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Home , Allotransplantation, Eurotransplant, Organ trade, Skin grafting

and the future of human transplants

Owen Morgan, Alan Cann1*

1Department of Neuroscience, Psychology and Behaviour, School of Biological Sciences, University of Leicester, Leicester,

*Corresponding Author

Email: [email protected]

This report was submitted as a final year dissertation for the School of Biological Sciences, University of Leicester, UK.

Abstract

A shortage of organ donors has been an issue since the inception of human . Despite attempts to increase the number of donated organs, the demand for transplants now far exceeds the number of organs available for transplantation. This continuing deficit has questioned whether current sources of organs for human transplantation are currently still viable and importantly for the predicated future increases in demand. Improvements with transplantation over the past few decades has resulted is organ transplants being associated with high survival rates and quality of life making transplantation the optimal solution to chronic organ dysfunction, increasing the demand for transplants. This study investigated organ transplantation in the United Kingdom (UK), of America (US) and (representing , , , , , the , and from 2012 ) to look for widespread trends in organ transplantation. Analyses of data from the sources shows clear deficits in the number of organ donations compared to the increasing demand for transplants. In all cases studied, less than a third of patients received their required transplant each year. The large difference between donation and transplantation suggests that a new source of organs for human transplants is required, with xenotransplantation (transplanting organs, tissues, or cells between different species) offering a more immediate and extensive solution to this problem compared to current alternatives.

Keywords organ shortage, xenotransplantation, genetic modification, transplantation

Xenotransplantation and the future of human organ transplants 2

Table of contents

1. A Brief Historical Introduction………………………………………………………………… 3

1.1 Current Problems with Organ Transplantation…………………………………………… 5

1.1.1 Shortage of transplantable organs……………………………………………………… 5

1.1.2 Long term outcomes……………………………………………………………………… 12

1.1.3 Minimising the use of immunosuppressant drugs………………………………..……. 15

2. Xenotransplantation as the solution?……………………………………………………….. 16

2.1 Pigs as the Source of Organs……………………………………………………………... 16

2.2 Early Problems with Xenotransplantation………………………………………………… 17

2.3 Solving Early Problems……...……………………………………………………………... 18

2.4 Problems Still to Address…………………………………………………………………... 20

2.5 Current Status of Major Organs…………………………………………………………… 22

2.5.1 The Heart……………………………………………………………...... 22

2.5.2 The Kidneys…………………………………………………………………………….. 24

2.5.3 The ……………………………………………………………...... 26

2.6 Islet Xenotransplantation……………………………………………………………………. 26

3. The Ethics and Safety of Xenotransplantation…………………………………………….. 28

3.1 Ethics of the pig-to-primate model………………………………………………………… 28

3.2 Ethics of breeding pigs for the purpose of xenotransplantation………………………... 29

3.3 Safety of xenotransplantation……………………………………………………………… 29

3.4 Ethical advantages of increasing the number of organs………………………………... 30

4. Discussion ……………………………………………………………………………………...... 30

4.1 Why an alternative to is needed…………………………………….. 30

4.2 The Future of Xenotransplantation………………………………………………………... 30

4.3 Alternatives to xenotransplantation……………………………………………………….. 31

4.4 The future of organ transplantation……………………………………………………….. 32

References…………………………………………………………………………………………….. 34

Appendix………………………………………………………………………………………………. 46

Xenotransplantation and the future of human organ transplants 3

1. A Brief Historical Introduction

Organ transplantation is the process of removing an organ from one individual and then surgically inserting it into another. Throughout the 20th century many pioneers experimented with organ transplantation to reach our current level of skill and knowledge.

After several years of varied unsuccessful attempts, the first truly successful human organ transplant occurred in 1954 when transplanted a from a living donor (Murray et al., 1956). The kidney survived for 8 years, until the recipient died of a heart attack. The success of this procedure was the result of the donor and recipient being identical twins, this meant that a major problem with transplantation was avoided: the immunological rejection of the organ. It was not until the 60’s that the first immunosuppressants were used to overcome this problem (Barker and Markmann, 2013). Major developments included the discovery and usage of the dual therapy of prednisone and together and then, several years later, cyclosporine which reduced rejection rates. Cyclosporine was first used in 1980 and was considered the standard baseline immunosuppressant until being partly replaced by in 1989, which is still used today (Barker and Markmann, 2013). During this period of immunosuppressant drug discovery, major advances where made in organ transplantation. After several failed attempts, Thomas Starzyl finally carried out the first truly successful liver transplant in 1967 (using azathioprine and prednisone) with the patient surviving over a year (Starzl et al., 1968). In the same year, the first successful heart transplant was carried out by Christian Barnard (using immunosuppressants including azathioprine and prednisone), although the patient died 18 days later of pneumonia, the operation was a success showing that was possible (Barnard, 1967). Figure 1.1 shows a brief summary of the first major transplants carried out in the world and in the UK.

Figure 1.1 – A brief overview of the history of organ transplantation. Top: First major organ transplants carried out in the world. Bottom: First major organ transplants carried out in the UK.* Represents the first time that transplant was carried out successfully, with prior attempts being deemed unsuccessful due to the patients dying very soon or the organ being non-functional after transplantation. World Data from: Murray et al., 1956, Kelly et al., 1966, Barnard, 1967, Starzl et al., 1968, Cooper, 1986, Patterson et al., 1988 and Nadalin et al., 2006. UK Data from: NHS, 2015b and NHS, 2015c.

Xenotransplantation and the future of human organ transplants 4

Developments in , organ preservation and compatibility testing have allowed for the successful transplantation of the majority of human organs, from both living and deceased donors. Therefore, in less than a hundred years we have reached a stage where organ transplantation has gone from being considered within the realm of fiction to become a routine procedure. In 2014 alone, 4,431 transplants were performed in the UK including kidney, liver, heart, and pancreas transplants (figure 1.2) (NHS, 2015). In addition, certain tissues can also be donated and transplanted. However despite these advances there are still problems with organs transplantation, mainly in finding enough suitable organs for transplantation. By the end of 2014, 6,943 patients were left waiting for a transplant (NHS, 2015). Donor shortage is not just a recent problem, it has been a problem since transplantation was pioneered during which xenotransplantation (transplantation across the species barrier) was attempted as the solution. But attempts at transplanting chimpanzee and baboon kidneys into humans in the 1960s-70s were largely unsuccessful (Reemtsma et al., 1964 and Starzl et al., 1964). However recent advances in genetic modification have led to xenotransplantation being reassessed as a potential source of transplantable organs.

Figure 1.2 – Transplants carried out by the NHS in 2014, by organ type. The amount of transplants carried out of each organ type, as a percentage of total transplants in 2014. Cardiothoracic transplants include heart, lung (single and double) and combined heart and lung transplants. In some cases kidney, pancreas and liver transplants included an intestinal transplant as well. Figure produced using data from NHS, 2015.

Xenotransplantation and the future of human organ transplants 5

1.1 Current Problems with Organ Transplantation

1.1.1 Shortage of transplantable organs

A significant organ shortage is currently the major problem with organ transplantation. Advances in techniques and immunosuppression have greatly improved the post-transplant outcomes. The increased survival rates and quality of life now associated with organ transplants has resulted in transplantation becoming the solution to chronic organ dysfunction. Although patients with kidney failure can be maintained with , this is associated with reduced long-term survival compared to transplantation (Rao et al., 2007).

The long term trends in organ transplantation between 1988 and 2015 in the US (figure 1.3) demonstrate clearly that the demand for organ transplants has increased substantially with organ donors unable to match this demand (OPTN, 2016). In less than three decades the demand for organs for human transplantation has increased by over 700% in the US, which clearly is not being met by current (which has less than doubled in the same time). However this trend of a large difference between the number of transplants and the increasing number of patients on the waiting list is not US specific. The UK shows a similar pattern (figure 1.4), with a large difference between demand and supply resulting in only 27.1% of patients being transplanted in 2014. This is due to the number of patients on the waiting list increasing at a higher rate than the number of donors. Together figure 1.3 and 1.4 show that the transplantation rate is not controlled by the number of patients on the waiting list, but is limited by the number of organs retrieved from donors. However despite a common trend of high demand and low supply, countries differ in the percentage of patients transplanted and how this varies between years (figure 1.5). The trend observed in US is the result of waiting list numbers continually increasing whilst the number of donors remained fairly constant. Whereas the changes in percentage transplanted for Eurotransplant are the result of fluctuations in waiting list numbers, with donor numbers remaining fairly constant. However it is clear that in all the countries studied the current sources of organs are not matching the demand, with all having a transplantation rate of less than a third of those that desperately need a transplant. This unfortunately results in patients dying before they can be transplanted. In 2014/15, over 2.6% of patients (429 individuals) died whilst on the waiting list in the UK (NHS, 2015). A further 4.9% of patients (807 individuals) were removed from the list due to deteriorating health and becoming ineligible for transplant, many of these would have died soon after.

