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Journal of Virology, March 2001, p. 2972-2981, Vol. 75, No. 6
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.6.2972-2981.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells

John L. Jakubczak,1 Michele L. Rollence,1 David A. Stewart,1 Jonathon D. Jafari,1 Dan J. Von Seggern,2 Glen R. Nemerow,2 Susan C. Stevenson,1,* and Paul L. Hallenbeck1

Genetic Therapy, Inc./A Novartis Company, Gaithersburg, Maryland 20878,1 and Department of Immunology, The Scripps Research Institute, La Jolla, California 920372

Received 3 October 2000/Accepted 20 November 2000

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.beta gal.Delta F, an E1-, E3-, and fiber-deleted adenoviral vector encoding beta -galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.

* Corresponding author. Mailing address: Genetic Therapy, Inc./A Novartis Company, 9 West Watkins Mill Rd., Gaithersburg, MD 20878. Phone: (301) 258-4830. Fax: (301) 258-4680. E-mail: sue.stevenson{at}pharma.novartis.com.

Journal of Virology, March 2001, p. 2972-2981, Vol. 75, No. 6
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.6.2972-2981.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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