Proteins linked to a protein transduction domain efficiently transduce pancreatic islets.
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 beta-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-X(L) and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-alpha in a pancreatic beta-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.
Embury
J
J
Diabetes Research Institute, University of Miami School of Medicine, Miami, Florida 33136, USA.
Klein
D
D
Pileggi
A
A
Ribeiro
M
M
Jayaraman
S
S
Molano
R D
RD
Fraker
C
C
Kenyon
N
N
Ricordi
C
C
Inverardi
L
L
Pastori
R L
RL
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001786:Blood Glucose / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000601:surgery*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D006678:HIV / Q000235:genetics; D006943:Hyperglycemia / Q000628:therapy; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology*; D016381:Islets of Langerhans Transplantation / Q000473:pathology / Q000502:physiology*; D008297:Male; D051379:Mice; D016513:Mice, SCID; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods*; D014183:Transplantation, Heterologous / Q000502:physiology; D051020:bcl-X Protein; D001616:beta-Galactosidase / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus
D051017:Apoptosis Regulatory Proteins; C494812:Bcl2l1 protein, mouse; C494813:Bcl2l1 protein, rat; D001786:Blood Glucose; D015696:Gene Products, tat; C490444:Pea15 protein, mouse; C490443:Pea15 protein, rat; D010750:Phosphoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein; D054322:tat Gene Products, Human Immunodeficiency Virus; D001616:beta-Galactosidase; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases