Xenotransplantation: Still a hope?

1. Xenotransplantation: Still a hope? Emanuele Cozzi University Hospital - Padua

2. An approach to the problem of Xenograft rejection Recipient Donor Production of “engineered” animals

3. Xenotransplantation: Key issues • Immunology • Physiology • Biosafety • Ethics and regulations

4. Xenotransplantation: Key issues and genetic engineering • Immunology • Physiology • Biosafety • Ethics and regulations

5. Clinical Xenotransplantation: Key issues • Immunology • Physiology • Biosafety • Ethics and regulations

6. To deal with immunological issues 3 approaches have been followed • Pharmacological immunosuppression • Tolerance inducing regimens • Genetic engineering of the pig

7. Recommendations of the ISHLT Xenotransplantation Advisory Committee • A clinical trial should be considered when: • ~ 60% survival has been achieved of a life-supporting organ in a NH primate for at least 3 months • Some animals should achieve a longer survival ( 6 months) • In the absence of life-threatening complications from IS [Cooper et al:JHLT 2000, 19:1125]

8. Pharmacological immunosuppression • Anti CD154 has been used as part of the immunosuppression in the study with the longestsurvivor(median 63 days; range: 16-179d) . • Perhaps antiCD40 may lead to similar results? • All grafts underwent AHXR which was characterized by the presence of thrombotic micoangiopathy (little cellular infiltrate; no obvious interstitial hemorrhage) [Kuwaki et al, Nat Med 2005; Shimizu et al A J Pathol 2008]

9. Pharmacological immunosuppression In contrast, a clinical applicable regimen was used in the study with the longest median survival (median 96 days; range: 15-137d) of heterotopic hearts: [Anti CD20 + ATG] + Tacro+ Rapa + steroids+TPC +SPX • No consumptive coagulopathy • treatable infections • Rejection in 2 cases only! [McGregor et al, J Thorac Cardiovasc Surgery 2005]

10. Longest survival of primates transplanted with porcine organs or cells • 179 days for a heterotopic heart xenograft • 56 days for an orthotopic heart xenograft • 90 days for a life supporting kidney xenograft • >180 days in at least series of islet xenografts

11. Six-month survival in islet pig-to-primate xenotransplantation • Hering et al [nature Med. 2006] • Cardona et al [Nature Med. 2006] • Larsen et [ATC, San Francisco 2007] • Gianello et al [IXA-IPITA-CTS, Minneapolis 2007]

12. Six month survival in islet pig-to-primate xenotransplantation • Hering et al [nature Med. 2006] • Cardona et al [Nature Med. 2006] • Anti-CD25+ anti-CD154 + FTY + RAD+ LFM • Anti CD25 + anti CD154 + sirolimus + Belatacept • Larsen et [ATC, San Francisco 2007] • Anti CD25 + anti CD40 + sirolimus + Belatacept • Gianello et al [IXA-IPITA-CTS, Minneapolis 2007] • Encapsulation with alginate, no immunosuppression

13. Six month survival in islet pig-to-primate xenotransplantation • Hering et al [nature Med. 2006] • Cardona et al [Nature Med. 2006] • Anti-CD25+ anti-CD154 +FTY+ RAD+LFM • Anti CD25 + anti CD154 + sirolimus +Belatacept • Larsen et [ATC, San Francisco 2007] • Anti CD25 + anti CD40 + sirolimus +Belatacept • Gianello et al [IXA-IPITA-CTS, Minneapolis 2007] • Encapsulation with alginate, no immunosuppression In all cases, WILD TYPE islets were used

14. 600 400 200 0 Longestsurvival of porcine islet xenografts (II) -4 0 4 8 12 14 20 24 Weeks Dufrane et al, Minneapolis 2007

15. Longestsurvival of porcine islet xenografts [BSM]+FTY720+RAD+ABI793+LFM Hering et al, Nature Medicine 2006

16. To deal with immunological issues 3 approaches have been followed • Pharmacological immunosuppression • Tolerance inducing regimens • Genetic engineering of the pig

17. Tolerance inducing regimens • Co-transplantation of vascularised thymic tissue with GalT-KO kidneys using a tolerance inducing regimen • prolonged survival of functioning kidney xenografts, without the development of anti-donor T cell responses or of induced antibody (median: 69; range 31-83 days) [Yamada et, Nat Med 2005]

18. To deal with immunological issues 3 approaches have been followed • Pharmacological immunosuppression • Tolerance inducing regimens • Genetic engineering of the pig

19. Improving graft survival and genetic engineering of the pig Better comprehension of the rejectionmechanisms Identification of new targets for specific intervention Genetic engineering of the pig genome

