title: Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters.
abstract: Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.
authors: Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.
authors: Birgit
authors: B
authors: Dreier
authors: Annemarie
authors: A
authors: Honegger
authors: Christian
authors: C
authors: Hess
authors: Gabriela
authors: G
authors: Nagy-Davidescu
authors: Peer R E
authors: PR
authors: Mittl
authors: Markus G
authors: MG
authors: Grütter
authors: Natalya
authors: N
authors: Belousova
authors: Galina
authors: G
authors: Mikheeva
authors: Victor
authors: V
authors: Krasnykh
authors: Andreas
authors: A
authors: Plückthun
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics* / Q000378:metabolism; D017089:Ankyrin Repeat / Q000235:genetics; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D020836:Protein Structure, Quaternary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C508053:ERBB2 protein, human