Bipartite Vector Encoding hVEGF and hIL-1Ra for Ex Vivo Transduction Into Human Islets

Mol Pharm. Jan-Feb 2009;6(1):274-84. doi: 10.1021/mp800183b.

Abstract

Ex vivo gene transfer can improve the outcome of islet transplantation for treating type I diabetes. Earlier we have shown coexpression of human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra) after transfection of plasmid DNA encoding these two genes. Due to poor transfection efficiency of plasmid DNA and the better known islet transduction efficiency of adenoviral (Adv) vectors, in this study, we constructed Adv-hVEGF-hIL-1Ra by cloning hVEGF and hIL-1Ra coding sequences and polyA signal under separate cytomegalovirus (CMV) promoters in Adenoquick plasmid (Ad 13.1). There was dose and time dependent expression of these genes after transduction of Adv-hVEGF-hIL1Ra into human islets. The mRNA expression of hVEGF and hIL-1Ra was more than 100 times higher than that of the nontransduced and bipartite plasmid transfected control islets. Transduced islets were viable as evidenced by insulin release upon glucose challenge. Coexpression of hVEGF and hIL-1Ra by islets showed decrease in caspase-3 activity and apoptosis induced by a cocktail of inflammatory cytokines such as TNF-alpha, IL-1beta and IFN-gamma. Compared to nontreated or Adv-LacZ transduced islets, transduction of islets with Adv-hVEGF-hIL-1Ra prior to transplantation under the kidney capsules of diabetic NOD-SCID mice reduced the blood glucose levels, and increased serum insulin and c-peptide levels. Immunohistochemical staining of the islet bearing kidney sections at day 20 after transplantation was positive for human insulin, hVEGF and von Willebrand factor. These results indicate that the bipartite Adv vector efficiently expresses both growth factor and antiapoptotic genes, decreases apoptosis and improves the outcome of islet transplantation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenoviridae / genetics
  • Animals
  • Apoptosis
  • Genetic Vectors / genetics*
  • Humans
  • Immunohistochemistry
  • Interleukin 1 Receptor Antagonist Protein / genetics
  • Interleukin 1 Receptor Antagonist Protein / metabolism*
  • Islets of Langerhans / cytology
  • Islets of Langerhans / metabolism*
  • Islets of Langerhans Transplantation
  • Mice
  • Tissue Culture Techniques
  • Transgenes / genetics
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism*

Substances

  • Interleukin 1 Receptor Antagonist Protein
  • Vascular Endothelial Growth Factor A