The resounding success of a new immunosuppressive regimen known as the Edmonton protocol demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. However, under the Edmonton protocol, a single donor does not provide enough islets to attain the insulin independence of a transplant recipient. This limitation is mainly caused by islet apoptosis triggered during isolation. In this study, we describe a highly efficient system of transiently transferring anti-apoptotic proteins into pancreatic islets, thus opening an exciting new therapeutic opportunity to improve the viability of transplantable islets. We fused β-galactosidase to the 11–amino acid residues that constitute the protein transduction domain (PTD) of the HIV/TAT protein and transduced pancreatic islets ex vivo with this fusion protein in a dose-dependent manner with >80% efficiency. We observed that transduction of the anti-apoptotic proteins Bcl-XL and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-α in a pancreatic β-cell line, indicating that TAT/PTD anti-apoptotic proteins retained their biological activity. Finally, we demonstrated that TAT-fusion proteins did not affect the insulin secretion capability of islets, as determined by glucose static incubation and by reversion of hyperglycemia in diabetic immunodeficient mice.
Address correspondence and reprint requests to R.L. Pastori, Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Ave., Miami, FL 33136. E-mail:.
Received for publication 22 December 2000 and accepted in revised form 15 May 2001. Posted on the World Wide Web at www.diabetes.org/diabetes on 21 June 2001.
AFC, 7-amino-4-trifluoromethyl coumarin; CHX, cycloheximide; DED, death effector domain; DEVD, Asp-Glu-Val-Asp; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; FBS, fetal bovine serum; FITC, fluorescein isothyiocyanate; FMK, fluoromethyl ketone; IEQ, islet equivalent; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; PTD, protein transduction domain; SI, stimulation index; TNF-α, tumor necrosis factor-α; VAD, Val-Ala-Asp.