Generation of recombinant adenoviruses and lentiviruses expressing angiogenic and neuroprotectivefactors using Gateway cloning technology

Abstract

The critical aspect in gene and gene-cell therapy is tofind an optimal vector - a carrier of genetic information.Viruses represent a natural biological system for genetransfer into eukaryotic cells. One of the most effectiveand proven vectors for delivery of recombinant nucleic acidsinto mammalian cells are adenoviruses and lentiviruses. Inthis study using the Gateway cloning we have constructedadenoviral and lentiviral vectors encoding angiogenic andneuroprotective factors: various isoforms of vascularendothelial growth factor vegf121, vegf165, vegf189; basicfibroblast growth factor fgf2; glial cell-derived neurotrophicfactor gdnf. The efficiency of transduction of HEK293A cellline with generated recombinant viruses and expression ofrecombinant proteins were confirmed by immunofluorescentanalysis.

Keywords

adenovirus / lentivirus / Gateway cloning / vascular endothelial growth factor / basic fibroblast growth factor / glial cell-derived neurotrophic factor

References

[1] Leker R.R., Shohami E. Cerebral ischemia and trauma-different etiologies yet similar mechanisms: neuroprotective opportunities. Brain Res. Brain Res. Rev. 2002; 39(1): 55-73.

[2] Abe K. Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury. J. Cereb. Blood Flow Metab. 2000; 20(10): 1393-408.

[3] Leker R.R., Soldner F., Velasco I. et al. Long-lasting regeneration after ischemia in the cerebral cortex. Stroke 2007; 38(1): 153-61.

[4] Beck K.D., Valverde J., Alexi T. et al. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain. Nature 1995; 373(6512): 339-41.

[5] Tomac A., Lindqvist E., Lin L.F. et al. Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. Nature 1995; 373(6512): 335-9.

[6] Dore-Duffy P., Wang X., Mehedi A. et al. Differential expression of capillary VEGF isoforms following traumatic brain injury. Neurol. Res. 2007; 29(4): 395-403.

[7] Lafuente J.V., Argandona E.G., Mitre B. VEGFR-2 expression in brain injury: its distribution related to brain-blood barrier markers. J. Neural. Transm. 2006; 113(4): 487-96.

[8] Papavassiliou E., Gogate N., Proescholdt M. et al. Vascular endothelial growth factor (vascular permeability factor) expression in injured rat brain. J. Neurosci. Res. 1997; 49(4): 451-60. 9. Sköld M.K., Gertten C. VEGF and VEGF receptor expression after experimental brain contusion in rat. Neurotrauma 2005; 22(3): 353-67.

[9] Vorburger S.A., Hunt K.K. Adenoviral gene therapy. Oncologist 2002; 7(1): 46-59.

[10] Yang X., Boehm J.S., Yang X. et al. A public genome-scale lentiviral expression library of human ORFs. Nat. Methods. 2011; 8(8): 659-61.

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