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Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors

Gene Therapy volume 9, pages 227–231 (2002)Cite this article

Abstract

The presence of adenoviral cis-elements interfering with the activity of tissue-specific promoters has seriously impaired the use of transcriptional targeting adenoviruses for gene therapy purposes. As an approach to overcome this limitation, transcription terminators were previously employed in cultured cells to insulate a transgene promoter from viral activation. To extend these studies in vivo, we have injected into heart and skeletal muscle, adenoviruses containing the human growth hormone terminator and the cardiac-specific α-myosin heavy chain promoter (αMyHC) driving the chloramphenicol acetyltransferase (CAT) reporter gene. Promoterless CAT constructs were also tested to study interfering viral transcription and terminator activity. Here we demonstrate that the presence of a terminator can produce undesirable effects on the activity of heterologous promoters. Our analysis shows that in particular conditions, a terminator can reduce the tissue specificity of the transgene promoter. By RNAse protection assay performed on cardiac myocytes, we also show that adenoviral elements can direct high levels of autonomous transcription within the E1A enhancer region. This finding supports the model that passive readthrough of the transgene promoter is responsible for loss of selective expression.

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References

  1. Curiel D.T. . Considerations and challenges for the achievement of targeted gene delivery Gene Therapy 1999 6: 1497 1497

    Article  CAS  PubMed  Google Scholar 

  2. Wickham T.J. . Targeting adenovirus Gene Therapy 2000 2000: 7 7

    Google Scholar 

  3. Silman N.J., Fooks A.R. . Biophysical targeting of adenovirus vectors for gene therapy Curr Opin Mol Ther 2000 2: 524 524

    CAS  PubMed  Google Scholar 

  4. Siders W.M., Halloran P.J., Fenton R.G. . Transcriptional targeting of recombinant adenoviruses to human and murine melanoma cells Cancer Res 1996 56: 5638 5638

    CAS  PubMed  Google Scholar 

  5. Rothmann T. et al. Heart muscle-specific gene expression using replication defective recombinant adenovirus Gene Therapy 1996 3: 919 919

    CAS  PubMed  Google Scholar 

  6. Larochelle N. et al. Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer Gene Therapy 1997 4: 465 465

    Article  CAS  PubMed  Google Scholar 

  7. Kim S. et al. Transcriptional targeting of replication-defective adenovirus transgene expression to smooth muscle cells in vivo J Clin Invest 1997 100: 1006 1006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Inesi G. et al. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes Am J Physiol 1998 274: C645 C645

    Article  CAS  PubMed  Google Scholar 

  9. Griscelli F. et al. Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors Hum Gene Ther 1998 9: 1919 1919

    Article  CAS  PubMed  Google Scholar 

  10. Imler J.L. et al. Targeting cell-specific gene expression with an adenovirus vector containing the lacZ gene under the control of the CFTR promoter Gene Therapy 1996 3: 49 49

    CAS  PubMed  Google Scholar 

  11. Ring C.J. et al. Use of transcriptional regulatory elements of the MUC1 and ERBB2 genes to drive tumour-selective expression of a prodrug activating enzyme Gene Therapy 1997 4: 1045 1045

    Article  CAS  PubMed  Google Scholar 

  12. Shi Q., Wang Y., Worton R. . Modulation of the specificity and activity of a cellular promoter in an adenoviral vector Hum Gene Ther 1997 8: 403 403

    Article  CAS  PubMed  Google Scholar 

  13. Hatfield L., Hearing P. . Redundant elements in the adenovirus type 5 inverted terminal repeat promote bidirectional transcription in vitro and are important for virus growth in vivo Virology 1991 184: 265 265

    Article  CAS  PubMed  Google Scholar 

  14. Miralles V.J., Cortes P., Stone N., Reinberg D. . The adenovirus inverted terminal repeat functions as an enhancer in a cell-free system J Biol Chem 1989 264: 10763 10763

    CAS  PubMed  Google Scholar 

  15. Leza M.A., Hearing P. . Cellular transcription factor binds to adenovirus early region promoters and to a cyclic AMP response element J Virol 1988 62: 3003 3003

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Sassone-Corsi P. et al. Far upstream sequences are required for efficient transcription from the adenovirus-2 E1A transcription unit Nucleic Acids Res 1983 11: 8735 8735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hearing P., Shenk T. . The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element Cell 1983 33: 695 695

    Article  CAS  PubMed  Google Scholar 

  18. Vassaux G., Hurst H.C., Lemoine N.R. . Insulation of a conditionally expressed transgene in an adenoviral vector Gene Therapy 1999 6: 1192 1192

    Article  CAS  PubMed  Google Scholar 

  19. Steinwaerder D.S., Lieber A. . Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo Gene Therapy 2000 7: 556 556

    Article  CAS  PubMed  Google Scholar 

  20. Katz E.B. et al. Cardiomyocyte proliferation in mice expressing alpha-cardiac myosin eavy chain-SV40 T-antigen transgenes Am J Physiol 1992 262: H1867 H1867

    CAS  PubMed  Google Scholar 

  21. Gaudin C. et al. Overexpression of Gs alpha protein in the hearts of transgenic mice J Clin Invest 1995 95: 1676 1676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kitsis R.N. et al. Hormonal modulation of a gene injected into rat heart in vivo Proc Natl Acad Sci USA 1991 88: 4138 4138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Proudfoot N.J. . Transcriptional interference and termination between duplicated alpha-globin gene constructs suggests a novel mechanism for gene regulation Nature 1986 322: 562 562

    Article  CAS  PubMed  Google Scholar 

  24. Sambrook J., Fritsch E.F., Maniatis T. . Molecular Cloning: A Laboratory Manual 2nd edn Cold Spring Harbor Laboratory Press 1989

    Google Scholar 

  25. Kass-Eisler A. et al. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo Proc Natl Acad Sci USA 1993 90: 11498 11498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chan J.S. et al. Molecular cloning and expression of the rat angiotensinogen gene Ped Nephrol 1990 4: 429 429

    Article  CAS  Google Scholar 

  27. Schaack J., Langer S., Guo X. . Efficient selection of recombinant adenoviruses by vectors that express beta-galactosidase J Virol 1995 69: 3920 3920

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Buvoli M., Buvoli A., Leinwand L.A. . Suppression of nonsense mutations in cell culture and mice by multimerized suppressor tRNA genes Mol Cell Biol 2000 20: 3116 3116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chomczynski P., Sacchi N. . Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 1987 162: 156 156

    Article  CAS  PubMed  Google Scholar 

  30. Proudfoot N.J., Whitelaw E. . Termination and 3’ end processing of eukaryotic RNA Hames BD, Glover DM (eds); Transcription and Splicing IRL Press 1988 pp 97–129

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Acknowledgements

We would like to thank J Carnes and N Madigan for help with viral injections and A Buvoli for comments on the manuscripts. This work was supported by NIH grant HL50560.

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Author notes

  1. S Bialik

    Present address: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, USA

    M Buvoli, SJ Langer & LA Leinwand

  2. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA

    S Bialik

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  1. M Buvoli
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  3. S Bialik
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  4. LA Leinwand
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Buvoli, M., Langer, S., Bialik, S. et al. Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors. Gene Ther 9, 227–231 (2002). https://doi.org/10.1038/sj.gt.3301640

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