmentally stable chromatin structure in the human beta-globin gene cluster. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1359–1363. doi: 10.1073/pnas.83.5.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fraser C. C., Szilvassy S. J., Eaves C. J., Humphries R. K. Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1968–1972. doi: 10.1073/pnas.89.5.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gelinas R., Frazier A., Harris E. A normal level of beta-globin expression in erythroid cells after retroviral cells transfer. Bone Marrow Transplant. 1992;9 (Suppl 1):154–157. [PubMed] [Google Scholar]
  16. Grabowski P. J., Nasim F. U., Kuo H. C., Burch R. Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1991 Dec;11(12):5919–5928. doi: 10.1128/mcb.11.12.5919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grosveld F., van Assendelft G. B., Greaves D. R., Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 1987 Dec 24;51(6):975–985. doi: 10.1016/0092-8674(87)90584-8. [DOI] [PubMed] [Google Scholar]
  18. Hu W. S., Temin H. M. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. doi: 10.1126/science.1700865. [DOI] [PubMed] [Google Scholar]
  19. Hughes P. F., Thacker J. D., Hogge D., Sutherland H. J., Thomas T. E., Lansdorp P. M., Eaves C. J., Humphries R. K. Retroviral gene transfer to primitive normal and leukemic hematopoietic cells using clinically applicable procedures. J Clin Invest. 1992 Jun;89(6):1817–1824. doi: 10.1172/JCI115786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Humphries R. K., Eaves A. C., Eaves C. J. Self-renewal of hemopoietic stem cells during mixed colony formation in vitro. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3629–3633. doi: 10.1073/pnas.78.6.3629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Karlsson S., Bodine D. M., Perry L., Papayannopoulou T., Nienhuis A. W. Expression of the human beta-globin gene following retroviral-mediated transfer into multipotential hematopoietic progenitors of mice. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6062–6066. doi: 10.1073/pnas.85.16.6062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim K. J., Kanellopoulos-Langevin C., Merwin R. M., Sachs D. H., Asofsky R. Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol. 1979 Feb;122(2):549–554. [PubMed] [Google Scholar]
  23. 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]
  24. Luo G. X., Sharmeen L., Taylor J. Specificities involved in the initiation of retroviral plus-strand DNA. J Virol. 1990 Feb;64(2):592–597. doi: 10.1128/jvi.64.2.592-597.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maniatis T. Mechanisms of alternative pre-mRNA splicing. Science. 1991 Jan 4;251(4989):33–34. doi: 10.1126/science.1824726. [DOI] [PubMed] [Google Scholar]
  26. McIvor R. S. Deletion in a recombinant retroviral vector resulting from a cryptic splice donor signal in the Moloney leukemia virus envelope gene. Virology. 1990 Jun;176(2):652–655. doi: 10.1016/0042-6822(90)90039-t. [DOI] [PubMed] [Google Scholar]
  27. Miller A. D., Bender M. A., Harris E. A., Kaleko M., Gelinas R. E. Design of retrovirus vectors for transfer and expression of the human beta-globin gene. J Virol. 1988 Nov;62(11):4337–4345. doi: 10.1128/jvi.62.11.4337-4345.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Moschonas N., de Boer E., Flavell R. A. The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences. Nucleic Acids Res. 1982 Mar 25;10(6):2109–2120. doi: 10.1093/nar/10.6.2109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mulligan R. C. The basic science of gene therapy. Science. 1993 May 14;260(5110):926–932. doi: 10.1126/science.8493530. [DOI] [PubMed] [Google Scholar]
  31. Ney P. A., Sorrentino B. P., McDonagh K. T., Nienhuis A. W. Tandem AP-1-binding sites within the human beta-globin dominant control region function as an inducible enhancer in erythroid cells. Genes Dev. 1990 Jun;4(6):993–1006. doi: 10.1101/gad.4.6.993. [DOI] [PubMed] [Google Scholar]
