Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus

Gene Therapy volume 8, pages 846–854 (2001)Cite this article

Abstract

Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus–adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 × 108p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in ∼100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Mullbacher A, Bellett AJ, Hla RT . The murine cellular immune response to adenovirus type 5 Immunol Cell Biol 1989 67: 31–39

    Article  PubMed  Google Scholar 

  2. Yang Y, Ertl HC, Wilson JM . MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses Immunity 1994 1: 433–442

    Article  CAS  PubMed  Google Scholar 

  3. Yang Y et al. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy Proc Natl Acad Sci USA 1994 91: 4407–4411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Juillard V et al. Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector Eur J Immunol 1995 25: 3467–3473

    Article  CAS  PubMed  Google Scholar 

  5. Yang Y et al. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis Nat Genet 1994 7: 362–369

    Article  CAS  PubMed  Google Scholar 

  6. Engelhardt JF, Ye X, Doranz B, Wilson JM . Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver Proc Natl Acad Sci USA 1994 91: 6196–6200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Armentano D et al. Characterization of an adenovirus gene transfer vector containing an E4 deletion Hum Gene Ther 1995 6: 1343–1353

    Article  CAS  PubMed  Google Scholar 

  8. Krougliak V, Graham FL . Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants Hum Gene Ther 1995 6: 1575–1586

    Article  CAS  PubMed  Google Scholar 

  9. Wang Q, Jia XC, Finer MH . A packaging cell line for propagation of recombinant adenovirus vectors containing two lethal gene-region deletions Gene Therapy 1995 2: 775–783

    CAS  PubMed  Google Scholar 

  10. Fang B et al. Lack of persistence of E1- recombinant adenoviral vectors containing a temperature-sensitive E2A mutation in immunocompetent mice and hemophilia B dogs Gene Therapy 1996 3: 217–222

    CAS  PubMed  Google Scholar 

  11. Gorziglia MI et al. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy J Virol 1996 70: 4173–4178

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang Q et al. Persistent transgene expression in mouse liver following in vivo gene transfer with a delta E1/delta E4 adenovirus vector Gene Therapy 1997 4: 393–400

    Article  CAS  PubMed  Google Scholar 

  13. Amalfitano A et al. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted J Virol 1998 72: 926–933

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Hartigan-O'Connor D, Amalfitano A, Chamberlain JS . Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase J Virol 1999 73: 7835–7841

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Mitani K, Graham FL, Caskey CT, Kochanek S . Rescue, propagation, and partial purification of a helper virus- dependent adenovirus vector Proc Natl Acad Sci USA 1995 92: 3854–3858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Fisher KJ et al. Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis Virology 1996 217: 11–22

    Article  CAS  PubMed  Google Scholar 

  17. Kochanek S et al. A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase Proc Natl Acad Sci USA 1996 93: 5731–5736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lieber A, He CY, Kirillova I, Kay MA . Recombinant adenoviruses with large deletions generated by Cre- mediatedexcision exhibit different biological properties compared withfirst-generation vectors in vitro and in vivo J Virol 1996 70: 8944–8960

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Parks RJ et al. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal Proc Natl Acad Sci USA 1996 93: 13565–13570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Parks RJ, Graham FL . A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging J Virol 1997 71: 3293–3298

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Schiedner G et al. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity published erratum appears in Nat Genet 1998 Mar;18(3): 298) Nat Genet 1998 18: 180–183

    Article  CAS  PubMed  Google Scholar 

  22. Morsy MA et al. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene Proc Natl Acad Sci USA 1998 95: 7866–7871

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Parks RJ et al. Effects of stuffer DNA on transgene expression from helper-dependent adenovirus vectors J Virol 1999 73: 8027–8034

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Morral N et al. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons Proc Natl Acad Sci USA 1999 96: 12816–12821

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Morsy MA, Caskey CT . Expanded-capacity adenoviral vectors – the helper-dependent vectors Mol Med Today 1999 5: 18–24

