2013 - IXA 2013 Congress


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Joint IXA/JEXSWINE Plenary Session: Update on Genetic Engineering- Tg Pigs & Tissue Engineering - Part 1 (Sponsored by Astellas)

37.1 - Part 1: Tg Pigs - Breeding strategies for multi-GM pigs

Presenter: Eckhard, Wolf, Melbourne, Germany
Authors:

 

Genetically engineered donor pigs with improved pancreatic islets

Nikolai Klymiuk, Annegret Wünsch, Andrea Bähr, Barbara Keßler, Simone Renner, Lelia Wolf-van Bürck*, Jochen Seissler*, Eckhard Wolf

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Germany; *Diabetes Zentrum, Medizinische Klinik und Poliklinik IV - Campus Innenstadt, Klinikum der LMU München, Munich, Germany

The transplantation of Langerhans islets to diabetic recipients might provide a long-lasting treatment for diabetes and has been implemented with considerable success, but the shortage of human organ donors restricts its future application to a very small proportion of potential recipients. For overcoming this limitation, the usage of Langerhans islets from a different source such as the pig pancreas represents an attractive alternative. Transplantation of encapsulated porcine pancreatic islets to type 1 diabetic patients with severe unaware hypoglycemia has already entered clinical studies, but encapsulation may shorten the lifespan of the islets. To overcome the rejection of pig islets by human T cells, we generated transgenic pigs expressing the optimized CTLA-4Ig variant LEA29Y in the pancreatic beta-cells. Neonatal islet cell clusters (ICCs) from INS-LEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rg null mice. Cloned LEA-tg pigs are healthy and exhibit a strong beta-cell specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. This study provided the first proof-of-principle report on transgenic pigs with beta-cell specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression (Klymiuk et al., Diabetes 61:1527-32, 2012).

To prevent apoptosis of beta-cells during isolation of Langerhans islets from the donor pancreas, in vitro cultivation, and the hypoxic stress phase in the early post-transplantation period, we generated transgenic pigs expressing the X-linked inhibitor of apoptosis (XIAP) under the control of the porcine INS promoter. So far, 10 INS-XIAP transgenic founders have been produced by somatic cell nuclear transfer using pools of stable transfected donor cells. Engineered porcine islets which are less prone to undergo apoptosis as compared to wild-type pig islets should be interesting for encapsulation or for application in specific devices such as the Beta-O2 system from Beta-O2 Technologies Ltd., Petach-Tikva, Israel, or the MAILPAN system developed by Defymed, Strasbourg, France.

To establish a large animal model for testing such devices, we created the first pig model for permanent neonatal diabetes (PNDM) by expression of C94Y mutant insulin in the beta-cells of transgenic pigs (Renner et al., Diabetes 62:1505-11, 2013). This model develops a stable diabetic phenotype without further interventions such as streptozotocin treatment or pancreatectomy and should thus be ideally suited for testing the functionality of islet transplantation therapies.

Grant support: Deutsche Forschungsgemeinschaft, Transregio-CRC 127.


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