A novel approach to xenotransplantation combining surface engineering and genetic modification of isolated adult porcine islets.
BACKGROUND
Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly[ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2.


METHODS
Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining. Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SCID mice reconstituted with human lymphocytes (5 x 10 8 /intraperitoneally/15 days before PIT).


RESULTS
No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end"-capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P <.05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P <.001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100% of the animals given islets modified with DSPEG + AdBcl-2 during the observation time.


CONCLUSIONS
Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.
Contreras
Juan L
JL
Department of Surgery, Division of Human Gene Therapy and Gene Therapy Center, University of Alabama at Birmingham, USA.
Xie
Dong
D
Mays
Jimmy
J
Smyth
Cheryl A
CA
Eckstein
Christopher
C
Rahemtulla
Firoz G
FG
Young
Carlton J
CJ
Anthony Thompson
J
J
Bilbao
Guadalupe
G
Curiel
David T
DT
Eckhoff
Devin E
DE
D000818:Animals; D017209:Apoptosis / Q000276:immunology; D002470:Cell Survival / Q000276:immunology; D002478:Cells, Cultured; D015870:Gene Expression; D018014:Gene Transfer Techniques; D019254:Genes, bcl-2 / Q000235:genetics*; D007515:Islets of Langerhans / Q000276:immunology; D016381:Islets of Langerhans Transplantation / Q000276:immunology*; D023421:Models, Animal; D011092:Polyethylene Glycols / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013552:Swine; D014183:Transplantation, Heterologous / Q000276:immunology*
D013501:Surface-Active Agents; D011092:Polyethylene Glycols