Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability

Background We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native...

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Bibliographic Details
Published in:The journal of gene medicine 2002-07, Vol.4 (4), p.356-370
Main Authors: Magnusson, Maria K., See Hong, Saw, Henning, Petra, Boulanger, Pierre, Lindholm, Leif
Format: Article
Language:English
Subjects:
Ad5 vectors
Adenoviridae
Animals
cell-targeting
COS Cells
fiber gene
fiber solubility
Fluorescent Antibody Technique
Gene therapy
Gene Transfer Techniques
Genetic Vectors
Humans
Immunoglobulin Fragments - genetics
Ligands
protein folding
virus viability
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Summary:Background We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions. Methods Polypeptide ligands of cell surface molecules, including single‐chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity. Results A major limiting factor for recovering viable recombinant Ad5 carrying fiber‐fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber‐ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number. Conclusions Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber‐ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors. Copyright © 2002 John Wiley & Sons, Ltd.
ISSN:1099-498X
1521-2254
DOI:10.1002/jgm.285