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A ligand-pseudoreceptor system based on de novo designed peptides for the generation of adenoviral vectors with altered tropism

 
 
Affiliation:
National Research Council Canada; NRC Biotechnology Research Institute
Language:
English
Type:
Article
Published in:
The Journal of Gene Medicine
Date:
2008
Pages :
355-367
NRCC #:
47814
NPArC #:
3539700
Keywords:
bio; Cell Line; Cells; Epidermal Growth Factor; Ligands; methods; Peptides; pha; Transgenes; adenovirus; retargeting; packaging cells; pseudoreceptor; gene therapy; fiber
Abstract:
Background: Delivery of transgenes into specific tissues by adenovirus vectors (AdVs) relies on ablations of their natural tropism and on introduction of a new tropism. If the interaction with its natural receptor is ablated, a new packaging cell line is required to produce the AdV. In the present study, we have used two de novo designed peptides (E-Coil and K-Coil) that interact with each other with high affinity to establish a new receptor-ligand system for the propagation of retargeted AdVs. Methods: We produced a cell line (293E) expressing on its surface a pseudoreceptor containing the E-Coil. An AdV (AdFK4m/GFP) lacking the interaction with the primary receptor for adenovirus (CAR) and containing the K-Coil inserted at the fiber C-terminus was constructed and tested using two strategies: (1) an RGD motif (Arg-Gly-Asp) was inserted into the HI-loop of the fiber; (2) AdFK4m/GFP was conjugated to a bispecific adaptor for the epidermal growth factor receptor (EGFR). Results: AdFK4m/GFP infected 293E cells more efficiently than cells lacking the pseudoreceptor. The transduction was due to the K-Coil/E-Coil specific interaction since it was competed by addition of soluble K-Coil, but not soluble fiber. We demonstrated that the modified AdV was retargeted toward αv integrin by inclusion of the RGD motif, or toward EGFR using the bispecific adaptor. Conclusions: We have established a new system to produce AdVs ablated of natural tropism. This system should permit the retargeting of AdVs by inserting new ligandswithin the fiber or through the interactionwith bispecific adaptors.
 
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