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A lentiviral vector-based adenovirus fiber-pseudotyping approach for expedited functional assessment of candidate retargeted fibers.

Journal article
Authors Taco G Uil
Jeroen de Vrij
Jort Vellinga
Martijn J W E Rabelink
Steve J Cramer
On Ying A Chan
Margherita Pugnali
Maria K Magnusson
Leif Lindholm
Pierre Boulanger
Rob C Hoeben
Published in The journal of gene medicine
Volume 11
Issue 11
Pages 990-1004
ISSN 1521-2254
Publication year 2009
Published at Institute of Biomedicine, Department of Microbiology and Immunology
Pages 990-1004
Language en
Links dx.doi.org/10.1002/jgm.1395
Subject categories Microbiology in the medical area

Abstract

BACKGROUND: Many studies aimed at retargeting adenovirus (Ad) rationally focus on genetic modification of fiber, which is the primary receptor-binding protein of Ad. Retargeted fibers ultimately require functional validation in the viral context. METHODS: Lentiviral vectors (LV) were used to express fiber variants in cells. Infections with a fiber gene-deleted Ad vector yielded fiber-pseudotyped viruses. An enzyme-linked immunosorbent assay and slot blot-based assays probed target binding-ability of retargeted fibers. Differential treatments with an alkylating agent prior to western blot analysis allowed for examination of intra- and extracellular redox states of fibers. RESULTS: In the present study, LV-based fiber-pseudotyping of Ad is presented as an accelerated means to test new fibers. LV-mediated gene transfer yielded stable and uniform populations of fiber variant-expressing cells. These populations were found to effectively support fiber-pseudotyping of Ad. As a secondary objective of the study, we functionally assessed a chimeric fiber harboring a tumor antigen-directed single-chain antibody fragment (scFv). This fiber was shown to trimerize and achieve a degree of binding to its antigenic target. However, its capsid incorporation ability was impaired and, moreover, it was unable to confer a detectable level of target binding upon Ad. Importantly, subsequent analyses of this fiber revealed the improper folding of its scFv constituent. CONCLUSIONS: LV-based fiber-pseudotyping was established as a convenient method for testing modified fibers for functionality within Ad particles. Furthermore, a new chimeric fiber was found to be inadequate for Ad retargeting. The folding difficulties encountered for this particular fiber might be generally inherent to the use (i.e. for genetic Ad capsid incorporation) of complex, disulfide bridge-containing natural ligands.

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