  32. Novak U., Harris E. A., Forrester W., Groudine M., Gelinas R. High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells. Proc Natl Acad Sci U S A. 1990 May;87(9):3386–3390. doi: 10.1073/pnas.87.9.3386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Palacios R., Steinmetz M. Il-3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo. Cell. 1985 Jul;41(3):727–734. doi: 10.1016/s0092-8674(85)80053-2. [DOI] [PubMed] [Google Scholar]
  34. Philipsen S., Talbot D., Fraser P., Grosveld F. The beta-globin dominant control region: hypersensitive site 2. EMBO J. 1990 Jul;9(7):2159–2167. doi: 10.1002/j.1460-2075.1990.tb07385.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Plavec I., Papayannopoulou T., Maury C., Meyer F. A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells. Blood. 1993 Mar 1;81(5):1384–1392. [PubMed] [Google Scholar]
  36. Pruzina S., Hanscombe O., Whyatt D., Grosveld F., Philipsen S. Hypersensitive site 4 of the human beta globin locus control region. Nucleic Acids Res. 1991 Apr 11;19(7):1413–1419. doi: 10.1093/nar/19.7.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pulsinelli G. A., Temin H. M. Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer. J Virol. 1991 Sep;65(9):4786–4797. doi: 10.1128/jvi.65.9.4786-4797.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ryan T. M., Behringer R. R., Martin N. C., Townes T. M., Palmiter R. D., Brinster R. L. A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice. Genes Dev. 1989 Mar;3(3):314–323. doi: 10.1101/gad.3.3.314. [DOI] [PubMed] [Google Scholar]
  40. Szilvassy S. J., Fraser C. C., Eaves C. J., Lansdorp P. M., Eaves A. C., Humphries R. K. Retrovirus-mediated gene transfer to purified hemopoietic stem cells with long-term lympho-myelopoietic repopulating ability. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8798–8802. doi: 10.1073/pnas.86.22.8798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Talbot D., Philipsen S., Fraser P., Grosveld F. Detailed analysis of the site 3 region of the human beta-globin dominant control region. EMBO J. 1990 Jul;9(7):2169–2177. doi: 10.1002/j.1460-2075.1990.tb07386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tuan D. Y., Solomon W. B., London I. M., Lee D. P. An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "beta-like globin" genes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2554–2558. doi: 10.1073/pnas.86.8.2554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tuan D., London I. M. Mapping of DNase I-hypersensitive sites in the upstream DNA of human embryonic epsilon-globin gene in K562 leukemia cells. Proc Natl Acad Sci U S A. 1984 May;81(9):2718–2722. doi: 10.1073/pnas.81.9.2718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tuan D., Solomon W., Li Q., London I. M. The "beta-like-globin" gene domain in human erythroid cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6384–6388. doi: 10.1073/pnas.82.19.6384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Turhan A. G., Humphries R. K., Phillips G. L., Eaves A. C., Eaves C. J. Clonal hematopoiesis demonstrated by X-linked DNA polymorphisms after allogeneic bone marrow transplantation. N Engl J Med. 1989 Jun 22;320(25):1655–1661. doi: 10.1056/NEJM198906223202504. [DOI] [PubMed] [Google Scholar]
  46. Walters M., Kim C., Gelinas R. Characterization of a DNA binding activity in DNAse I hypersensitive site 4 of the human globin locus control region. Nucleic Acids Res. 1991 Oct 11;19(19):5385–5393. doi: 10.1093/nar/19.19.5385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Warner N. L., Moore M. A., Metcalf D. A transplantable myelomonocytic leukemia in BALB-c mice: cytology, karyotype, and muramidase content. J Natl Cancer Inst. 1969 Oct;43(4):963–982. [PubMed] [Google Scholar]
  48. Wieringa B., Hofer E., Weissmann C. A minimal intron length but no specific internal sequence is required for splicing the large rabbit beta-globin intron. Cell. 1984 Jul;37(3):915–925. doi: 10.1016/0092-8674(84)90426-4. [DOI] [PubMed] [Google Scholar]

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