Proteins Linked to a Protein Transduction Domain Efficiently Transduce Pancreatic Islets

Abstract

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.

Footnotes

  • 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: rpastori{at}med.miami.edu.

    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.

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