Retroviral display of urokinase-binding domain fused to amphotropic envelope protein |
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Authors: | Boucquey Antoine Vilhardt Frederik Mitrovic Tatjana Franco Dominique Weber Anne Horellou Philippe |
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Institution: | EMI 00-20, INSERM and Université Paris XI, Bat Grégory Pincus, 80 rue du Général Leclerc, 94276 Le Kremlin Bicêtre Cedex, France. |
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Abstract: | Tumors frequently express urokinase (uPA) receptor (uPAR). To investigate whether uPAR can efficiently target cancerous cells using amphotropic retroviral vectors, we generated a retrovirus displaying the amino-terminal fragment (ATF) of uPA as an N-terminal extension of viral envelope protein. We also made use of a "two-step strategy" by inserting a uPA cleavage site between the ATF moiety and the envelope. We measured the ability of ATF-bearing chimeric envelopes to infect huPAR-overexpressing Madin-Darby canine kidney (MDCK) and control MDCK II cells. The ATF-viruses infected both MDCK cell lines with an equivalent efficiency, suggesting that the chimeric viruses were not sequestered by uPAR and infect cells preferentially via the Pit-2 receptor. The addition of a uPA cleavage site increased the infection level of huPAR-MDCK cells by 2-fold when uPA was present in the infection medium. Surprisingly, ATF-env viruses infected huPAR-MDCK cells 5.5-fold more efficiently in the presence of exogenous uPA. This stimulatory effect of uPA on infection of huPAR-MDCK cells by the ATF-env virus was completely abolished by methyl-beta-cyclodextrin, suggesting that this effect involves the caveolar endocytosis pathway. |
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Keywords: | Gene therapy Retrovirus Targeting Urokinase receptor Tumor Caveolae Endocytosis |
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