Xenotransplantation and the future of human organ transplants 6

Figure 1.3 – Long term trends in organ transplantation in the US. ‘Donors’ is the number of people (living and deceased) that donated at least one organ, ‘Transplants’ is the number of patients that received a transplant and ‘Waiting List’ is the number of patients on the waiting list at some point during the year. The difference between donors and transplants is due multiple organs being collected from a single donor. The difference between transplants and waiting list represents the number of people that did not receive their required transplant. Percentage transplanted was calculated by dividing the number of people that received their transplants by the total waiting per year. From 1988 to 2015, the percentage of patients transplanted has decreased from 84.0% to only 25.4%. Graph produced using data from OPTN, 2016.

Figure 1.4 – Number of organ donors, patients transplanted and patients on the active waiting list from 2005 to 2014 in the UK. ‘Donors’ is the number of people (living and deceased) that donated at least one organ, ‘Transplants’ is the number of patients that received a transplant and ‘Waiting List’ is the number of patients on the waiting list at some point during the year. The difference between donors and transplants is due multiple organs being collected from a single donor. The difference between transplants and waiting list represents the number of people that did not receive their required transplant for some reason. In 2014, 2,374 people donated organs resulting in 4,431 transplants being carried out from a waiting list of16, 374 people. Graph produced using data from NHS, 2006-2015.

Xenotransplantation and the future of human organ transplants 7

Figure 1.5 – The percentage of patients that received their required transplant during the year between 2005 and 2014 in the UK, US and Eurotransplant. The number of people that received their transplant is divided by the total waiting per year, including those that remained on the waiting list and those that were removed. In 2014 the percentage of patients that received their required transplant was; 27.1% in the UK, 23.2% in the US and 30.1% for Eurotransplant. Graph produced using data from NHS, 2006-2015 for UK, OPTN/SRTR, 2016 for US and Eurotransplant, 2006-2015 for Eurotransplant

Worldwide, the percentage of older people in the population is increasing which will continue to increase the demand for transplantable organs. In 2012, the United Nations estimated that by 2050 22% of the world’s population will be 60 years or over, doubling from 2012, with estimates of over 2 billion people worldwide being over 60 by 2050 (United Nations, 2012). This aging population is the result of improving living conditions and greater access to health care, decreasing mortality and increasing life expectancy (WHO, 2012). However, the aging process is associated the development of illnesses and diseases which will, amongst other problems, increase the demand for organ transplants. Heart disease (and risk factors such as and coronary artery disease), chronic and an increasing prevalence of chronic liver disease are all associated with increasing age with the solution being transplantation (Frith et al., 2009, Tonelli and Riella, 2014 and Vigen et al., 2012). Over the past 10 years, the aging populations of the US and UK have increased the demand for organ transplantation in each country (figures 1.6 and 1.7). In both, the number of patients waiting for transplant of a major organ has increase since 2003 so that the majority of patients requiring a transplant are within this age group. This demand will only increase as the populations continue to age, with estimates of over 21.5 million people over 60 in the UK (30% of population) by 2050, and over 107 million in the US (27% of population) (United Nations, 2012).

Xenotransplantation and the future of human organ transplants 8

Figure 1.6 – Percentage of patients on the waiting list over 50 in the US. Waiting list is the number of patients waiting for transplant at any time in the year. From 2003, the number of patients over 50 waiting for a transplant has increased by 11.7% for kidneys, 3.7% for hearts and 16.7% . Graph produced using data from OPTN/SRTR, 2016.

Figure 1.7 – Percentage of patients of the waiting list over 50 in the UK. Waiting list is the number of patients still waiting for transplant at the end of the year. ‘Cardiothoracic’ includes waiting lists for heart, lung and heart/ transplants From 2003, the number of patients over 50 waiting for a transplant has increased by 14.2% for kidneys, 7.4% for cardiothoracic and 13.7% livers. Graph produced using data from NHS, 2004-2015.

Xenotransplantation and the future of human organ transplants 9

Despite the percentage of patients transplanted in the UK remaining below 30% over the past 10 years, the sources of organs used has changed in an attempt to increase the size of the ‘donor pool’ (figure 1.8). The number of organs from each donor type differs, with an average of 3.8 organs from donors after (DBD), 2.7 organs from donors after circulatory death (DCD) and either a single kidney or part of their liver from living donors in the UK in 2014 (NHS, 2015). Although donors after circulatory death provided the majority of organs during the conception of transplantation, their use was largely abandoned due to high rates of ischemic organ and surgical damage compared to DBD organs. However the need for transplantable organs has led to an increase in DCD usage (NHS, 2002-2015). Despite this, organs from DCD still experience a higher rate of damaged due to hypoxia, hypotension and ischemia associated with the dying process and the mandatory period of five minutes after death before organ harvesting (Morrissey and Monaco, 2014). In addition, potential donors not progressing to death within a suitable timeframe leads to organs being unsuitable and a high rate of surgical damage leads to a large number of organs from DCD being discarded.

Organs from living donors are associated with better outcomes, however the number of organs that can be donated and the currently very small number of ‘altruistic’ (donating an organ to an unknown recipient) donors limits the impact of living donation in increasing the size of the donor pool. Living organ donation is largely confined to donating either a kidney or part of the liver, in 2014 over 96% (1,052 individuals) of living donors donated a kidney and only 40 individuals donated part of their liver (NHS, 2015). In addition, the majority of living organ donations are to specific recipients known to the donors (NHS, 2007-2015). Despite ‘altruistic’ living donation being formally introduced in 2006 and the numbers growing, it is still considered an uncommon practice with only 107 kidney and 1 liver transplants the result of altruistic donation in 2014 (NHS, 2015). If the number of ‘altruistic’ organ donors could be increased then so would the supply of organs that can be donated by living donors, reducing the demand from deceased donors. However it is uncertain how this could be achieved, potentially by introducing financial compensation for the donors but this surrounded by ethical issues. But this is not to dismiss the role of living donors in organ transplantation, as a significant number of kidney transplants are the result of living organ donation (figure 1.9). However this figure shows that the UK and US show a trend of a decreasing number transplants from living donors. In the UK this is the result a lower percentage overall of living donor transplants due to a higher rate of deceased donor transplants. But in the US this trend is the result of a gradually decreasing number of living donor transplants as well as an increasing number of deceased donor transplants.

Xenotransplantation and the future of human organ transplants 10

Figure 1.8 – Changes in organ donation in the UK since 2001. DBD: donors after brain death and DCD: donors after circulatory death. In 2001, there were 735 DBD, 42 DCD and 381 living donors. In 2014, there were 772 DBD, 510 DCD and 1092 living donors. Graph produced using data from NHS, 2002-2015.

Figure 1.9 – The percentage of kidney transplants conducted from living organ donors per year from 2002 and 2014 in the UK, US, EU and Eurotransplant. Percentage calculated by dividing the number of living donor kidney transplants by the total number of kidney transplants during the year. No EU data available prior to 2008 and no Eurotransplant data available prior to 2005. In 2014 the percentage kidney transplants from living donor was; 33.7% in the UK, 31.1% in the US, 21.7% in the EU and 28.7% for Eurotransplant. Graph produced using data from NHS, 2002-2015 for UK, OPTN/SRTR, 2016 for US, EDQM, 2009-2015 for EU and Eurotransplant, 2006-2015 for Eurotransplant.

