**3. Immunity and xenotransplantation**

The immune system is designed to recognize and eliminate harmful pathogens, while remaining unresponsive to host cells and beneficial microbes. The immune system can be divided into innate and adaptive responses, an interdependent set of activities which both contribute to immunity. The innate response is more immediate, broadly recognizing conserved microbial elements, such as cell wall polysaccharides, and activating a variety of cell types which attack the invading pathogens [19]. The adaptive immune response, which is typically initiated by innate response mechanisms, leads to more precise antigen-specific antibodies and immune cells that continue to control and eliminate pathogens. In addition, the adaptive response creates long-lasting immune "memory" for rapid and specific protection against future infections, as demonstrated by vaccines [20].

Despite being described as separate systems, the innate and adaptive immune responses are highly interdependent and create a layered set of defenses with increasing specificity for pathogens over time [21]. Under normal circumstances, any individual function may not eliminate a given target with 100% efficiency, but when used together in a redundant fashion can prevent nearly all infection. Although the specificity of the immune response indirectly helps to avoid recognition of host tissues, additional tolerance mechanisms are required to restrain the immune system to prevent autoreactivity. Disruptions of the balance between

**35**

*Genome Engineering for Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.84782*

recipients, requiring more and different solutions.

of the engineering strategies being employed.

able for pigs [23].

accessible for use in pigs.

**4. Genome engineering and xenotransplantation**

immunity and tolerance can lead to the immune system destroying host tissues (autoimmunity) or allowing repeated severe infections (immunodeficiency) [22]. The transplantation of foreign cells or tissues into a human host can trigger a hostile response from the immune system, leading to immune rejection. The extraordinary precision of the immune system can distinguish even minor differences between donor and recipient, so that even organs from closely related donors may be rejected. Although immunosuppressive drugs can reduce the chance of rejection of human donor organs, the massive amount of immunogenic material found in a whole organ presents an ongoing risk which requires monitoring. Because of the greater genetic differences between pigs and humans, the vigor of the rejection response is much stronger than occurs between human donors and

A major advantage in using pigs for xenotransplantation is the potential to manipulate the porcine genome to create donor organs that are more compatible for human patients. However, the scope of the engineering challenge in xenotransplantation is extremely large, involving a variety of genes and pathways. With so many potential targets for genetic modification, an assortment of different genome engineering strategies have been applied, including editing or deletion of porcine genes and insertion of human or engineered genes. Because of the great diversity of genome modification efforts being carried out in xenotransplantation research, representative approaches will be highlighted here as examples of the general types

Historically, mice have been subject to more and different genetic modifications than any other mammalian species, and many of the protocols described here were first developed in mice. Aside from their well-established and convenient husbandry, small size, and rapid generation times, mice also have a variety of technological advantages for genome manipulation and production. Although genetic modification has been demonstrated for multiple agricultural species, including pigs, the scale and complexity possible with mice has, until recently, not been avail-

One advantage for the creation of mice with multiple genetic modifications is the availability of embryonic stem (ES) cells, which can be cultured *in vitro* for many generations and subject to repeated transfections and selections without loss of competence for production of viable mice [24]. By contrast, pig ES cells have been much more difficult to create, and have not been routinely used for genetic manipulation and production of animals [25]. Cloning of genetically-modified pigs has required use of primary cells, typically fetal, which can be passaged only a short time *in vitro* before losing their competence to produce viable embryos [26]. Therefore, the complex multi-site modifications and selections used in mice are not

Mouse ES cells not only allow more straightforward and efficient genome engineering, but also facilitate large scale production of cloned mice. The mouse ES cells typically employed for genetic modification can be injected into very early stage embryos (blastocysts) and will aggressively populate the inner cell mass, creating viable chimeric mice which are almost entirely ES-cell derived. Since the ES cells will also contribute to the germ cells of the chimeric mice, the progeny will be highly likely to receive the genetic modifications made to the ES cells [27]. Without readily available porcine ES cells, pig cloning instead relies upon somatic cell nuclear transfer (SCNT), similar to the protocols used to create the sheep

