1. KIDNEY XENOTRANSPLANTATION - THE NEXT GREAT BREAKTHROUGH IN NEPHROLOGY? David K.C. Cooper MD, PhD, FRCS Thomas E. Starzl Transplantation Institute University of Pittsburgh

2. COMPETING INTERESTS I am Chairman of the Scientific Advisory Board of Revivicor, Inc., Blacksburg, VA, but I have no financial interest in the company and do not receive any remuneration whatsoever.

3. REVIVICOR Small biotechnology company (25 staff) that concentrates its effort on the genetic engineering of pigs for medical purposes: 1. Xenotransplantation 2. Human immunoglobulin (also for biodefense)

4. ACKNOWLEDGEMENTS Many colleagues at STI, Allegheny General Hospital, and Revivicor

6. HISTORY OF XENOTRANSPLANTATION

9. XENOTRANSPLANTATION: THE PIG AS THE ORGAN-SOURCE

10. XENOTRANSPLANTATION Advantages 1: 1. Unlimited supply of donor organs. 2. Organs available electively. 3. Avoids effects of brain death. 4. Infection-free donors.

11. XENOTRANSPLANTATION Advantages 2: 1. Borderline candidates 2. “Cultural” barriers to deceased donation (e.g. Japan) 3. Diabetes mellitus/cell transplants

12. CLINICAL PIG-TO-HUMAN XENOTRANSPLANTATION Organs (kidney, heart, liver, lung) Islets Corneas Neuronal cells Hepatocytes Skin Red blood cells

13. XENOTRANSPLANTATION: BARRIERS

14. BARRIERS TO XENOTRANSPLANTATION IMMUNOLOGIC Physiologic Safety (risk of infection) Ethical Regulatory / Legal

15. BARRIERS TO XENOTRANSPLANTATION “Immunologic” includes: 1. Innate immune response (e.g., antibody, complement, macrophages) 2. Adaptive immune response (e.g., T and B cells) 3. Coagulation dysfunction (e.g., thrombin, platelets) 4. Inflammation

16. PIG ORGAN XENOTRANSPLANTATION IN NONHUMAN PRIMATES

17. PIG-TO-BABOON KIDNEY TX (DAY 0)

18. PIG-TO-BABOON KIDNEY TX (HAR)

20. PORCINE HUMAN αGal NeuGc ß4GalNT2 ABH NeuAc

21. XENOTRANSPLANTATION: EXPERIMENTAL PROGRESS

23. Solution 1 Gene-knockout of known antigenic targets for human anti-pig antibodies, e.g., αGal, NeuGc, ß4GalNT2

24. Human antibody binding to the AECs *p<0.05 (n=6) * * * * * * IgMIgG

25. Solution 2 Pigs transgenic for a human protein, e.g., complement- regulatory, coagulation- regulatory, anti-inflammatory, immunosuppressive agent

26. Isotype GE pig AECs Human AECs Surface expression on genetically-engineered (GE) pig and human aortic endothelial cells (AECs) CD55 (DAF)CD141 (TBM)CD46CD55 (DAF)CD141 (TBM)CD46

28. GENETICALLY-ENGINEERED PIGS CURRENTLY AVAILABLE Revivicor has produced pigs with 20 different genetic manipulations Some pigs have 7 modifications Worldwide, 40 different manipulations

29. ELICITED ANTI-PIG ANTIBODIES Unless prevented by immunosuppressive therapy, after exposure to a pig organ or cells, anti-pig antibodies can increase >10-fold

30. PROGRESS IN PIG-TO-NHP KIDNEY TX Progress in immunosuppressive therapy: Conventional (e.g., tacrolimus, steroids) Costimulation blockade (Genetically-engineered pigs)

31. PROGRESS IN PIG-TO-NHP KIDNEY TX The Emory group had one monkey surviving >10 months with a life- supporting pig kidney graft (The NIH group had two baboons surviving >1 year after a heterotopic heart graft)

32. PIG-TO-NHP RENAL XENOTX GTKO/hDAFRhesus macaque: T cell depletion, anti-CD154, MMF/steroids

35. BARRIERS TO XENOTRANSPLANTATION Immunologic PHYSIOLOGIC Safety (risk of infection) Ethical Regulatory / Legal

36. B9313 - INCREASE IN SIZE OF KIDNEY

37. BARRIERS TO XENOTRANSPLANTATION Immunologic Physiologic SAFETY (RISK OF INFECTION) Ethical Regulatory / Legal

38. SAFETY OF XENOTRANSPLANTATION Concern regarding potential transfer of infectious microorganisms to (1) recipient, (2) public

39. SAFETY OF XENOTRANSPLANTATION ‘Remaining’ potential risk: Porcine endogenous retroviruses (PERVs)

40. BARRIERS TO XENOTRANSPLANTATION Immunologic Physiologic Safety (risk of infection) ETHICAL REGULATORY / LEGAL

41. GENETICALLY-ENGINEERED PIGS 1. No unacceptable implications for the health and welfare of the pig 2. No serious ethical objections to the genetic procedure - brain (pig or human) - reproduction of one species by the other The Netherlands

43. “There are many ways of losing money. Women are the most fun. Gambling is the fastest. Research is the most certain.” Lord Hives Chairman of Rolls Royce

44. GENETICALLY-ENGINEERED PIGS Recent new technologies, e.g., Zinc finger nucleases TALENS CRISPR/Cas9 will facilitate the production of pigs with multiple genetic modifications

46. History tells us that procedures that were inconceivable yesterday, and are barely achievable today, often become routine tomorrow. Thomas E. Starzl, 1982

47. POTENTIAL ALTERNATIVES TO XENOTRANSPLANTATION 1. Human stem cells 2. Regenerative medicine 3. Cell-based mechanical devices

48. FIRST CLINICAL TRIAL ? Patients highly-sensitized to alloantigens (high PRA) ? Patients with problems of vascular access for dialysis

51. “It is often sufficient to know, in the large, that a thing may be possible” Littre, 1710 Royal Academy of Science Paris

52. One day “making a pig of yourself” could have a whole new meaning

53. THANK YOU