20. Genetic engineering of the pig : Possible targets of intervention • Complement regulation (CD55, CD46, CD59) • Immunogenicity (αGALT-KO) • Coagulation (CD39, TM, TFPI, TF-KO, TM...) • Immunomodulation (CTLA4Ig, HLA-G) • Ischemia/IRI (CD39, A20, VEGF...) [adapted from D’Apice et al, Xenotransplantation 2008]

21. Genetic engineering • Substantial improvements in the technology: • Identification of novel targets of intervention • Cloning of the pig • Multigene constructs (adaptation of FMV 2A syst.) • Very recent identification of pig embryonic stem (ES) cells [D’Apice et al, Xenotransplantation 2008]

22. Clinical Xenotransplantation: Key issues • Immunology • Physiology • Biosafety • Ethics and regulations

23. Pig-to-primate physiological compatibility • Molecular incompatibilities have been demonstrated between pig and man (coagulation, liver) • A consumptive coagulopathy is invariably observed following xenotransplantation. However, we now know that DIC is NOT the rule in primate xenotransplantation (neither in baboons, nor in cynos) • In all cases, to date no insormountable physiological or anatomical incompatibilities have been reported (life-supporting renal/cardiac models)

24. Clinical Xenotransplantation: Key issues • Immunology • Physiology • Biosafety • Ethics and regulations

25. Potential microbial problems in xenotransplantation Potentially problematic microbes are those: • difficult to eliminate • maintained in a latent or intracellular state in an asymptomatic host • Porcine endogenous retrovirus (PERV) • Herpesviruses • Circovirus etc. • as yet unidentified

26. PERVs: important aspects • In a retrospective study in 160 patients exposed to living porcine tissues, no signs of PERV-related infection (current, past or latent) or disease were observed. • A line of minipigs carrying PERV that do not replicate in vitro in human cells has been reported • PERV replication inhibited by antiviral agents (including AZT) • (Anti-PERV vaccine prior to exposure to porcine tissues) • Genetically engineering of the donor • siRNA • (PERV- knock out pigs)

27. Xenotransplantation and Zoonoses • Pig breeding and close monitoring in SPF facilities • The search for potential unknown pathogens remains a central issue. • latest advances in biomolecular technology are proving to be very useful in the timely identification of unknown microorganism potentially present in human tissues. • These include: • PCR • Differential display screening • Microarray • Application of molecular engineering to safety aspects

28. Unknown viruses in allotransplantation A New Arenavirus in a Cluster of Fatal Transplant-Associated Diseases [Gustavo Palacios et al. N Engl J Med 2008;Volume 358:991-998] kidney liver kidney Organ donor

29. Clinical Xenotransplantation: Key issues • Immunology • Physiology • Biosafety • Ethics and regulations

30. Position paper of the Ethics Committee of the International Xenotransplantation Association (2003) • Respect for the person and informed consent • The problem of “xenotourism” • Securing benefit over harm through pre-clinical studies • Ethics and the use of animals • Clinical trials if appropriate regulatory oversight by national body and IRB • Favourable risk/benefit assessment (for the patient and for society)

31. Regulatory aspects: key documents • U.S. Public Health Service (PHS) Guideline on Infectious Disease Issues in Xenotransplantation [January 19, 2001] • Guidance for Industry - Source Animal, Product, Preclinical and Clinical Issues Concerning the Use of xenotransplantation Products in Humans – FDA April 2003 • Recommendation Rec(2003)10 of the Committee of Ministers to EU member states on xenotransplantation • Canada (2001) • Australia (2003) • New Zealand regulatory framework for xenotransplantation • WHO

32. Regulatory aspects: the role of WHO • WHO Assembly adopted in May 2004 the Health Assembly Resolution WHA57.18 that: • Urges Member States to "allow xenotransplantation only when effective national regulatory control and surveillance mechanisms overseen by National Health Authorities are in place“ • Requires the Director-General of the WHO to support Member States in the development and regulation of xenotransplantation. • Refers to the concept of “harmonisation” [“cooperation” of the EU rec (2003)10] • Global Consultation on Regulatory Requirements for Xenotransplantation, held in Changsha (China)

33. Are we currently performing clinical xenotransplantation trials?

34. Clinical xenotransplantation activities • We are aware of 2 biotech companies “interacting” at the FDA level (University of Minneapolis and UPMC) • Conditional approval of a xenotransplantation trial by the New Zealand Goverment (LCT) • An ongoing clinical trial in Russia (LCT)

35. Conclusions Considerable progress has been achieved in the field of solid organ and cell xenotransplantation. We have a better understanding of the rejection process and refined our genetic engineering techniques. This has lead to novel immunosuppressive strategies and the generation of new lines of engineered animals Islet xenografts certainly work longterm and solid xenograft may soon work just as well. However, current EFFICACY DATA do not justify progression to the clinic. For the time beeing we have to design, under the guidance of the WHO, a globally applicable ethical and regulatory framework, to allow safe initiation of xenotransplantation trials.