Division of Transplantation
University of Illinois at Chicago
A novel, non-viral mRNA delivery system for targeted protein translation in human islets
Diana Gutierrez1,2, Martha Bissing2, Enza Marchese2, Yong Wang2, José Oberholzer1,2.
1Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, United States; 2Department of Surgery, University of Illinois at Chicago, Chicago, IL, United States
Our group described that human islet cells can be driven to proliferate by expressing specific cell cycle proteins via adenoviral vectors . Adenoviral vectors have several limitations including poor penetration into intact islets; only the most superficial cell layers get infected. Due to safety concerns associated with using viral systems clinically to expand human islet cells for transplant purposes, significant emphasis has been placed on producing a safe and effective non-viral delivery system for gene therapy. To obtain this goal, we propose the use of an innovative technology that utilizes gold nanoparticles (AuNPs) as a non-viral method of delivery. Our laboratory was one of the first to describe the use of AuNPs in human islets and observe AuNPs can penetrate into the core of islets and deliver a gene to the vast majority of the cells, without damaging the cells . AuNPs proved to be a biocompatible delivery system both in vitro and in vivo .
Thus far, gene therapy and molecular biology have focused primarily on delivering DNA of a specific gene into cells. The risk of this approach is that the DNA can be incorporated into the genome and lead to random chromosomal damages with the risk of generation of cancerous cells. This risk could be significantly reduced, or likely eliminated, with the use of mRNA. In general, mRNA is too unstable to be used as a molecular tool to overexpress specific proteins. With advances in nanotechnology, and better understanding of the translation process, methods have been developed that allow for expression of specific proteins by intracellular delivery of protein-encoding modified mRNA .
We used AuNPs conjugated to modified mCherry mRNA as a proof of concept to increase expression of a specific protein, the red fluorescent mCherry. AuNPs were conjugated to mCherry mRNA via a short, non-coding oligo-DNA. We investigated the AuNP-oligoDNA-mRNA construct delivery efficacy and its impact on islet functionality.
Gold nanoparticle transfection efficacy, optimal concentration, and size association were determined in human islets using confocal microscopy. The impact of AuNP-oligoDNA-mRNA on islet function was determined by measurement of intracellular calcium signaling using fura-2/AM and mitochondrial potentials using rhodamine 123 dye during islet microperifusion assays as previously described . Insulin was measured using an enzyme-linked immunosorbent assay (ELISA).
Confocal images showed strong red fluorescence of AuNPs (7 nm) conjugated with mCherry mRNA at a concentration of 20 nM and 50 nM after 24 hours of incubation, indicating correct translation of the mCherry mRNA. The glucose stimulated intracellular calcium was comparable between the groups. In addition, mitochondrial potential indices demonstrated similarity between the control group and both sized AuNPs at the two different concentrations (p>0.05). In conclusion, during the study of AuNP-oligoDNA-mRNA we observed appropriate protein expresion and islet function was not impaired.
José Oberholzer. Chicago Diabetes Project. University of Illinois at Chicago Islet Isolation Team. Oberholzer Lab.
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11:00 - 13:30
|Prospects for Clinical Islet Xenotransplantation (CIX)||Commentary on Current status of clinical islet allotransplantation by Camillo Ricordi||Room 109|
11:00 - 12:30
|Topics in Experimental and Clinical Islet Transplantation||A novel, non-viral mRNA delivery system for targeted protein translation in human islets||Plenary Room 1|
17:30 - 18:30
|Joint Poster Session||Autologous islet cell transplantation following duodenum sparing total pancreatectomy in situs inversus totalis to treat chronic pancreatitis||Main Foyer 1 & 2|