#### *Genome Engineering for Xenotransplantation DOI: http://dx.doi.org/10.5772/intechopen.84782*

*Genetic Engineering - A Glimpse of Techniques and Applications*

primate organs [14].

organs into patients throughout the nineteenth and early twentieth centuries [3–7]. After World War II, organ transplantation from living humans was considered too high risk, and cadaveric organs were insufficient in both quality and number to meet clinical needs. Development of immunosuppressive drugs suggested the possibility that organs from more closely-related mammals could potentially be used in humans. As a consequence, xenotransplantation efforts shifted to the use of organs from primates in human patients. In the 1960s, experiments by Reemstma et al. [8, 9], Hardy et al. [10], and Starzl et al. [11, 12] showed that while it was technically possible to transplant animal organs into humans, there were still too many clinical challenges at that time for the approach to be viable. More research was required to understand and overcome the barriers to the practical application of xenotransplantation in humans. One of the major advances in xenotransplantation research in the past few decades has been the focus on the use of pigs as donors [13]. This was based, in part, on purely practical considerations. Unlike primates, pigs are an agricultural species for which large scale breeding is well-established. In addition, the evolutionary distance between humans and pigs reduces the risk of transmission of zoonoses from pig organs to patients compared with primate organs. Most importantly, the use of porcine organs does not present the same ethical barriers as the use of non-human

Although the anatomy and physiology of pig organs is closely analogous to that of humans, the advantages of porcine organ production and availability do not address the critical issue of incompatibilities of non-human tissues and organs with the human immune system [15]. Significant advancements have been made in recent years in understanding the molecular mechanisms of xenorejection responses, and a variety of genetic modifications have been made to overcome these mechanisms. Experiments transplanting pig organs into non-human primates have demonstrated a progressive improvement in organ survival and function as new genetics and drug regimens have been implemented [16]. The FDA is currently developing guidelines for clinical xenotransplantation [17], and efforts to initiate

The immune system is designed to recognize and eliminate harmful pathogens, while remaining unresponsive to host cells and beneficial microbes. The immune system can be divided into innate and adaptive responses, an interdependent set of activities which both contribute to immunity. The innate response is more immediate, broadly recognizing conserved microbial elements, such as cell wall polysaccharides, and activating a variety of cell types which attack the invading pathogens [19]. The adaptive immune response, which is typically initiated by innate response mechanisms, leads to more precise antigen-specific antibodies and immune cells that continue to control and eliminate pathogens. In addition, the adaptive response creates long-lasting immune "memory" for rapid and specific protection against

Despite being described as separate systems, the innate and adaptive immune responses are highly interdependent and create a layered set of defenses with increasing specificity for pathogens over time [21]. Under normal circumstances, any individual function may not eliminate a given target with 100% efficiency, but when used together in a redundant fashion can prevent nearly all infection. Although the specificity of the immune response indirectly helps to avoid recognition of host tissues, additional tolerance mechanisms are required to restrain the immune system to prevent autoreactivity. Disruptions of the balance between

clinical trials in the near term have been announced [18].

future infections, as demonstrated by vaccines [20].

**3. Immunity and xenotransplantation**

**34**

immunity and tolerance can lead to the immune system destroying host tissues (autoimmunity) or allowing repeated severe infections (immunodeficiency) [22].

The transplantation of foreign cells or tissues into a human host can trigger a hostile response from the immune system, leading to immune rejection. The extraordinary precision of the immune system can distinguish even minor differences between donor and recipient, so that even organs from closely related donors may be rejected. Although immunosuppressive drugs can reduce the chance of rejection of human donor organs, the massive amount of immunogenic material found in a whole organ presents an ongoing risk which requires monitoring. Because of the greater genetic differences between pigs and humans, the vigor of the rejection response is much stronger than occurs between human donors and recipients, requiring more and different solutions.