Xenotransplantation and the future of human organ transplants 11

Although there is a high demand for organ transplants, not all organs from deceased donors are transplanted (figure 1.10). Reasons for not being transplanted (and retrieved) include unsuitability of the donor (medical, non-medical or age) or of the donated organ (such as poor function and damage during harvesting) (NHS, 2015). These highlight clear limitations with deceased organ donation, where the decision to utilise a donated organ and the outcomes associated with its transplantation are limited by the cause of death and state of the donor organs after death. The cause and progression of death must be such that the organs are still suitable for transplantation. In 2014, only 52% of the organs offered by deceased donors in the UK were actually transplanted (NHS, 2015). An ’opt-in’/deemed consent system, where no objection to organ donation is assumed, has been considered to increase the number of donors. However as the majority of donation refusals are the result of donor/organ unsuitability, it is unlikely that deemed consent would greatly increase the number of transplants (at least to match the demand) (NHS, 2006-2015). Therefore, current sources of organs need to be expanded or new sources need to be found to match the demand.

Figure 1.10 – Deceased organ donation and utilisation in the UK (2014-15). Offered for transplantation is the number of organs that met the initial suitability criteria and were offered for transplantation. % retrieved is the number of organs retrieved for transplantation as percentage of those offered. % transplanted is the number of organs transplanted as a percentage of those offered. Graph produced using data from NHS, 2015.

Xenotransplantation and the future of human organ transplants 12

1.1.2 Long term outcomes – the effect of donor type and the need for re- transplantation

The donor shortage and transplant demand is amplified by the need for re-transplantation, as long-term outcomes of transplantation are still limited. The post-transplant outcomes demonstrate the need for re-transplantation of patients, as a substantial proportion of transplanted organs fail within 5 years (figure 1.11).

In the case of kidney, liver and pancreas transplants, the only long-term treatment for failure in another transplant. For heart transplants, graft survival is directly linked to patient survival. In kidney (and liver) transplantation, organs from living donors have better outcomes than from deceased donors (figures 1.12 and 1.13). Ten years after there is a substantial difference in graft failure between living and deceased donors, with 58.6% of kidneys from deceased donors and 41.8% from living donors failing within 10 years (figure 1.11). Large differences in percentage graft failure/survival between the two donor types are present within the first few months post-transplant, with the difference increasing with time after transplantation (figures 1.12 and 1.13). The reduced outcomes of organs transplantation and the differences between living and deceased donor transplants have been strongly associated with ischemia-reperfusion injury (IRI), which is currently a frequent and unavoidable problem of organ transplantation influencing short- and long-term graft outcomes (Salvadori et al., 2015). IRI is the result of blood flow disturbances during the process of organ donation, preservation and transplantation. During the process of donation, blood supply to the organ is cut off causing a shortage of oxygen and nutrients leading to ischemic damage. Further damage to the organ occurs when blood supply is returned, due to the activation of the innate and adaptive immune responses which can result in graft rejection (Bonventre and Yang, 2011 and Yellon and Hausenloy, 2007). Despite the use of immunosuppressants, T-cell-mediated is still the cause of a large part of transplant morbidity and graft loss (Loewendorf and Csetec, 2013). IRI has been shown to be associated with development of progressive scaring (fibrosis) of the graft leading to chronic dysfunction and rejection (Curci et al., 2014).

In kidneys from living donors, ischemia-reperfusion injury is less frequent and less severe than from deceased donors (Salvadori et al., 2015). Despite organs from living donors also undergoing a period of ischemia during harvesting, the disturbances associated with death are not present and the time between donation and transplantation is shorter. With deceased organ donors blood flow disturbances occur prior to the process of donation (due to donor death), resulting in ischemia and activation of immune responses causing organ damage before harvesting (Salvadori et al., 2015). In addition, the increased time between donation and transplantation of deceased donor organs leads to increased ischemic damage. Therefore IRI contributes to the differences in outcomes of transplants from living and deceased organ donors (see figures 1.12 and 1.13). The outcomes of organs transplantation are limited by the injury and stress responses initiated by the process of harvesting/transplantation and directly as the result of donor brain or circulatory death, resulting in tissue damage (Wood and Goto, 2012). This also includes the immune response of the recipient to the tissue damage (and associated molecules released) after transplantation. Ischemia-reperfusion injury is associated with an increased incidence of acute and chronic rejection. These two types of rejection differ, although injury sustained during an acute rejection episode may be a cofactor in chronic rejection. Although the

Xenotransplantation and the future of human organ transplants 13

complete mechanisms of chronic rejection are not currently know, chronic rejection involves the responses of the innate and adaptive immune systems (to IRI) leading to late graft dysfunction and rejection (Kwun and Knechtle, 2009). Chronic rejection is still a major problem for organ transplantation as it reduces long-term survival and increases the need for re-transplantation. The use of immunosuppressants has also been associated with reducing the long-term outcomes of organ transplantation.

Figure 1.11 – Percentage graft survival among adult kidney, liver, heart and pancreas transplant recipients over 5 years. Average percentage graft survival of adult kidney, liver and pancreas transplants recipients transplanted in 2008 in the US. Average percentage graft survival of adult heart transplant recipients transplanted from 2006 to 2008 in the US. In 2008, 22,257 adult kidney, 5,706 adult liver and 1,204 adult pancreas transplants were conducted in the US. From 2006-2008, 5,558 adult heart transplants were conducted in the US. * Average survival from both living and deceased donors. ** Patient survival among adult heart transplant recipients. Percentage graft survival recorded every month after transplantation. After 60 months (5 years) the average percentage graft survival was; 80.9% for kidney, 70.3% for liver, 75.3% for heart and for 69.1% pancreas transplants. Graph produced using data from

OPTN/SRTR, 2015.

Xenotransplantation and the future of human organ transplants 14

Figure 1.12 – Percentage graft failure among adult kidney transplant recipients over 10 years . Average percentage graft failure calculated using data from US adult patients that received kidney transplants between 1991 and 2013. Percentage graft failure recorded 6 months, 1 year, 3 years, 5 years and 10 years after transplantation. Graph produced using data from OPTN/SRTR, 2015.

Figure 1.13 – Percentage graft survival among adult kidney transplant recipients over 5 years. Average percentage graft survival of adult kidney transplant recipients transplanted in 2008 in the US. In 2008, 16,582 kidney transplants from deceased donors and 5,675 from living donors were conducted in the US. Percentage graft survival recorded every month after transplantation. After 60 months (5 years) the average percentage graft survival from deceased donors was 76.0% and from living donors was 85.9%. Graph produced using data from OPTN/SRTR, 2015.

Xenotransplantation and the future of human organ transplants 15

1.1.3 Minimising the use of immunosuppressant drugs

The use of immunosuppressant drugs has been a staple part of organ transplantation, however their long-term use has negative side effects on the recipient. Immunosuppressants are used to prevent organ rejection in transplanted patients by suppressing their immune system. But their use is associated with an increased risk of and cancer (malignancy) with many additionally increasing cardiovascular risk (Lodhi et al., 2011 and Piselli et al., 2014). The most common side effects of immunosuppressive drugs include; bacterial , viral infections, nephrotoxicity (with chronic kidney failure), development of , hypertension, and the development of cancers (Scherer et al., 2007). Unsurprisingly, suppressing the immune system leaves the patient vulnerable to infection, increasing the risk of bacterial and viral infections which can be life-threatening (Ahmed et al., 2008 and Fishman, 2007). This includes infections associated with and the hospital setting, but also infections by opportunistic pathogens due to the long-term use of immunosuppressants post-transplantation. The risk of infection is highest in the period after transplantation when immunosuppression is strongest, with the decrease in infection risk reflecting the decrease in immunosuppression post-transplant. One of the most common causes of morbidity and mortality in transplanted patients is cardiovascular disease (Hsu and Katelaris, 2009). The use of glucocorticoids and/or calcineurin inhibitors as part of the immunosuppressant therapy has been shown to increase cardiovascular risk (Hsu and Katelaris, 2009). The increased risk is the result of hyperglycaemia, hyperlipidaemia, hyperuricemia and hypertension due to the use of immunosuppressants.

