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Multi-transgenic pigs for vascularized pig organ xenografts

David Ayares¹, Carol Phelps¹, Todd Vaught¹, Suyapa Ball¹, Michael Mendicino¹, Jagdeece Ramsoondar¹, Jeff Monahan¹, Amy Delong¹, Anneke Walters¹, Amy Dandro¹, Angelica Giraldo¹, Thomas Starzl², Yifan Dai¹, David Cooper²

¹Revivicor Inc, Blacksburg, VA; ²Starzl Transplant Institute, University of Pittsburgh, Pittsburgh, PA; United States

Background: Organs from transgenic pigs with Gal-transferase knockout (GTKO) combined with complement inhibitor genes (hCD46 or DAF) are resistant to hyperacute rejection and most anti-pig antibody mediated rejection. Delayed xenograft rejection including thrombosis, inflammation, and cell-mediated rejection may be addressed by further genetic modification including over-expression of anti-coagulation genes (thrombomodulin, CD39), and down-regulation of SLA Class-II using a dominant-negative mutant of CIITA transactivator (CIITA-DN).

Methods: Using breeding and cloning, GTKO/CD46/DAF pigs were generated as base genetics. A constitutive CAG promoter vector was used to clone and express the CIITA-DN transgene, which was subsequently transfected into either WT or GTKO/CD46/DAF fibroblasts and used to produce cloned pigs. CIITA-DN was analyzed by FACS and MLR assays. Endothelial-specific vectors using either TIE-2 or ICAM-2 expression systems were used to build human CD39 and human thrombomodulin (TM) transgenes. Expression of CD39TIE and TMICAM was analyzed by RT-PCR and IHC in endothelial cell lines, cloned fetuses, and live pigs.

Results: Viable multi-transgenic pigs were produced including: GTKO/CD46/DAF, GTKO/CD46/DAF/CIITA-DN, GTKO/CD46/TMICAM, and GTKO/CD46/CD39TIE. In all cases, founder animals were euthanized and examined for strong transgene expression in target tissues/organs (heart, kidney, liver, lung). Expression was quantified by real-time-PCR, IHC, and by FACS analysis for endo-specific transgenes in isolated aortic endothelial cells. Bioactivity of CIITA-DN was demonstrated by lack of cell surface SLA class II expression on PAEC, and inhibition of human CD4+ T cells by MLR. Founders with optimal expression were subsequently used for re-cloning to generate animals for testing in non-human primates.

Conclusions: Building on a GTKO/CD46 background, transgenic pigs were produced that additionally express an inhibitor of cellular rejection (CIITA-DN), as well as, targeted endo-specific expression of anti-thrombosis/anti-inflammatory genes (hCD39 and hTM). Testing of these multi-transgenic pig lines in NHP systems will provide insights into utility and efficacy for xeno-organ transplants.