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Gal-knockout pigs transgenic for hCD46 and islet-specific TFPI/pCTLA4Ig have normal glucose metabolism and isolated islets exhibit normal glucose and arginine responses and should be considered as donors for islet xenotransplantation

Martin Wijkstrom¹, Suzanne Bertera², Burcin Ekser¹, Cassandra Long¹, Hidetaka Hara¹, Mohamed Ezzelarab¹, Eefje Dons¹, Goutham Kumar¹, Dirk van der Windt¹, Tatyana Votjakova², Carol Phelps³, David Ayares³, David KC Cooper¹, Rita Bottino²

¹Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA; ²Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA; ³Revivicor, Blacksburg, VA; United States

Background: Streptozotocin-diabetic nonhuman primates were rendered normoglycemic long-term by islet transplantation from pigs transgenic for the human complement-regulatory protein, hCD46. Further genetic modifications may decrease immediate islet destruction and reduce both the innate and adaptive immune responses, allowing for clinical application. A new strain of α1,3-galactosyltransferase gene-knockout (GTKO) pigs was established transgenic for hCD46 and additionally expressing islet-specific tissue factor pathway inhibitor (TFPI) and porcine CTLA4Ig under the control of an insulin promoter. This new transgenic pig strain was evaluated as potential islet source by studying islet function.

Methods: Fasting glucose and acute C-peptide responses to arginine in GTKO/hCD46 or GTKO/hCD46/TFPIins/pCTLA4Igins were tested in adult pigs (200-250kg). Isolated islets were tested by glucose/theophylline perifusion assay or in vivo (diabetic NODscid mice transplanted with 800 islets). Islet viability was also assessed by determining islet mitochondrial respiratory activity by measuring the rates of oxygen consumption and by calculation of respiratory control parameters from mitochondrial resting and ATP-producing respiratory states.

Results: Fasting blood glucose was <90mg/dL. Fasting C-peptide level was 0.40-0.63ng/mL at baseline, and increased by 1.0-1.2ng/mL after arginine challenge. Islet isolation yielded 315,000-350,000IEQ (purity, 60-70%; good viability and mitochondrial function) with ~80% recovery after culture. Perifusion assay showed adequate insulin release to glucose, further potentiated by theophylline, with stimulation indices of 3.5-5.8 and 4.7-9.9, respectively. In vivo islet function after transplantation in diabetic immunodeficient mice showed ability to normalize blood glucose levels <200mg/dL, followed by return to hyperglycemia following graft-bearing nephrectomy. The islets demonstrated good mitochondrial functions as measured by respiratory parameters (maximal to resting respiration ratio was 9.92).

Conclusions: Our study demonstrates that adult GTKO/hCD46/TFPIins/pCTLA4Igins pigs exhibited normal glucose metabolism. Successful islet isolation was achieved. As more of these pigs become available, they will be tested in a nonhuman primate model and for potential protection from IBMIR.