Patients that undergo organ transplantation are at an increased risk of developing cancers compared to the general population (Piselli et al., 2014). The frequency of malignancies increases with intensity and duration of immunosuppressant therapy, due to the impairment of the immune system (reduced immunosurveillance) but also immunosuppressant drugs having direct effects promoting cancer formation (Campistol et al., 2012). The impairment of the immune system leaves the patient vulnerable to cancers caused by opportunistic infectious agents (virus-related malignancies). The most frequent post-transplant malignancies are non-melanoma cancers and post-transplant lymphoproliferative disorders (PTLDs) largely associated with Epstein-Barr virus infection (Piselli et al., 2014). A higher intensity and/or duration of immunosuppression is associated with a higher risk of PTLDs and skin cancers in all types of transplants, with the reduction or removal of immunosuppression used as a successful treatment for early forms of both types of post- transplant malignancies (Geisller and Schlitt, 2004 and Taylor et al., 2005). However the removal of immunosuppression will increase the risk of graft rejection. Transplanted patients have a 3 times greater risk of cancer compared to the general population (age- and sex- matched), which has led to malignancy becoming a main cause of patient death with graft function (Vajdic and Leeuwen, 2009). It is expected that post-transplant malignancy will increase to the be the main cause of death in transplanted patients due to the longer life expectancy and ageing of recipients (Piselli et al., 2014). In addition some commonly used immunosuppressants (calcineurin inhibitors, such as cyclosporin and tacrolimus) are nephrotoxic causing damage to kidneys (Lodhi et al., 2011). This side effect does not only reduce long term outcomes of kidney transplants, as many patients after a non-kidney transplants exhibit /damage (Bloom and Reese, 2007). Therefore a large number of patients often require a kidney transplant after the transplantation of another organ, further increasing the demand. The overall problems associated with

Xenotransplantation and the future of human organ transplants 16 immunosuppressant drugs warrant an effort to minimise their use and/or to look into alternative methods to prevent graft rejection. There are clearly significant problems with the current practices of allotransplantation (transplantation between individuals of the same species), primarily a substantial shortage of transplantable organs. However could organs from another species be used for human transplantation to match the increasing demand?

2. Xenotransplantation as the solution?

There is clearly a large demand for transplantable human organs and it seems very unlikely that allotransplantation will ever meet this demand. Therefore we need to look for alternative sources of organs for human transplantation, xenotransplantation could be a solution in the not too distant future. Xenotransplantation is the process of transplanting organs, tissues, or cells between different species (Cooper and Ayares, 2011). The main benefits of xenotransplantation over current allotransplantation practices are an unlimited supply of organs, tissues and cells (allowing patients that currently would be declined to receive a transplant), and by using live donors xenotransplantation avoids the damaging effects of death on organs from deceased donors (see figures 1.12 and 1.13) (Cooper and Ayares, 2011).

2.1 Pigs as the Source of Organs

Although non-human primates (NHPs) are the most closely related species to humans and hence might seem to be the ideal source of organs, they are not considered suitable for clinical xenotransplantation (see figure 2.1 for reasons) resulting in the pig becoming the species of choice. Progress has been made using the pig-to-NHP model (pig organs transplanted into an NHP) and in vitro models to identify any physiological incompatibilities that would result in pig organs being deemed unsuitable for human transplantation (Ibrahim et al., 2006). Although there are differences, available evidence suggests that the heart, kidney, liver or lungs of a pig could support a human. However the lack of any long term studies and limitations of the models used may affect this conclusion. The shorter lifespan of pigs (15-20 years) has raised questions about the long term effects of xenotransplantation, however evidence from allotransplantation suggests that the organ would age at the rate of recipient (Ibrahim et al., 2006). However, the millions of years of evolution that have separated pigs and humans has resulted in immunological differences between the two species and overcoming these differences is still a problem for xenotransplantation (Michel et al., 2015). The ability to genetically modify (the process of directly altering an organism’s genetic material) pigs has resulted in significant progress in solving these differences (Nagashima et al, 2012).

Xenotransplantation and the future of human organ transplants 17

Figure 2.1 – The main benefits and concerns of using pigs or non-human primates (NHPs) as the source of organs for clinical xenotransplantation. The number of concerns of using non- human primate organs has led to pig organs being used for xenotransplantation, largely due their ability to fulfil criteria that the NHPS could not. Figure produced using information from Ekser et al., 2015 and Yang and Sykes, 2007.

2.2 Early Problems with Xenotransplantation

Initial attempts at xenotransplantation with un-modified pig organs into NHPs were unsuccessful, with the vast majority of organs dying within a day and many in a matter of hours (Lambrigts et al., 1998). This rapid loss of the transplanted organ is the result of hyperacute rejection, where natural (preformed) anti-pig antibodies of the primate bind to the vascular endothelial cells of the graft causing complement-mediated injury (Cooper et al., 2015). This results in thrombosis, haemorrhaging and oedema, which ultimately leads to rejection of the organ. It has since been determined that the carbohydrate galactose-α-1, 3- galactose (Gal), expressed on the pig vascular membranes is the major target recognised and bound to by natural anti-antibodies (most importantly IgM and IgG) (Cooper et al., 2015). The production of these anti-Gal antibodies is due loss of expression of Gal in primates millions of years ago (Gal is present in all other mammals) (Byrne et al., 2015). Therefore in NHPs (and humans), the natural antibodies recognise the presence of Gal on pig organs as foreign antigens and ultimately reject the graft. If acute rejection can be prevented, then acute humoral xenograft rejection occurs within days or weeks (Ekser and Cooper, 2010). This form of rejection is associated with natural antibody binding (anti- nonGal or low levels of anti-Gal antibodies) and the innate immune system, resulting in complement-mediated injury. As part of the innate immune system, natural killer (NK) and macrophages are involved in acute humoral xenograft rejection but their exact role is currently unknown (Cooper et al., 2015). Following the prevention of acute and acute humoral xenograft rejection, the graft succumbs to acute cellular rejection (usually as the result of inadequate immunosuppression). This rejection is the result of primate T-cells infiltrating the graft cells, leading to the T-cell-dependent production of antibodies against the graft, leading to rejection (Ekser and Cooper, 2010). Therefore, in contrast to allotransplantation where rejection is the result of cell mediated immunity, the majority of graft rejection in xenotransplantation is the result of a rapid antibody-mediated response (although cell mediated immunity is involved if this is overcome) (Cooper and Ayares, 2011).

Xenotransplantation and the future of human organ transplants 18

2.3 Solving Early Problems – Genetic Modification of Pigs

Xenotransplantation of organs is now not significantly limited by hyperacute, acute humoral xenograft and acute cellular rejection due to genetic modification of pigs and improvements in immunosuppression (Ekser et al., 2012). Pigs for xenotransplantation have been genetically modified by removing (‘knocking-out’) pig genes that would trigger an immune response in humans and inserting human genes into the pig genome (Mohiuddin et al., 2014). The rapid rejection of un-modified pig organs was the result of antigens expressed on the surface of vascular cells, leading to complement-mediated injury and rejection within hours-days. The attempts in the 1990’s at preventing this involved genetically modifying pigs by inserting transgenes for human complement regulatory proteins (hCRPs) into the pig genome (Cooper and Ayares, 2011). Complement regulatory proteins are expressed in order to regulate complement system activation and protect host cells/tissues from damage (Meri and Jarva, 2013). Pigs express their own complement proteins, however the expression of hCRPs protects the xenograft from the effects of the human complement system avoiding the complement-mediated injury associated with early rejection in the pig-to-NHP model (Cooper et al., 2015). Expression of the hCRPs: CD46, CD55 or CD59 individually increased graft survival, and the expression of multiple hCRPs further increased graft survival (Griesemer et al., 2014). However this method of avoiding rejection was not as successful as initially thought (Cooper and Ayares, 2011).

Once the expression of the carbohydrate galactose-α-1, 3-galactose (Gal) was identified as a major cause of hyperacute rejection, attempts were made to prevent Gal expression. This was a achieved by ‘knocking-out’ the gene (possible due to improvements in technology) for the enzyme α-1,3-galactosyltransferase which synthesises Gal. Inactivation of this enzyme meant that the expression of Gal was removed and the first α-1,3-galactosyltransferase gene-knockout (GTKO) pigs were produced in 2003 (Phelps et al., 2003). Transplantation of GTKO pig organs increased graft survival significantly, with organs surviving longer than with hCRP expression (Cooper et al., 2016). Survival times were increased further by expressing hCRPs in GTKO (GTKO/hCRP) pigs than either alone, which further decreased the incidence of hyperacute rejection (Ekser et al., 2012). Although GTKO/hCRP pigs were created to overcome the antibody-mediated cause of rejection, investigations have shown that these pigs exhibit a reduced T-cell response (Ekser et al., 2012). However it is unlikely that this reduced response is enough to reduce immunosuppression for clinical use, further genetic medication will be required to reduce T-cell-dependent response which is currently controlled by intensive experimental immunosuppressive regimens (further discussed). Although early rejection is now a rare occurrence, graft survival is still limited by other factors.

