Author:

Keywords:

Animals, Cell Aggregation, Cell Death, Cell Line, Cell Separation, Cell Survival, Cytokines, Flow Cytometry, Humans, Insulin, Islets of Langerhans, Islets of Langerhans Transplantation, Lentivirus, Male, Mice, Rats, Rats, Wistar, Time Factors, Transduction, Genetic, Transgenes, Science & Technology, Life Sciences & Biomedicine, Cell & Tissue Engineering, Medicine, Research & Experimental, Transplantation, Cell Biology, Research & Experimental Medicine, type 1 diabetes, pancreatic beta-cells, pseudoislet aggregates, gene therapy, lentiviral vector, islet transplantation, PANCREATIC BETA-CELLS, GENE-THERAPY, VIRAL VECTORS, MESSENGER-RNA, EXPRESSION, APOPTOSIS, DEATH, ADENOVIRUS, DELIVERY, GRAFTS, Insulin Secretion, 06 Biological Sciences, 10 Technology, 11 Medical and Health Sciences, Neurology & Neurosurgery, 31 Biological sciences

Abstract:

Islet transplantation is a promising treatment in type 1 diabetes, but the need for chronic immunosuppression is a major hurdle to broad applicability. Ex vivo introduction of agents by lentiviral vectors-improving beta-cell resistance against immune attack-is an attractive path to pursue. The aim of this study was to investigate whether dissociation of islets to single cells prior to viral infection and reaggregation before transplantation would improve viral transduction efficacy without cytotoxicity. This procedure improved transduction efficacy with a LV-pWPT-CMV-EGFP construct from 11.2 +/- 4.1% at MOI 50 in whole islets to 80.0 +/- 2.8% at MOI 5. Viability (as measured by Hoechst/PI) and functionality (as measured by glucose challenge) remained high. After transplantation, the transfected pseudoislet aggregates remained EGFP positive for more than 90 days and the expression of EGFP colocalized primarily with the insulin-positive beta-cells. No increased vulnerability to immune attack was observed in vitro or in vivo. These data demonstrate that dispersion of islets prior to lentiviral transfection and reaggregation prior to transplantation is a highly efficient way to introduce genes of interest into islets for transplantation purposes in vitro and in vivo, but the amount of beta-cells needed for normalization of glycemia was more than eightfold higher when using dispersed cell aggregates versus unmanipulated islets. The high price to pay to reach stable and strong transgene expression in islet cells is certainly an important cell loss.