Abstract
Purpose: :
Upregulation of VEGF (vascular endothelial growth factor) in the eye leads to uncontrolled retinal vessel growth in diseases like AMD (age related macula degeneration) or DR (diabetic retinopathy). Repeated injections of anti-angiogenic molecules like Lucentis® (Ranibizumab) or Avastin® (Bevacizumab) are the state of the art treatment for these disorders. The aim of this study was to develop a gene addition therapy, with which anti-VEGF molecules are produced over long time periods in the eye. To achieve this, we expressed the soluble isoform of the VEGF receptor 1 (sFlt1) under the control of the Tetracyclin-inducible TetOn-promotor and the F(ab)-fragment Ranibizumab (Ra01) under the control of a CMV promoter in two different eukaryotic cell lines and analyzed the biological activity.
Methods: :
The sequences of both chains of Ranibizumab were synthesized and cloned into the same, IRES (internal ribosomal entry site) containing, expression vector. The sFlt1 cDNA was amplified from corneal extracts and cloned into a TetOn expression vector. HEK293 and HeLa cell lines were transfected with these constructs and the expression of sFlt1 was induced with the Tetracyclin-derivate Doxycyclin. The gene-product containing supernatand was collected after 24 hours of expression. HUVEC (human umbilical vein endothelial cells) migration assays and HUVEC tube formation assays were performed to determine the biological activity of the expressed molecules compared to Lucentis® and Avastin®. The concentration of the expressed molecules was determined by ELISA.
Results: :
In the HUVEC tube formation assay, Ra01 showed a reduction in tube formation (inhibition of VEGF) of 67%, the induced sFlt1 57% and the non-induced sFlt1 a reduction of 6%, compared to Lucentis® (100ng) of 45% and Avastin® (100ng) of 51%. The migration assay showed an inhibition of VEGF-induced HUVEC migration of 15% with expressed sFlt1 and a decrease of 40% with Ra01, compared to Lucentis (100ng) with 47%.
Conclusions: :
The expression of sFlt1 can be controlled using the TetOn system, which produces sufficient amounts of sFlt1 to inhibit VEGF in the tube formation assay compared to Lucentis® or Avastin®. Ra01 can be assembled into a heterodimer in eukaryotic cells and shows a VEGF inhibiting activity comparable or slightly better than Lucentis® or Avastin®. These results provide the basis for a gene-addition therapy to inhibit VEGF in pathologies like AMD or DR.
Keywords: vascular endothelial growth factor • gene transfer/gene therapy • neovascularization