There are currently around 40 genetic modification that have been created in pigs, with a number of pigs expressing multiple (up to 6) different modifications (Cooper et al., 2016). Figure 2.2 shows the timeline of the genetically modified pigs that are discussed in this project. Developments in technology have enabled pigs with multiple genetic modifications to be produced more quickly and at a lower cost, further developments will almost certainly result in the more rapid progress of xenotransplantation.

Xenotransplantation and the future of human organ transplants 19

Figure 2.2 – Timeline of important genetic modifications generated in pigs. The timeline shows the publication dates of papers in which certain important genetic modifications where first generated in pigs, as there is currently around 40 different individual modifications this timeline represents only those that are discussed in this project. Pigs with multiple genetic modifications can be generated by breeding pigs with different modifications. The importance of the GTKO modification has led to it to be present in the vast majority of transgenic pigs generated for xenotransplantation. Figure produced using references cited on timeline.

Abbreviations:

• GTKO: α1,3-galactosyltransferase gene-knockout • hTM: human thrombomodulin gene expressed • CMAHKO: cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene-knockout • MHCC1KO: MHC class I gene-knockout • β4GalNT2KO: β1,4 N-acetylgalactosaminyl transferase gene-knockout • hCTLA4Ig: human cytoxic T-lymphocyte associated antigen4-Immunoglobulin gene expressed

Xenotransplantation and the future of human organ transplants 20

2.4 Problems Still to Address

Acute rejection has largely been prevented however; the presence of nonGal antigens, coagulation (blood clotting) dysfunction between pig and primate and the reliance on strong experimental immunosuppression are reducing graft survival and the clinical applications of xenotransplantation. Graft survival is now limited by the development of thrombotic microangiopathy, where fibrin deposition and platelet aggregation blocks the small blood vessels of the graft resulting in ischemic damage and death of cell/tissues (Cooper et al., 2015). The cause of thrombotic microangiopathy is currently thought to be the result of coagulation dysfunction between pig and primate, and activation of graft vascular endothelial cells by antibodies and/or innate immune cells (Cowan and Robson, 2015). With endothelial activation resulting in the cells changing to a state that promotes blood clotting. Several known incompatibilities between the species coagulation/anticoagulation factors contribute to an inability to maintain the anticoagulant state, leading to fibrin and platelet deposition (Cowan et al., 2011). For example, pig thrombomodulin does not function correctly with primate proteins (Cooper et al., 2016). Advanced thrombotic microangiopathy can result in consumptive coagulopathy, where vessels throughout the body begin to clot resulting in multiple organ damage, heavy and fatality (Cooper et al., 2016). Genetic modification is gradually overcoming the problem of coagulation dysfunction by inserting ‘anticoagulant’ or ‘anti-thrombotic’ genes into the pig genome. Expression of the human thrombomodulin (hTM) gene to reduce blood coagulation has been shown to increase graft survival in heart xenotransplantation (Mohiuddin et al., 2014).

Although the production of GTKO pigs has largely removed Gal expression, pig organs still express other antigens (non-Gal antigens) which are the target of other natural antibodies. These antibodies are likely responsibly for the rare cases of early immune injury reported and endothelial cell activation associated with thrombotic microangiopathy (Byrne et al., 2015). Two potential targets of human anti-non-Gal antibodies have been identified: N- glycolylneuraminic acid (NeuGc) and β1,4 N-acetylgalactosaminyltransferase (B4GALNT2) (Cooper et al., 2016). Other pig antigens have also been identified as potential targets of non-Gal antibodies (Griesemer et al., 2014). Humans do not express NeuGc and hence produce natural antibodies against it, however both pigs and NHPs express NeuGc and hence do not produce anti-NeuGc antibodies (Cooper et al., 2016). Therefore anti-NeuGc antibodies have no role in the immune response in the pig-to-NHP model, but NeuGc is likely to have a role in human xenotransplantation and the effects of which are unknown. ‘Knocking-out’ the gene for the enzyme (CMAH) that synthesises NeuGc, has allowed for the production of pigs that do not express either NeuGc or Gal (GTKO/CMAHKO) (Lutz et al., 2013). In vitro experiments with human samples have shown that these ‘double knock-out’ pig cells exhibit less human antibody binding that GTKO cells (Estrada et al., 2015). The second target (B4GALNT2) was more recently identified, and hence although pigs that do not express Gal, NeuGc and B4GALNT2 have been produce, its effect on graft survival is unknown. However some NHPs have been shown to produce natural antibodies against B4GALNT2, therefore the pig-to-NHP model can used to determine its effects on survival (Estrada et al., 2015). The current types of graft rejection are summarized in figure 2.3, showing what stages of rejection have been largely solved and which are still limiting xenograft survival.

Xenotransplantation and the future of human organ transplants 21

Figure 2.3 – Summary of the current types of graft rejection in xenotransplantation. The figure shows the types of rejection observed and when they occur, in terms of post-transplant time and relative to other types of rejection. Also shows the types of rejection that have largely been prevented via genetic modification or immunosuppression. Figure produced using information from: Cooper et al., 2015, Ekser and Cooper, 2010 and Griesemer et al., 2014.

The T-cell (adaptive immune) response in xenotransplantation has largely been controlled by the use of immunosuppressants, however it is advantageous to long-term graft survival that immunosuppression is kept to a minimum and hence genetic modification is being investigated to prevent T-cell-mediated rejection (Higginbotham et al., 2015). Genetically modified pigs that express the gene for CTLA4-Ig, that inhibits T-cell activity, in skin, heart and kidney tissues have been produced (Wan et al., 2015).These pigs showed no sign of susceptibility to infection and prolonged skin xenograft survival was observed in pig-to-rat models, therefore CTLA4-Ig expression could be used overcome T-cells roles in graft rejection. Another approach has been to modified pigs to express a mutant human MHC class II transactivator gene, resulting in a reduce expression of antigens that activate T-cells and hence reducing the immune response. Pigs without MHC class I genes have recently been produced that shown no signs of susceptibility to infection but their effects as xenotransplant donors is currently being investigated (Reyes et al., 2014). The use an anti- CD154 monoclonal antibody has been highly successful in blocking the T-cell response and hence increasing graft survival times. However this agent is not suitable for clinical applications due to the high risk of blood vessel clot formation and post-transplant consumptive coagulopathy associated with its use (Bühler et al., 2000). Alternatives to this agent (anti-CD40) are being investigated, however minimising the use of immunosuppressants will minimise the risks/complications associated with them. Therefore it seems that the long term solution to the adaptive immune response is further genetic modification. Despite these problems, considerable progress has been made with xenotransplantation as shown by the significant increases in xenograft survival of major organs.

Xenotransplantation and the future of human organ transplants 22

2.5 Current Status of Major Organs

2.5.1 The Heart

Xenotransplantation of non-life-supporting heterotopic and life-supporting orthotopic (replaces recipient’s heart) hearts have been studied using the pig-to-NHP model. Heterotopic grafts are used to observe the effects of the immune system on the graft, allowing immunosuppression therapies and further pig genetic modifications to be determined. To date, the longest survival is more than 500 days using a heart from a GTKO/CD46/hTM pig with anti-CD40 (beneficial alternative to anti-CD154) as part of the immunosuppression therapy (Mohiuddin et al., 2014). Three (of five) grafts in this study where still functioning at the time of publication, unpublished data by Mohiuddin et al. has stated that graft survival has been increased to 945 days. Maximum life-supporting graft survival is currently 57 days with GTKO/CD46 pigs, although the graft experience the effects of the immune system the graft was not rejected (Byrne et al., 2011). Subsequent studies have been held back by ‘perioperative cardiac xenograft dysfunction’, where a high percentage of grafts are lost within 48 hours due to postoperative complications (e.g. ischemic injury) (Michel et al., 2015). However these maximum graft survival times represent a significant increase to those reported in 1998, when heterotopic hearts lasted only up 29 days and orthotopic hearts up to 19 days (figures 2.4 and 2.6) (Cooper et al., 2014). On average, long-term graft survival has increased each year, with figures 2.5 and 2.7 showing the increases in maximum graft survival of all publications and longest graft survival per year (shown by linear regressions). These increases with subsequent years provide encouraging evidence that modified pig hearts may one day be transplanted into humans.

