title: Functional Characterization of AAV-Expressed Recombinant Anti-VEGF Single-Chain Variable Fragments In Vitro.
abstract: OBJECTIVE
  Most retinal neovascular disorders are caused by upregulation of vascular endothelial growth factor (VEGF) expression. These disorders are treated with repeated injections of anti-VEGF molecules, which may have severe side effects. The expression of anti-VEGF molecules by the retina itself in a controlled manner following adeno-associated viral (AAV) gene transfer could be a replacement of this therapy.
  
  
  METHODS
  The open reading frames (orf) of the light and the heavy chain of ranibizumab were cloned into an expression plasmid separated by an internal ribosomal entry site (IRES). The construct was mutated to generate ranibizumab single-chain variable fragments (scFv). Expression was verified by western blotting and the concentrations were measured with a custom-made ranibizumab ELISA. Biological activity, VEGF-binding properties, and the doxycycline-dependent induction of anti-VEGF expression were tested. An AAV2/5 vector was generated containing the optimal variant Ra02.
  
  
  RESULTS
  Ra01-Ra05 molecules were detected in the cell culture medium. While the VEGF-binding affinity was significantly lower for Ra01 and Ra02 compared to Lucentis(®), the inhibition of cell migration was comparable and the maximum inhibition of Ra01 and Ra02 was reached at lower doses. The expression of Ra01 and Ra02 was shown to be regulable with the TetOn-system(®) as plasmid (Ra01, Ra02) and AAV vector construct (Ra02).
  
  
  CONCLUSIONS
  Ra01 and Ra02 can be produced in eukaryotic cells after AAV-mediated gene transfer in a regulable manner in vitro and display comparable biological activity as Lucentis. These results are the basis for in vivo studies in human VEGF-overexpressing mice, a model for human neovascular disorders.
authors: Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany.
authors: Tobias
authors: T
authors: Wimmer
authors: Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany.
authors: Birgit
authors: B
authors: Lorenz
authors: Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany.
authors: Knut
authors: K
authors: Stieger
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D000069579:Ranibizumab / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D015861:Retinal Neovascularization / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000276:immunology
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000069579:Ranibizumab