Growth factor purification and delivery systems (PADS) for therapeutic angiogenesis

Abstract

Background: Therapeutic angiogenesis with vascular endothelial growth factor (VEGF), delivered either vi recombinant protein infusion or via gene therapy, has shown promise in preclinical models of various disease including myocardial infarction, renovascular disease, preeclampsia, and neurodegenerative disorders. However dosing, duration of expression, and tissue specificity are challenges to VEGF gene therapy, and recombinant VEG delivery suffers from extremely rapid plasma clearance, necessitating continuous infusion and/or direct injection a the site of interest Methods: Here we describe a novel growth factor purification and delivery system (PADS) generated by fusion o VEGF 121 to a protein polymer based on Elastin-like Polypeptide (ELP). ELP is a thermally responsive biopolyme derived from a five amino acid repeat sequence found in human tropoelastin.VEGFPADS were constructed by fusio of the ELP coding sequence in-frame with the VEGF 121 coding sequence connected by a flexible di-glycine linker In vitro activity of VEGFPADS was determined using cell proliferation, tube formation, and migration assays wit vascular endothelial cells. Pharmacokinetics and biodistribution of VEGFPADS in vivo were compared to free VEGF i mice using quantitative fluorescence techniques Results: ELP fusion allowed for recombinant expression and simple, non-chromatographic purification of th ELP-VEGF 121 chimera in yields as high as 90 mg/L of culture and at very high purity. ELP fusion had no effect o the VEGF activity, as the VEGFPADS were equally potent as free VEGF12in stimulating HUVEC proliferation, tub formation, and migration. Additionally, the VEGFPADS had a molecular weight five-fold larger than free VEGF 121 which lead to slower plasma clearance and an altered biodistribution after systemic delivery in vivo Conclusion: PADS represent a new method of both purification and in vivo stabilization of recombinant growt factors. The use of this system could permit recombinant growth factors to become viable options for therapeuti angiogenesis in a number of disease models.