Figure 2.4 – All non-life-supporting heterotopic heart xenotransplants published from 1998 to 2013 . The graph shows the average and longest maximum graft survival achieved per year. For 1998, the average maximum survival was 14 days with the longest survival of 29 days achieved. For 2013, the average maximum survival was ~145.3 days with the longest survival of 380 days achieved. No studies on heterotopic heart xenotransplantation were published in 2001 and only one paper was published in 2002 and 2009. Graph produced with data edited from Cooper et al., 2014.

Xenotransplantation and the future of human organ transplants 23

Figure 2.5 – Trends in non-life-supporting heterotopic heart xenotransplantation (1998- 2013) . Each black dot is the maximum graft survival achieved per paper. Average/blue dots represent the average maximum graft survival of all publications per year. Max/red dots represent the paper that had the longest graft survival for that year. No studies on heterotopic heart xenotransplantation were published in 2001 and only one paper was published in 2002 and 2009. Graph produced with data edited from Cooper et al., 2014.

Figure 2.6 – All life-supporting orthotopic heart xenotransplants published from 1998 to 2013 . The graph shows the average and longest maximum graft survival achieved per year. For 1998, the average maximum survival was ~12.3 days with the longest survival of 19 days achieved. For 2013, only one paper was published in which the heart survived for 57 days. No studies on orthotopic heart xenotransplantation were published in 1999, 2001-2004, 2006, 2008-2010, 2012 and 2013. Graph produced with data edited from Cooper et al., 2014.

Xenotransplantation and the future of human organ transplants 24

Figure 2.7 – Trends in life-supporting orthotopic heart xenotransplantation (1998-2013). Each black dot is the maximum graft survival achieved per paper. Average/blue dots represent the average maximum graft survival of all publications per year. Max/red dots represent the paper that had the longest graft survival for that year. No studies on orthotopic heart xenotransplantation were published in 1999, 2001-2004, 2006, 2008-2010, 2012 and 2013 and only one paper was published in 2011. Graph produced with data edited from Cooper et al., 2014.

2.5.2 The Kidneys

Two recent studies have reported the survival of functional xenotransplanted pig kidneys for over 125 days, representing a significant increase in graft survival (figure 2.8). A life supporting kidney from a pig with six genetic modifications (GTKO/CD46/CD55/hTM/ EPCR/CD39) transplanted into a baboon survived for 136 days, using anti-CD40 as part of the immunosuppression therapy (Iwase et al., 2015). The kidney functioned normally and death was due to an infection but not graft failure. Antibody-mediated injury was not observed and there were minimal signs of consumptive coagulopathy. In the other study, kidneys from GTKO/CD55 pigs were still functioning normally in two rhesus macaques (126 and 133 days) at the time of publication however anti-CD154 was used (Higginbotham et al., 2015b). These results show promise for kidney transplantation by significantly increasing survival from 90 days (more than 30 days was unusual, figure 2.8) and reducing previous concerns about incompatibilities between pig and primate kidneys (Iwase and Kobayashi, 2015). Although achieving long term graft survival has been problematic, experiments with kidney xenotransplantation since 1998 have shown that long term kidney survival has increased each year (figure 2.9). On average, maximum graft survival for all publications has increased per year and longest graft survival per year has also increased but at a slower rate (shown by linear regressions in figure 2.9). Further difficulties with maintaining long term survival of kidney graft is that they develop complications of consumptive coagulopathy more rapidly than heart xenografts.

Xenotransplantation and the future of human organ transplants 25

Figure 2.8 – All life-supporting kidney xenotransplants published from 1998 to 2013. The graph shows the average and longest maximum graft survival achieved per year. For 1998, the average maximum survival was 24 days with the longest survival of 35 days achieved. For 2013, the average maximum survival was ~22.5 days with the longest survival of 55 days achieved. No studies on life supporting kidney xenotransplantation were published in 2008. Graph produced with data edited from Cooper et al., 2014.

Figure 2.9 – Trends in life-supporting kidney xenotransplantation (1998-2013). Each black dot is the maximum graft survival achieved per paper. Average/blue dots represent the average maximum graft survival of all publications per year. Max/red dots represent the paper that had the longest graft survival for that year. No studies on life supporting kidney xenotransplantation were published in 2008. Graph produced with data edited from Cooper et al., 2014.

Xenotransplantation and the future of human organ transplants 26

2.5.3 The Liver

The longest survival was achieved in 2011, where a life supporting GTKO pig liver was transplanted into a baboon and survived for 9 days. As part of the study two other graft survived 6 and 8 days, in all three the livers functioned normally and graft survival was not limited by rejection (Kim et al., 2012). The baboons died due to bleeding and infection, thrombocytopenia (slow clotting) and consumptive coagulopathy resulting in fatal bleeding are considered the current major limitations of liver xenotransplantation (Cowan and Robson, 2015). The survival and normal function of the recipient baboons shows that the pig liver is able to carry out at least the vital functions required, however long term effects are unknown.

The delicate nature of the lungs has made them especially prone damage and rejection from the primate immune system, resulting in graft survival being limited to hours (Kubicki et al., 2015).

2.6 Islet Xenotransplantation

Although the xenotransplantation of organs is a number of years away from clinical trials, transplantation of pig pancreatic islets as a treatment for type 1 diabetes (in which, β cells of the pancreatic islets no longer produce insulin) has shown more promise. By 2015, over 3.45 million people in the UK have been diagnosed with diabetes, approximately 10% of which with type 1 (Diabetes UK, 2015). In 2012, it was estimated that the UK spent £1.802 billion treating type 1 diabetes and its complications (Kanavos et al., 2012). Although type 1 diabetes can be treated with insulin injections, the long-term cure is a pancreas transplant or transplantation of the islets themselves. However, both of these are limited by the short supply of human organs but the xenotransplantation of pancreatic islets from pigs could be a potential long-term treatment for type 1 diabetes. Natural pig insulin is functional in humans (differs from human insulin by only one amino acid), however some immunological differences between the species still need to be resolved and there is debate over the age of pig donors to use (Windt et al., 2012). Adult pigs have the advantages (over foetal and neonatal pigs) of containing high numbers of developed islets that will function only hours after transplantation. However these islets are more difficult to isolate from the pig and the high costs of maintaining the animals in a pathogen free environment to adulthood (over 2 years) is not desirable (Windt et al., 2012). Ethically, there is also the issue of the number pigs required, with estimates of considerably lower numbers of adult pigs required to obtain enough islets for functional insulin production in humans (Hering et al., 2006 and Thompson et al., 2011).

Genetic modification has made significant progress in solving problems with immunological differences (GTKO and hCRPs insertion), however a major problem currently is the IBMIR (Instant Blood Mediated Inflammatory Reaction) which leads to the rapid loss (within minutes) of a large number of transplanted islets (Park et al., 2015). Further genetic modifications to reduce incompatibilities between the two species coagulate systems and/or transplanting the islets to site where they are not immediately exposed could eventually solve this problem (Berman et al., 2009 and Windt et al., 2012). Additionally, the use of the clinically unsuitable immunosuppressant anti-CD154 to prevent the T-cell response is another obstacle that needs to be overcome before human applications. A recent study achieved 6 month functional (controlled blood glucose in diabetic primates) islet survival in 4

Xenotransplantation and the future of human organ transplants 27

(of 5) primates, with the longest still functional at the time of publication (603 days) (Shin et al., 2015). Although this studied used anti-CD154, its shows the feasibility and potential for islet xenotransplantation if an alternative immunosuppressant can be found and is close to fulfilling a proposed standard for human clinical trials (Hering et al., 2009). An alternative to long-term immunosuppression is encapsulating the islets, which allows small molecules in and out of the encapsulate membrane but prevents cells of primate immune system from reaching the islets (preventing the antibody or T-cell response). Two studies have shown functional encapsulated islets survival for up to 6 months in the pig-to-NHP model, however it is still unknown if the encapsulated islets will continue to function long-term (Dufrane et al., 2006 and Dufrane et al., 2010).

