Liver Xenotransplantation Comprehensive Development Program A. Joseph Tector MD PhD Indiana University School of Medicine Clarian Transplant Institute

Need for Alternative Source of Organs 98,000 Americans are waiting for a solid organ transplant 46,000 have died waiting for a transplant since 2000

Xenotransplantation Unlimited source of donor organs for all types of transplants Potential to genetically engineer donor animals to eliminate immunological barriers

Problems with Xenotransplantation Immunological barriers Hyperacute rejection Acute vascular rejection Cellular rejection Physiological barriers Animal proteins may or may not work with human counterparts Coagulation proteins

Problems with Xenotransplantation Risks of zoonotic infection Porcine Endogenous Retrovirus (PERV) Finding an indication with immediate chance to benefit patient

The Case Against Cardiac and Renal Xenografts Dialysis can be tolerated for years Assist devices can bridge a patient for 12-18 months Xenograft would need to function at least 6 months to be considered for a clinical trial

The Case for Liver Xenotransplantation Temporary liver support is lacking Liver xenograft could be used to bridge patients for a period of days and be a success

The Patient

The Solution

Three Weeks Following Pig Liver Perfusion Followed by Human Liver Transplant

Recipients with Liver Failure are Less Capable of Rejecting a Liver Xenograft

The Program

Genetic Engineering of Donor Pigs

Advantages of Genetic Engineering Decrease response of the human immune system Minimize zoonotic infectious risk Eliminate physiologic incompatibilities

Nuclear Transfer Procedure Enucleated Oocyte Fused dublet Nucleated Oocyte Dublet Nuclear Donor Somatic Cell Activation CLONE

Enucleation 1st polar body

Enucleation 1st polar body Metaphase plate

Dublet Preparation Nuclear donor Enucleated oocyte or Cytoplast

Product of Genetic Engineering

Better Way to Make Pigs Bank fetal fibroblasts Make genetic modifications on fibroblast from latest generation of donor pig Eliminate expensive and time consuming breeding programs

Better Way to Make Pigs Eliminate need for large herds and big farms New pig every 6 months instead of 2-3 years

Gal Knockout Construct LH PURO RH SalI BstXI AscI MluI

Human CD39/CD59 Construct

Human CD46/CD47 Construct

Next Steps in Donor Engineering Introduce GTKO, CD/39/59, and CD46/47 constructs into fetal fibroblasts (1 month) SCNT to produce pigs (5 months) Transgenes to minimize risks of zoonotic infections Transgenes to decrease cellular immunity

Xenograft Immunology Christopher Burlak PhD started July 1, 2008 PhD student working on non-gal xenoantigens Surgical Resident studying T- cell xenoimmunology

Zoonotic Considerations May 2008 - Hired viral forecaster Dr. Nathan Wolfe DsC, UCLA School of Epidemiology, as a consultant to setup guidelines for monitoring xenograft recipients, their personal contacts, and health care workers He is in the process of establishing an advisory board to provide guidance with regards to infectious risks

Operative Considerations in Pig to Human Liver Xenografts

Pig Liver in Situ

Human Cadaver

Suprahepatic Cava Anastamosis

Portal Venous and Hepatic Arterial Anastamoses

Next Steps We will transplant pig livers into brain-dead individuals We should have GTKO pigs in early January We could begin transplanting in late February or early March If the GTKO pig livers function for 24 hours we could begin to use these organs as a bridge to transplant for clinical liver transplantation