Cationic lipid and polymer-based gene delivery to human pancreatic islets.
Transplantation of pancreatic islets has great potential for treating Type I diabetes. Ex vivo gene therapy may promote re-vascularization or inhibit apoptosis of the islets and promote graft. In this study, we investigated the feasibility of non-viral gene delivery using Enhanced Green Fluorescent Protein (EGFP) and human Vascular Endothelial Growth Factor (hVEGF(165)) expression plasmids as model reporter and therapeutic genes. LipofectAMINE/pDNA and Superfect/pDNA complexes showed high transfection efficiency in rapidly dividing Jurkat cells, but low transfection in non-dividing human islets. LipofectAMINE/pCAGGS-hVEGF transfected islets showed relatively higher levels of hVEGF than in those transfected with LipofectAMINE/pCMS-EGFP complexes or 5% glucose. To exclude endogenously secreted hVEGF, real time RT-PCR experiment was repeated using pCAGGS vector-specific forward primer and hVEGF gene-specific reverse primer. In this case, both non-transfected islets and the islets transfected with LipofectAMINE/pCMS-EGFP complexes showed negligible amplification of hVEGF. On glucose challenge, insulin release from LipofectAMINE/pCAGGS-hVEGF transfected human islets increased from 10.78 +/- 4.56 to 65 +/- 5 ng/ml, suggesting little adverse effect on islet beta cell response to glucose challenge. The low transfection efficiency is due to the islets being a cluster of approximately 1000 non-dividing cells. This underscores the importance of experimentation with the actual human islets.
Mahato
Ram I
RI
Department of Pharmaceutical Sciences, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA. rmahato@utmem.edu
Henry
James
J
Narang
Ajit S
AS
Sabek
Omaima
O
Fraga
Daniel
D
Kotb
Malak
M
Gaber
A Osama
AO
D001483:Base Sequence; D002412:Cations; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000008:administration & dosage / Q000235:genetics; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000008:administration & dosage / Q000235:genetics; D007515:Islets of Langerhans / Q000378:metabolism*; D019169:Jurkat Cells; D008055:Lipids / Q000008:administration & dosage*; D008164:Luminescent Proteins / Q000008:administration & dosage / Q000235:genetics; D008222:Lymphokines / Q000008:administration & dosage / Q000235:genetics; D011108:Polymers / Q000008:administration & dosage*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D002412:Cations; D017931:DNA Primers; D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008055:Lipids; D008164:Luminescent Proteins; D008222:Lymphokines; D011108:Polymers; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D049452:Green Fluorescent Proteins