Long-term gene expression and metabolic control exerted by lentivirus-transduced pancreatic islets.

BACKGROUND

Genetic modification of non-human islets before transplantation may provide means by which they can escape immunity and, thus, be used in a human host. To accomplish this, efficient gene transfer methods are needed. Lentiviral vectors are transgene vehicles capable of stably transducing a variety of primary, post-mitotic cells including islets.

METHODS

We investigated whether lentiviral transduction impaired rat pancreatic islet function long term. Following transduction, the gross morphology, viability and in vitro functionality of islets were evaluated by microscopy, adenylate nucleotide and insulin secretion assays, respectively. Further, in vivo functionality of transduced islets was assessed by transplanting the islets under the kidney capsule of diabetic nude mice.

RESULTS

All transduced islets contained green fluorescent protein (GFP)-positive cells. In single cell suspensions prepared from transduced islets, 33+/-8% (n = 3) of dispersed islet cells were GFP-positive. The ADP/ATP ratio was 0.07+/-0.01 for transduced islets and 0.06+/-0.01 for controls (normal range <0.11). No morphological changes were observed in transduced islets. Further, basal insulin secretion was comparable between the two islet groups. When transduced and non-transduced islets were challenged with insulin secretagogues, they showed similar increases in insulin release. Transduced and non-transduced islets were equally effective in normalizing blood glucose when transplanted into diabetic nude mice. Euglycemia was maintained for 8 weeks until the graft-bearing kidney was removed. Intense green fluorescence was seen in removed islet grafts. Histology revealed preserved islet morphology, with abundant insulin-producing cells, few apoptotic cells and infiltrating leukocytes in both transduced and non-transduced grafts.

CONCLUSIONS

Lentivirus transduction does not affect islet morphology or function. Lentiviral vectors will allow genetic modifications to be performed in islets before transplantation--modifications that can improve engraftment and/or prevent xenograft rejection.