    Article  CAS  PubMed  Google Scholar 

  26. Sandig V et al. Optimization of the helper-dependent adenovirus system for production and potency in vivo Proc Natl Acad Sci USA 2000 97: 1002–1007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hofmann C et al. Efficient gene transfer into human hepatocytes by baculovirus vectors Proc Natl Acad Sci USA 1995 92: 10099–10103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Boyce FM, Bucher NL . Baculovirus-mediated gene transfer into mammalian cells Proc Natl Acad Sci USA 1996 93: 2348–2352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sandig V et al. Gene transfer into hepatocytes and human liver tissue by baculovirus vectors Hum Gene Ther 1996 7: 1937–1945

    Article  CAS  PubMed  Google Scholar 

  30. Graham FL . Covalently closed circles of human adenovirus DNA are infectious EMBO J 1984 3: 2917–2922

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. McGrory WJ, Bautista DS, Graham FL . A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5 Virology 1988 163: 614–617

    Article  CAS  PubMed  Google Scholar 

  32. Sauer B, Henderson N . The cyclization of linear DNA in Escherichia coli by site-specific recombination Gene 1988 70: 331–341

    Article  CAS  PubMed  Google Scholar 

  33. Sternberg N . Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs Proc Natl Acad Sci USA 1990 87: 103–107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Groner A, Granados RR, Burand JP . Interaction of Autographa californica nuclear polyhedrosis virus with two nonpermissive cell lines Intervirology 1984 21: 203–209

    Article  CAS  PubMed  Google Scholar 

  35. Carbonell LF, Miller LK . Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines Appl Environ Microbiol 1987 53: 1412–1417

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Hartig PC, Chapman MA, Hatch GG, Kawanishi CY . Insect virus: assays for toxic effects and transformation potential in mammalian cells Appl Environ Microbiol 1989 55: 1916–1920

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Hartig PC, Cardon MC, Kawanishi CY . Insect virus: assays for viral replication and persistence in mammalian cells J Virol Meth 1991 31: 335–344

    Article  CAS  Google Scholar 

  38. Hartig PC, Cardon MC, Kawanishi CY . Effect of baculovirus on selected vertebrate cells Dev Biol Stand 1992 76: 313–317

    CAS  PubMed  Google Scholar 

  39. Bett AJ, Haddara W, Prevec L, Graham FL . An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3 Proc Natl Acad Sci USA 1994 91: 8802–8806

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Barsoum J, Brown R, McKee M, Boyce FM . Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein Hum Gene Ther 1997 8: 2011–2018

    Article  CAS  PubMed  Google Scholar 

  41. DiLella AG et al. Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene Biochemistry 1986 25: 743–749

    Article  CAS  PubMed  Google Scholar 

  42. Ayres MD et al. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus Virology 1994 202: 586–605

    Article  CAS  PubMed  Google Scholar 

  43. Barsoum J . Concentration of recombinant baculovirus by cation-exchange chromatography Biotechniques 1999 26: 834–840

    Article  CAS  PubMed  Google Scholar 

  44. Guo ZS, Wang LH, Eisensmith RC, Woo SL . Evaluation of promoter strength for hepatic gene expression in vivo following adenovirus-mediated gene transfer Gene Therapy 1996 3: 802–810

    CAS  PubMed  Google Scholar 

  45. Berkner KL, Sharp PA . Generation of adenovirus by transfection of plasmids Nucleic Acids Res 1983 11: 6003–6020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lippe R, Graham FL . Adenoviruses with nonidentical terminal sequences are viable J Virol 1989 63: 5133–5141

    CAS  PubMed  PubMed Central  Google Scholar 

  47. O'Reilly DR . Use of baculovirus expression vectors Meth Mol Biol 1997 62: 235–246

    CAS  Google Scholar 

  48. Graham FL, Prevec L . Methods for construction of adenovirus vectors Mol Biotechnol 1995 3: 207–220

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grants DK51700 to RCE and DK5333 to VAK.

Author information

Authors and Affiliations

  1. Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY, USA

    N Cheshenko, N Krougliak, R C Eisensmith & V A Krougliak

  2. Department of Human Genetics, Mount Sinai School of Medicine, New York, NY, USA

    R C Eisensmith

Authors
  1. N Cheshenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. N Krougliak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R C Eisensmith
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. V A Krougliak
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cheshenko, N., Krougliak, N., Eisensmith, R. et al. A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Ther 8, 846–854 (2001). https://doi.org/10.1038/sj.gt.3301459

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301459

Keywords

This article is cited by

Search

Advanced search

Quick links