As with organ xenotransplantation, the transplantation of islets cells between species has made much progress (figure 2.10). On average, long term islet survival has increased each year since 1998, provide encouraging evidence that human trails with pig pancreatic islets may soon be approaching (shown by linear regressions in figure 2.11). Although widespread clinical trials with porcine islets have not begun, an early phase clinical trial with a small number of patients began in 2009 in New Zealand. Preliminary data is promising, with reduced hypoglycaemic episodes and prolonged reductions in insulin doses reported in patients (Garkavenko et al., 2011). There have clearly been substantial improvements with xenotransplantation over the past 20 years, however there are still problems with xenotransplantation that currently limit clinical application. In addition, potential ethical and safety concerns associated with xenotransplantation have to be considered.

Figure 2.10 – All pancreatic islet published from 1998 to 2013. The graph shows the average and longest maximum graft survival achieved per year. For 1999, the average maximum survival was 9 days with the longest survival of 12 days achieved. For 2013, the average maximum survival was ~149.2 days with the longest survival of 224 days achieved. No studies on pancreatic islet xenotransplantation were published in 1998 and only one paper was published in 2001 and 2004. Graph produced with data edited from Cooper et al., 2014.

Xenotransplantation and the future of human organ transplants 28

Figure 2.11 – Trends in pancreatic islet xenotransplantation (1998-2013). Each black dot is the maximum graft survival achieved per paper. Average/blue dots represent the average maximum graft survival of all publications per year. Max/red dots represent the paper that had t\he longest graft survival for that year. No studies on pancreatic islet xenotransplantation were published in 1998 and only one paper was published in 2001 and 2004. Graph produced with data edited from Cooper et al., 2014.

3. The Ethics and Safety of Xenotransplantation

3.1 Ethics of the pig-to-primate model

The most immediate ethical issue of xenotransplantation is the continued use of primates in the pig-to-primate model to determine the effects of the immune system on pig organs. The issues associated with animal experimentation are particularly prevalent with xenotransplantation research due to the use of non-human primates (mainly baboons and macaques) (Nuffield Council on Bioethics, 2005). These animals are sentient, with high cognitive capacities and are highly social forming complex groups in the wild. This means that, compared other animals used in research, there is a higher potential for them to experience pain and suffering as part of the research. This is of high concern due to the requirement to transplant a pig organ into the primate and the currently limited longevity associated with xenotransplantation. The living conditions and lack of sociability during the experimental period with also affect the animals used. The pig-to-primate model is continued to be utilised in order to obtain information that cannot come from alternatives and ensuring that pain and suffering to the animals involved is kept to a minimum. However many will still object solely on the basis that animals are being used for experimentation, but it is important to put this in the context of the advantages and benefits of human xenotransplantation. As with any animal used in research, there is the concern of the validity of results over whether they can be reliably applied to humans. It has been mentioned that the NHPs do not produce anti-NeuGc antibodies but humans do, and are likely to have role in human xenotransplantation (Cooper et al., 2016). However there is currently no alternative to humans that can be considered a ‘perfect model’ for testing the effects of clinical

Xenotransplantation and the future of human organ transplants 29 xenotransplantation. Therefore the pig-to-primate model (and other models) offers unique insights into xenotransplantation that could not be obtained alternatively, despite differences between the models and humans (Denner and Graham, 2015).

3.2 Ethics of breeding pigs for the purpose of xenotransplantation

There are ethical issues associated with the breeding of pigs to be killed for organ donation and that these animals are genetically modified organisms (GMOs). A primary concern of both is the health and welfare of the animals involved (Sautermeister, 2015). The production of GMOs is inefficient, with a large number of embryos required to produce a sufficient number of individuals that survive the process and carry the required modification (Ormandy et al., 2011). The requirement of large number of embryos, from which only a small number survive, raises ethical concerns. In addition, the effects of the modifications to each individual are not entirely known which may lead to unexpected side effects that would negatively affect an animal’s welfare (Laible, 2009). Further problems with GMOs include opinions over whether it is right/unnatural for humans to create modified organisms. The creation and breeding of genetically modified pigs goes against the principles of the Three Rs (Replacement, Reduction and Refinement) which aims to decrease the number of animals used in experimentation (Ormandy et al., 2011). In order for xenotransplantation to progress more genetic modifications will have to be produced requiring a larger number of pigs. These animals will be kept in conditions creating ethical issues, including the indoor facilities required to house them for the clinical applications of xenotransplantation. There are moral concerns to whether humans should create these animals for the purpose of killing them and the rights of these animals, but also ensuring that if animals have to be killed then it is done humanely (Ormandy et al., 2011). However it is worth considering the widely accepted practices currently used for food production when looking at the ethics of breeding for xenotransplantation. It is estimated that over 10 million pigs each year are killed for human consumption in the UK (HSA, 2016). The vast majority of these animals live in poorer conditions than those proposed for xenotransplantation and some may deem that organs for human transplantation is a greater benefit.

3.3 Safety of xenotransplantation

A concern of xenotransplantation has been the risk of diseases being transmitted from pigs to humans (zoonosis). However the majority of microorganisms that may present a risk to humans could be removed by housing and breeding pigs in pathogen-free indoor facilities (Denner and Tönjes, 2012). This would prevent pathogens from infecting the animals and hence being transferred, with xenografts, to humans. Protocols to monitor these facilities and screening organs before being transplanted could prevent any potentially harmful pathogens from coming into contact with human recipients. However, porcine endogenous retroviruses (PERVs) have in the past been considered a major potential risk. PERVs are present in the genome of all pigs (hence unaffected by pathogen-free facilities) but are not associated with any diseases in pigs. The fear of PERVs is that, if transfer to humans, they may be pathogenic to humans or recombine with HERVs (human endogenous retrovirus) to form a new potentially pathogenic virus (Denner and Tönjes, 2012). The current consensus is that PERVs are not likely to be pathogenic to humans, but if necessary PERV activation can be suppressed by genetic engineering using small interfering RNAs (Cooper et al., 2016 and Ramsoondar et al., 2009). Preliminarily data from human trails of islet xenotransplantation in

Xenotransplantation and the future of human organ transplants 30

New Zealand has shown no evidence of pig-to-human virus transmission (Garkavenko et al., 2011).

3.4 Ethical advantages of increasing the number of organs

There are currently are number of ethical issues associated with organ transplantation around the world, largely due to the shortage of transplantable organs. This shortage has led to the development of an international , where people travel abroad to pay for organ transplants. This has created problems for recipients, including reduced outcomes and higher medial compilations associated with transplantation and negative effects to the donor (Shimazono, 2007). Xenotransplantation could potentially remove/reduce these issues by greatly reducing the demand for transplantable organs.

4. Discussion

4.1 Why an alternative to allotransplantation is needed

This project has demonstrated that there is a high demand for organ transplants and sources of organ donation are not matching this demand. Attempts at increasing the number of transplantable organs from current sources have not been successful and current propositions to further increase donor numbers are highly unlikely to be sufficient enough to match the transplant demand. (NHS, 2006-2015). Deceased organ donation is limited by state of the donor and organs after death, this had led to a higher number of organs and potential donors being unsuitable for donation (NHS, 2006-2015). Therefore deemed consent systems for donation will unlikely result in substantial enough increases in donation, as only a small fraction of refusals are due to personal reasons (NHS, 2006-2015). Expanding the use of marginal deceased donors has been proposed to increase the number of donors, using elderly donors and donors with a history of medical conditions which are currently considered unsuitable (Saidi and Hejazii Kenari, 2014). Although recipients of marginal donor kidneys have higher survival rates than remaining on dialysis, the longer- term outcomes are reduced compared to those of standard organ transplants (Ojo et al., 2001). Therefore marginal donors are not ‘solving’ the organ shortage, they are simply reducing the outcomes of organ transplants and increasing re-transplantation rates. If the number of ‘altruistic’ living donors could be substantially increased, its scope is limited by the types of living donation meaning that deceased organ donors would still be required for the majority of organs (NHS, 2007-2015). The difference been organ donation and transplant demand is only going to increase further with a worldwide aging population.

