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A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob

Gene Therapy volume 8, pages 730–735 (2001)Cite this article

Abstract

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100–1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

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Acknowledgements

We would like to thank Jun Murai and Nobuko Heishi for technical assistance. We would like to thank Dr M Tada (Hokkaido University, Japan) and Dr JM Bergelson (The Children's Hospital of Philadelphia, PA) for kindly providing LN444 cells and mouse monoclonal antibody RmcB, respectively. This work was supported by grants from the Ministry of Health and Welfare in Japan and Grant-in-Aid for Scientific Research on Priority Areas (C). MAK was supported by NIH DK49022.

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Authors and Affiliations

  1. Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan

    H Mizuguchi, N Koizumi, T Hosono & T Hayakawa

  2. Department of Pharmaceutics, Showa Pharmaceutical University, Tokyo, Japan

    N Koizumi, N Utoguchi & Y Watanabe

  3. Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA, USA

    M A Kay

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Mizuguchi, H., Koizumi, N., Hosono, T. et al. A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob. Gene Ther 8, 730–735 (2001). https://doi.org/10.1038/sj.gt.3301453

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