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Adenoviral transfection of isolated pancreatic islets: a study of programmed cell death (apoptosis) and islet
1Department of Surgery, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Abstract:
Gene therapy provides a potential technique to modify immunity in vitro and therefore may prolong graft survival in vivo. However, viral infection and gene transfer may damage target cells and interfere with biologic function. Viruses, including adenovirus, are known to be capable of modulating apoptosis and initiating cell death by either inducing or suppressing specific processes, depending on the virus and cell system studied. The effect of adenovirus on islet cell viability and function has not been examined in detail. In this study, the dose-dependent effect of an adenoviral vector on islet cell death and glucose-stimulated insulin secretion (GSIS) was investigated to establish a therapeutic window for the dose of viral vector administered. Isolated pancreatic rat islets were incubated with an adenovirus expressing a beta-galactosidase gene (AdHCMVsp1LacZ) at different viral concentrations [multiplicity of infection (MOI) 1:10, 1:100, and 1:1000]. Transfection rate, in vitro and in vivo islet viability, and occurrence of programmed cell death were determined 1, 3, and 7 days after transfection. Islets, transfected at MOI 1:10 and 1:100, demonstrated apoptosis not significantly different from nontransfected controls. Islets, transfected at MOI 1:1000, demonstrated a significant increase in apoptosis at 24 hr, which decreased over 7 days of culture. The increase in apoptosis was not reflected by a significant decrease in in vitro GSIS of surviving islet cells, as assessed by stimulation index following in vitro perifusion. SCID or nude mice transplanted with AdlacZ-transfected islets (MOI 1:100 and 1:1000) remained normoglycemic for > or = 30 days. These results demonstrate that transfection of islets using adenoviral vectors can be manipulated such that efficient expression of the gene product encoded by the transfected gene (beta-galactosidase) can be achieved at lower transfecting concentrations of the adenoviral vector (MOI 1:10, 20.2%; MOI 1:100, 30.7%) while preserving islet function. This efficiency of transfection may allow pretransplant manipulation of isolated islet cells without vector-specific alteration of islet function. In cases where high virus concentrations are required for efficient gene transfer (adequate expression of the transgene product), a deleterious effect of the vector on islet cell function, with increased cell loss due to increased apoptotic events, is predicted. Using the AdlacZ vector, cell loss by apoptotic mechanisms appears limited to the first days following coculture with high viral concentrations, and does not appear to influence in vitro or in vivo cell function of the surviving islet cells.
Insights
Adenoviral vectors can modify islet cells for gene therapy, but high doses increase cell death. Lower doses achieve efficient gene transfer without harming islet function, preserving insulin secretion and graft survival.
Area of Science:
- Biotechnology
- Immunology
- Cell Biology
Background:
- Gene therapy offers potential for modifying immunity and prolonging graft survival.
- Viral vectors, like adenoviruses, can impact cell viability and apoptosis.
- The specific effects of adenoviruses on pancreatic islet cells require detailed investigation.
Purpose of the Study:
- To investigate the dose-dependent effects of adenoviral vectors on islet cell viability and function.
- To establish a safe therapeutic window for adenoviral vector administration in islet cell gene therapy.
- To assess the impact of adenoviral transfection on glucose-stimulated insulin secretion (GSIS) and in vivo graft survival.
Main Methods:
- Isolated rat pancreatic islets were transfected with an adenovirus expressing beta-galactosidase at varying multiplicities of infection (MOI).
- Transfection rates, in vitro and in vivo islet viability, and apoptosis were assessed at multiple time points.
- In vitro GSIS was measured using perifusion, and normoglycemia was monitored in immunocompromised mice transplanted with transfected islets.
Main Results:
- Transfection at MOI 1:10 and 1:100 showed no significant increase in apoptosis compared to controls.
- High MOI (1:1000) led to increased apoptosis within 24 hours, which subsided over 7 days.
- Despite transient apoptosis at high MOI, in vitro GSIS and in vivo normoglycemia (>30 days) were maintained in surviving islet cells.
Conclusions:
- Adenoviral vector transfection of islets can be optimized for efficient gene expression at lower doses (MOI 1:10-1:100) while preserving islet function.
- High viral concentrations may induce transient apoptosis but do not significantly impair the function of surviving islet cells.
- These findings support pre-transplant manipulation of islet cells using adenoviral vectors without compromising their therapeutic potential.