4.2 The Future of Xenotransplantation

Substantial advances have been made with xenotransplantation in the past 20 years, which have resulted in xenotransplantation being realistically considered as solution to the shortage of organs for human transplants. In addition to the practical problems discussed, there is also the need for a widely accepted consensus on the conditions that need to be achieved before clinical trials can be undertaken. The international xenotransplantation association has proposed conditions for clinical trials of porcine islets (Hering et al., 2016). This includes considerations on the requirements to justify clinical trials, the safety required to prevent disease transmission and an outline of the types of patients that would be suitable

Xenotransplantation and the future of human organ transplants 31 for clinical trials. In addition, the requirement that national regulatory frameworks that monitor xenotransplantation are established prior to clinical trials, which is reiterated by the World Health Organisation (WHO, 2011). This is to ensure that clinical trials are carried out safety and any concerns can be addressed correctly. A similar agreement needs to be established and implemented for organ xenotransplantation before clinical trials can be discussed. An important consideration when assessing the time until clinical applications of xenotransplantation is that recently published papers do not fully represent the current knowledge of xenotransplantation. There is a considerable time between the stages of genetic modification and publishing of papers with quantitative data, during which other research groups will have made additional beneficial advances (e.g. new genetic modifications/combinations of modifications). In addition, there have been significant improvements in genetic modification technology, increasing the efficiency and speed of modifying organisms and enhancing the progress of xenotransplantation (Nagashima et al, 2012 and Sachs and Galli, 2008). Therefore clinical application of xenotransplantation may be more immediate than suggested by current publications and progress over past decades.

4.3 Alternatives to xenotransplantation

Xenotransplantation is not the only potential alternative to allotransplantation, the production of artificial organs and laboratory grown organs have both been suggested. Of primary importance, these alternatives must be able to supply enough organs to match the increasing demand for transplants. Artificial organs have been used to replace (or assist) a natural organ’s function. However, despite being in a more advanced state than xenotransplantation, artificial organs are primarily used to sustain patients until they receive an allotransplant and are largely limited to cardiac devices (Mou et al., 2015). Both ventricular assist devices (VADs) and total artificial hearts are limited to maintaining patients to allotransplantation as long term use of either is associated with several detrimental problems that limit patient survival (haemorrhaging, thrombosis, infections, etc. (Mancini and Colombo, 2015, Quader et al., 2013 and Torregrossa et al., 2014). These problems of infection and the requirement of a portable power source for the organ limit the applications of any type of artificial organ and the production of artificial organs with more complex functions than the heart (such as the liver) have shown little progress (Pless, 2010). Xenotransplants have been proposed as a future ‘bridge’ to allotransplantation, where the xenograft is transplanted into patients temporarily whilst they wait for an allotransplant (Cooper, 2010 and Ekser et al., 2009). The survival times of certain organs in the pig- primate model has given feasibility to this idea however the effects of multiple of organ transplants in relative short succession is less than ideal.

Laboratory grow organs have the substantial advantage of effectively removing the immune response to the transplanted graft, improving long-term outcomes due the lack of immunosuppressant drugs required (Soto-Gutierrez et al., 2012). However the generation of functional organs, with complex 3D structures consisting of numerous cell types specifically organised, has not yet been achieved on a scale for human organ transplantation. Of the several methods proposed, the approach of decellularising a solid organ leaving only a ‘biological scaffold’ of the components of the extracellular matrix which is then repopulated with cells of the patient using stem cells (recellularised) is currently showing the most promise (although still problematic) (Soto-Gutierrez et al., 2012). Recent successes with this technique have been the generation rat organs (largely hearts, kidneys and livers) which

Xenotransplantation and the future of human organ transplants 32

have been semi-functional for a number of hours (Michel et al., 2015 and Stoltz et al., 2015). However, transitioning this method to the scale of generating human sized organs is currently a very difficult problem.

The most substantial problem for laboratory grown organs is finding a source of stem cells that would be sufficient enough to supply the demand for organ transplantation at the stage of clinical applications. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proposed as sources of stem cells, however they are both limited by substantial problems. ES cells are associated with serious ethical concerns due to being derived from human embryos and iPS cells are associated with promoting tumour growth (Wert and Mummery, 2003 and Ben-David and Benvenisty, 2011). Therefore there are currently still problems in finding a source of stem cell that can reliably generate large numbers of differentiated cells required for the production of laboratory grown organs. The use of stem cells to grown patient-specific organs offers extensive potential to reduce the organ shortage, however this field is still very much in its infancy with the large number current issues that still need to be addressed (see figure 4.1), meaning that the clinical applications of laboratory grown organs are still many years away (Stoltz et al., 2015).

Figure 4.1 – The main requirements to be achieved by laboratory grown organs for consideration of clinical applications. A summary of some of the main issues with laboratory grown organs which need to be solved before clinical applications can be realistically considered. This is not an exhaustive list of all the problems with grown organs and all the requirements for clinical applications. Figure produced using information from Michel et al., 2015, Soto-Gutierrez et al., 2012 and Stoltz et al., 2015.

4.4 The future of organ transplantation

The alternatives to allotransplantation discussed all have different advantages and problems that currently limit the clinical applications of each. The use of artificial organs is currently confined to shifting the demand for transplants as patients are maintained with a device, rather than effective reducing the organ shortage. This questions the future role of artificial organs in organ transplantation, however artificial organs/assist devices could be used to maintain patients until the transplantation of xenografts or grown organs. An ultimate goal of organ transplantation is the ability to prevent the immune response, removing the need for

Xenotransplantation and the future of human organ transplants 33 immunosuppressants and the negative long-term outcomes associated with them. This could be achieved with laboratory grown organs, however the relative infancy of this field means that clinical applications are still a considerable distance away. Therefore going forward xenotransplantation offers the more immediate and most widespread applications to reducing the organ shortage. However as continued research leads to the clinical applications of laboratory grown organs becoming more realistic, it may contribute to the organ shortage in the more distant future. Xenotransplantation and laboratory grown organs together may provide the solution, such as decellularising xenografts to create ‘biological scaffolds’ (Xenobioengineering) (Soto-Gutierrez et al., 2012). Whatever the future solution to the shortage of organ of human transplantation may be, and effort needs to be made to invest in alternatives to the unviable current practices of allotransplantation even though their clinical applications may be many years away.

Xenotransplantation and the future of human organ transplants 34

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Appendix – Data Collection

In order to assess the current problems with organ transplantation in general, data has been collected from three different groups in order to show that trends are not UK/country specific. UK data from the NHS, US data reported by the OPTN/SRTR and Eurotransplant data (representing Austria, Belgium, Croatia, Germany, Luxembourg, the Netherlands, Slovenia and from 2012 Hungary).

To obtain data for transplantation it involved researching bodies that controlled and recorded organ transplantation in each country. For the UK, the NHS has maintained detailed annual reports since 2001 (NHS, 2002-2015). These contained overall and organ specific data for organ donation, transplantation and waiting numbers. These reports also contained data for post-transplant outcomes, however they contained limited amounts of data on the long term (10 years) effects of transplantation. In order to see if the trends identified were UK specific or part of larger trend in the world, the UK data needed to compared against countries/regions with equally large populations such as the US or EU. Organ transplantation data is recorded by two bodies the OPTN ( & Transplantation Network) and SRTR (The Scientific Registry of Transplant Recipients) who have worked together to produce detailed annual reports since 2003 and have data from as early as 1988 available online (OPTN/SRTR, 2015-2016 and OPTN, 2016). These reports also provided data on the long term outcomes of organ transplantation of a large number of patients over many years. Finding data for Europe was more difficult, attempts at obtaining data from each country individually where not useful due to the limited number of years recorded and differences in data presentation and detail between countries. After contacting the library, who subsequently contacted other librarians throughout the UK and the rest of Europe, data for the EU was found in an annual report by the EDQM (European Directorate for the Quality of Medicines & HealthCare) (EDQM, 2009-2015). However the overall usefulness of this data was limited by differences in what transplant data was recorded and how it was then presented in difference countries. This meant that data under certain headings were missing from a number of countries, especially data about the number of people on the waiting list per year. Therefore data from Eurotransplant, representing 8 European countries, was used as well to compare trends with the UK and US (Eurotransplant, 2006-2015).

Data for survival times of xenotransplants are from Cooper et al., 2014, which was found after extensive searching through the literature. This paper contained all the xenotransplantation experiments that were published between 1998 and 2013, focusing on graft survival times of major organs and islets. The data presented was then edited (e.g. removing non-life supporting kidney transplants) and checked with the data of the original papers. However, as the majority of papers did not publish data of average survival times per experiment, the remaining reliable information was focused on maximum graft survival per experiment. From this I was able to compare the maximum graft survival per year published to determine whether survival times had changed between 1998 and 2013.