40 DAYS SURVIVAL AFTER ORTHOTOPIC CARDIAC XENOTRANSPLANTATION OF MULTI-TRANSGENIC PIG HEARTS IN A PIG-TO-BABOON MODEL WITH CD40MAB OR CD40L COSTIMULATION BLOCKADE AND XENOGRAFT PRESERVATION USING “STEENS” COLD BLOOD CARDIOPLEGIA PERFUSION

Paolo Brenner ^0,0; Tanja Mayr ^0,0; Bruno Reichart ⁰; Sonja Guethoff ^0,0; Stefan Buchholz ⁰; Alexander Dashkevich ⁰; Sebastian Michel ⁰; Isabelle Lutzmann ^0,0; Fabian Werner ^0,0; Andreas Bauer ⁰; Nikolai Klymiuk ⁰; Eckhard Wolf ⁰; Muhammad Mohiuddin ⁰; Keith Reimann ⁰; Walter Hermanns ⁰; David Ayares ⁰; Christopher McGregor ⁰; Christian Hagl ⁰; Stig Steen ⁰; Jan-Michael Abicht ⁰

²Dept. of Cardiac Surgery, Clinic of Grosshadern, University of Munich (LMU), Munich, United States; ³Dept. of Anaesthesiology, Clic of Grosshadern, University of Munch (LMU), Munich, United States; ⁴Walter-Brendel-Centre, University of Munich (LMU), Munich, United States; ⁵Dept. of Molecular Animal Breeding and Biotechnology, University of Munich (LMU), Munich, United States; ⁶MassBiologics, University of Massachusetts Medical School, Boston, MA, United States; ⁷Department of Veterinary Pathology, University of Munich (LMU), Munich, United States; ⁸Cardiothoracic Surgery Research Laboratory, NHLBI, NIH, Bethesda, MA, United States; ⁹Revivicor , Inc., Blackburg, MA, United States; ¹⁰Department of Cardiothoracic Surgery, University College, London, United States; ¹¹Department of Cardiothoracic Surgery, University of Lund, Lund, United States

Introduction: We used a non-toxic immunosuppression based on CD40/CD40L co-stimulation blockade in group G1 and additionally to improve primary xenograft function in a group G2 a new non-ischemic myocardial preservation technique to achieve a constant preclinical long-term survival in a life-supporting cardiac xenotransplantation model (pig-to-baboon).

Methods: According to the technique of Lower and Shumway 9 orthotopic (OHXTx) heart transplantations were performed in baboons with genetically-modified GalKO/hCD46/hTM transgenic pig hearts. The immunosuppression (IS) consisted of ATG, rituximab, MMF, tapered down cortisone and CD40 antibody or PASylated Fab-CD40L. Because a “perioperative cardiac xenograft dysfunction” (PCXD) played a critical role, cardioplegia with crystalloid solution (Bretschneider solution, 50ml/kg; G1: n=5) was replaced in the last 4 cases of G2 by Steen´s “non-ischemic preservation technique”: This 8°C cold myocardial perfusion solution consists of a  modified Krebs-Henseleit solution with albumin and 10% erythrocytes, hormones and vasoactive agents (Steen et al, 2016*). Under pressure, flow and temperature control heart is constantly perfused during explantation and storage time, intermittently during implantation, using an independent portable heart-lung-machine.

Results: Ischemic time ranged from 112-128 min. Survival in G1 with Bretschneider solution were 3, 1, 30, 1 and 1 day(s). Using non-ischemic preservation technique in G2 PCXD was not observed and the recipients survived 18, 1**, 27 and 40 days. Baboons mostly died of respiratory problems, renal and hepatic failure, one was sacrificed due to a major p.o. neurological deficit**, another died after hematothorax, but no hyperacute or delayed xenograft rejection was found. A problem after 3 weeks was a donor organ (over)growth. All long-term surviving baboons (especially in G2) were in good general conditions.

Conclusion: With conventional hypothermic, ischemic heart preservation techniques, perioperative cardiac xenograft dysfunction plays a detrimental role after orthotopic xHTx. This was prevented with the Steen’s “non-ischemic preservation technique, an important step on the way to a long-term survival of 2 -3 months as an important prerequisite for cardiac xenotransplantation in a clinical use.