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Journal of Virology, July 2002, p. 6893-6899, Vol. 76, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.14.6893-6899.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX

Igor P. Dmitriev,1 Elena A. Kashentseva,1 and David T. Curiel1,2*

Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery,1 Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-33002

Received 18 December 2001/ Accepted 5 April 2002

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

* Corresponding author. Mailing address: Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, Gene Therapy Center, University of Alabama at Birmingham, 901 19th St. South, BMR II-508, Birmingham, AL 35294-2172. Phone: (205) 934-8627. Fax: (205) 975-7476. E-mail: David.Curiel{at}ccc.uab.edu.

Journal of Virology, July 2002, p. 6893-6899, Vol. 76, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.14.6893-6899.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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