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Islet-specific expression of TFPI, CD39, and CTLA4Ig in transgenic pigs designed for xenoislet transplantation

David Ayares¹, Todd Vaught¹, Suyapa Ball¹, Jagdeece Ramsoondar¹, Jeff Monahan¹, Michael Mendicino¹, Anneke Walters¹, Amy Dandro¹, Angelica Giraldo¹, Suzanne Berterra², Rita Bottino², Massimo Trucco², Carol Phelps¹

¹Revivicor Inc, Blacksburg, Virginia, United States; ²Children’s Hospital, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Background: Pig islets with Gal-transferase knockout (GTKO) and/or constitutive expression of hCD46 have shown long-term function and successful restoration of insulin independence in nonhuman primate (NHP) models. An islet-specific expression system has been designed for expression of multiple immunomodulatory transgenes, for inhibition of thrombosis, inflammation, early islet loss, and rejection, towards improved xenoislet transplant outcomes.

Methods: Human CD39, human TFPI, and porcine CTLA4Ig transgenes were cloned individually into an islet-specific expression vector containing the rat insulin II promoter and mouse PDX-1 enhancer. All three vectors (CD39ins, TFPIins and CTLA4Igins) were transfected alone or co-transfected in combination into pig fibroblast cells which were already transgenic for hCD46 and homozygous GTKO. Southern (+) GTKO/hCD46/CD39ins, GTKO/hCD46/TFPIins/CTLA4Igins, and GTKO/hCD46/CD39ins/TFPIins/CTLA4Igins cell lines were used for nuclear transfer (NT) to produce pregnancies. Cloned fetuses were analyzed for expression in fetal organs (pancreas, heart, liver) by IHC, Western, and real-time PCR. Fetal fibroblasts with confirmed expression of the desired transgenes were used for NT to produce live pigs.

Results: Viable multi-transgenic pigs with three, four, or five different genetic modifications were produced. Three different pig lines were established: 1) GTKO/hCD46/CD39ins, 2) GTKO/hCD46/TFPIins/CTLA4Igins, and 3) GTKO/hCD46/CD39ins/TFPIins/CTLA4Igins. IHC revealed robust islet-specific expression of CD39, TFPI and CTLA4Ig in pancreas, with only background expression in heart and liver. Islets from pigs containing the TFPI transgene, when mixed with human blood, showed prolonged clotting times. Western analysis showed strong pCTLA4Ig expression only in the pancreas. To date, the three and four gene pig lines have been bred and produced viable offspring.

Conclusions: Building on GTKO/CD46 genetics, an islet-specific expression system was used to produce multi-transgenic pigs with CD39, TFPI, and CTLA4Ig to inhibit activation of tissue factor, inflammation, coagulopathy, and cellular rejection. Islets will be isolated from these pigs to evaluate efficacy in NHP models towards clinical application of xenoislet transplantation.