Adenovirus-Mediated Gene Transfer into Isolated Mouse Adult Pancreatic Islets: Normalβ-Cell Function Despite Induction of an Anti-Adenovirus Immune Response

Abstract

The in vitro purification of pancreatic islets offers an opportunity for their modification by ex vivo gene transfer. We investigated the efficiency and functional consequences of adenovirus-mediated gene transfer into adult murine pancreatic islets with a recombinant adenovirus encoding for the beta-galactosidase (beta-Gal) reporter gene. At 10(6) pfu/islet, almost all of the islets were transduced, but maximal transduction was obtained at 10(7) pfu/islet. Histochemical analysis of frozen islet sections showed that transduced cells were only located at the periphery of islets. Transduced islets showed normal insulin secretion in vitro, and were able to normalize in vivo the glycemia of streptozotocin-induced diabetes in syngeneic and allogeneic mice. beta-Gal expression in transduced islets was observed for at least 6 weeks in naive normal recipients and in immunodeficient mice, but was shortened in mice preimmunized to adenovirus. Nevertheless, islets maintained normal control of glycemia in all mice. An early leukocyte infiltrate was observed in syngeneic grafts of transduced islets, but no acceleration in rejection of fully MHC-incompatible islet grafts occurred. In summary, adenovirus-mediated gene transfer in adult mouse islets, although sparing most of the beta-cells, was highly efficient and did not impair insulin secretion by islets. The immune response to the adenovirus and/or to the transgene might be only partially responsible for the decreased expression over time of the transduced gene. Accordingly, adenovirus-mediated gene transfer might allow efficient expression of vectorized sequences with potential immunosuppressive effects in the islet microenvironment.

Elevated Vascular Endothelial Growth Factor Production in Islets Improves Islet Graft Vascularization

“…Thus, neither adenoviral-mediated transduction nor VEGF production in islet cells significantly affects the ability of ␤-cells to secrete insulin in response to glucose challenge. Consistent with these results are the previous observations that recombinant adenoviruses are highly efficient in transferring genes into isolated mouse islets without perturbing the function of islet cells (25)(26)(27). Effects of VEGF expression in islet grafts on glycemic control in diabetic mice.…”

Control of Voltage-gated Potassium Channel Kv2.2 Expression by Pyruvate-Isocitrate Cycling Regulates Glucose-stimulated Insulin Secretion

“…We were led to investigate this issue based on reports that Kv2.2 is expressed primarily in ␦-cells (42,57) and another recent study showing that global knock-out of Kv2.1 in transgenic mice results in enhanced GSIS in isolated islets and that Ad-siRNA-mediated knockdown of Kv2.2 in mouse islets enhances somatostatin but not insulin secretion (68). The latter findings should be interpreted with caution as mouse islets are reported to be refractory to penetration of recombinant adenoviruses to the ␤-cell core relative to rat islets (48,52), possibly explaining the absence of an effect of Kv2.2 knockdown on insulin secretion in the setting of the mouse islet. The current study used FACS-sorted mouse islet cells to demonstrate clear expression of Kv2.2 in ␤-cell-, ␣-cell-, and ␦-cell-enriched cell pools.…”

Baculovirus-mediated gene transfer into pancreatic islet cells.

“…For whole-islet transductions, the baculovirus, like adenovirus, only affects cells at the periphery of the islets (3)(4)(5). It is likely that the geometry of the islet and the existence of tight junctions between islet cells does not allow cells in the inner core of the islet to be exposed to viral particles.…”