This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parks, R. J.
Right arrow Articles by Graham, F. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parks, R. J.
Right arrow Articles by Graham, F. L.

 Previous Article  |  Next Article 

Journal of Virology, October 1999, p. 8027-8034, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Effects of Stuffer DNA on Transgene Expression from Helper-Dependent Adenovirus Vectors

Robin J. Parks,1,2,dagger Jonathan L. Bramson,2 Yonghong Wan,2 Christina L. Addison,1,Dagger and Frank L. Graham1,2,*

Departments of Biology1 and Pathology,2 McMaster University, Hamilton, Ontario L8S 4K1, Canada

Received 16 February 1999/Accepted 2 July 1999

We have analyzed transgene (lacZ) expression from a first-generation adenovirus (Ad) vector in comparison to helper-dependent (hd) Ads deleted for various portions of the viral coding sequences and generated by using the Cre/loxP helper-dependent system (R. J. Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996). An hd vector deleted for approximately 70% of the Ad genome (AdRP1001) provided levels and durations of transgene expression similar to those of a control first generation Ad vector containing an identical expression cassette. Deletion of all Ad sequences from the hdAd and replacement with a ~22-kb fragment of lambda DNA resulted in a decrease in the level and duration of lacZ expression which could not be reversed by the inclusion of a matrix attachment region. However, substitution of the lambda stuffer in the fully deleted hdAd with sequences from the human hypoxanthine-guanine phosphoribosyltransferase gene resulted in significantly improved transgene expression. In vitro assays for cytotoxic T lymphocytes (CTL) directed against putative peptides encoded by the vector backbone showed that, although CTL were generated against the vector containing the lambda DNA, no such CTL were generated against the vector containing the hypoxanthine-guanine phosphoribosyltransferase (HPRT) sequences. Surprisingly, the rate of loss of the HPRT- and lambda-containing vectors from mouse liver was similar, despite the differences in expression kinetics, indicating that the lambda stuffer-directed CTL were inefficient at eliminating the transduced cells. Thus, the nature of the DNA backbone of hdAds can have important effects on the functioning of the vector. Since most fully deleted vectors require "stuffer" DNA as part of the vector backbone to maintain optimum vector size, these observations must be taken into account in the design of hdAd vectors.

* Corresponding author. Mailing address: Department of Biology, McMaster University, 1280 Main St., West, LSB-430, Hamilton, Ontario L85 4K1, Canada. Phone: (905) 525-9140, ext. 23545. Fax: (905) 521-2955. E-mail: graham{at}mcmaster.ca.

dagger Present address: Centre for Molecular Medicine, Ottawa Hospital Research Institute, Ottawa, Ontario K1H 8L6, Canada.

Dagger Present address: Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109-0360.

Journal of Virology, October 1999, p. 8027-8034, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Ross, P. J., Kennedy, M. A., Parks, R. J. (2009). Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression. J. Virol. 83: 8409-8417 [Abstract] [Full Text]  
  • De Santa, F., Albini, S., Mezzaroma, E., Baron, L., Felsani, A., Caruso, M. (2007). pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation. Mol. Cell. Biol. 27: 7248-7265 [Abstract] [Full Text]  
  • Huh, M. S., Parker, M. H., Scime, A., Parks, R., Rudnicki, M. A. (2004). Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation. JCB 166: 865-876 [Abstract] [Full Text]  
  • Muruve, D. A., Cotter, M. J., Zaiss, A. K., White, L. R., Liu, Q., Chan, T., Clark, S. A., Ross, P. J., Meulenbroek, R. A., Maelandsmo, G. M., Parks, R. J. (2004). Helper-Dependent Adenovirus Vectors Elicit Intact Innate but Attenuated Adaptive Host Immune Responses In Vivo. J. Virol. 78: 5966-5972 [Abstract] [Full Text]  
  • Sargent, K. L., Meulenbroek, R. A., Parks, R. J. (2004). Activation of Adenoviral Gene Expression by Protein IX Is Not Required for Efficient Virus Replication. J. Virol. 78: 5032-5037 [Abstract] [Full Text]  
  • Ehrhardt, A., Xu, H., Dillow, A. M., Bellinger, D. A., Nichols, T. C., Kay, M. A. (2003). A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia. Blood 102: 2403-2411 [Abstract] [Full Text]  
  • Zobel, C., Cho, H. C., Nguyen, T.-T., Pekhletski, R., Diaz, R. J, Wilson, G. J, Backx, P. H (2003). Molecular dissection of the inward rectifier potassium current (IK1) in rabbit cardiomyocytes: evidence for heteromeric co-assembly of Kir2.1 and Kir2.2. J. Physiol. 550: 365-372 [Abstract] [Full Text]  
  • Ehrhardt, A., Xu, H., Kay, M. A. (2003). Episomal Persistence of Recombinant Adenoviral Vector Genomes during the Cell Cycle In Vivo. J. Virol. 77: 7689-7695 [Abstract] [Full Text]  
  • Roth, M. D., Cheng, Q., Harui, A., Basak, S. K., Mitani, K., Low, T. A., Kiertscher, S. M. (2002). Helper-Dependent Adenoviral Vectors Efficiently Express Transgenes in Human Dendritic Cells but Still Stimulate Antiviral Immune Responses. J. Immunol. 169: 4651-4656 [Abstract] [Full Text]  
  • Ehrhardt, A., Kay, M. A. (2002). A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo. Blood 99: 3923-3930 [Abstract] [Full Text]  
  • Schiedner, G., Hertel, S., Johnston, M., Biermann, V., Dries, V., Kochanek, S. (2002). Variables Affecting In Vivo Performance of High-Capacity Adenovirus Vectors. J. Virol. 76: 1600-1609 [Abstract] [Full Text]  
  • Hillgenberg, M., Tonnies, H., Strauss, M. (2001). Chromosomal Integration Pattern of a Helper-Dependent Minimal Adenovirus Vector with a Selectable Marker Inserted into a 27.4-Kilobase Genomic Stuffer. J. Virol. 75: 9896-9908 [Abstract] [Full Text]  
  • Croyle, M. A., Chirmule, N., Zhang, Y., Wilson, J. M. (2001). "Stealth" Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung. J. Virol. 75: 4792-4801 [Abstract] [Full Text]  
  • Rosenzweig, M., Connole, M., Glickman, R., Yue, S.-P. S., Noren, B., DeMaria, M., Johnson, R. P. (2001). Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34+ hematopoietic cells. Blood 97: 1951-1959 [Abstract] [Full Text]  
  • Russell, W. C. (2000). Update on adenovirus and its vectors. J. Gen. Virol. 81: 2573-2604 [Full Text]  
  • Sandig, V., Youil, R., Bett, A. J., Franlin, L. L., Oshima, M., Maione, D., Wang, F., Metzker, M. L., Savino, R., Caskey, C. T. (2000). Optimization of the helper-dependent adenovirus system for production and potency in vivo. Proc. Natl. Acad. Sci. USA 97: 1002-1007 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»