**5. Gene deletions**

As mentioned above, porcine cells produce molecules which are rapidly recognized by the human immune system and rejected. One straightforward approach to engineering the pig would be to simply eliminate the genes encoding reactive genes by either disrupting or removing the coding sequence. Several of the technical routes which can be employed to accomplish this are discussed below.

#### **5.1 Gene knockout**

Gene knockout (KO) approaches developed for use in mouse ES cells generally rely upon homologous recombination to replace a region of genomic DNA with a heterologous DNA sequence, which interrupts the function of the target gene [29]. To accomplish this, a DNA vector is generated with the heterologous DNA flanked on either side by sequences identical to regions flanking the genomic region to be eliminated. When introduced into mouse ES cells, the flanking sequences of the DNA vector first align with the cognate regions of the genome on either side of the target gene, after which the homologous recombination machinery replaces the genomic target with the heterologous DNA vector sequence found between the flanking sequences.

Because homologous recombination occurs at a relatively low rate, in order to identify properly targeted cells within the larger cell population, it is common to include a gene in the heterologous DNA to be inserted into the genome, which, once properly inserted, allows selection of the desired cells. For example, genes which confer resistance to drugs which kill mammalian cells (neomycin hygromycin or puromycin resistance), or genes encoding molecules that enable cells to be isolated via flow cytometry (green fluorescent protein or novel cell surface markers), allow isolation of even extremely rare targeted cells from a large mixed population [30].

The use of gene KO approaches was one of the earliest successes in pig genetic modification for xenotransplantation [31]. The porcine genome encodes proteins that can be substantially different from their human counterparts, or that carry additional modifications which are not present in humans and can induce immune responses. These molecules are collectively referred to as "xenoantigens" [32]. Some of the most reactive of these targets are carbohydrate molecules found as post-translational modifications to proteins observed in pig, but not human, cells. Human serum can contain high levels of pre-existing antibodies specific for these porcine-specific glycan epitopes, leading to the destruction of pig cells expressing these molecules through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) mechanisms [33]. It is not entirely clear why human serum carries antibodies to these particular carbohydrates; one proposal is that the xenoantigens are related to glycans found in the cell walls of pathogens, others suggest that the human dietary consumption of pork causes

**37**

**5.2 Gene editing**

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

tigen from being expressed by pig cells.

antibody generation to the porcine-specific molecules [34]. Because the novel carbohydrate structures are created by specific glycosylation enzymes, it is possible to eliminate the gene responsible for the enzymatic activity and prevent the xenoan-

The GGTA1 gene encodes the enzyme responsible for creating the highly reactive glycan Gal alpha (1,3) Gal epitope in pigs [35]. The KO of the GGTA1 gene is one of the earliest genetic modifications of pigs for application in xenotransplantation, and resulted in greatly reduced human antibody recognition of porcine cells [36, 37]. However, ablation of the GGTA1 gene alone did not completely eliminate porcine cell recognition by human serum antibodies. The enzymes responsible for other xenoantigens, such as CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase critical for Neu5Gc biosynthesis) and B4GALNT2 (beta 1,4 N-acetylgalactosaminyltran sferase), have been identified as sources of porcine-specific epitopes bound by antibodies found in human serum. In each case, the deletion of the gene responsible for creating the specific glycan leads to greatly decreased recognition of porcine cells by antibodies in human serum, and reduction in complement-mediated destruction [38, 39]. Another subset of xenoantigens is the swine leukocyte antigens (SLA), the physical and functional equivalent of the human leukocyte antigens (HLA) [40]. Much like the case for human HLA, the SLA genes are highly diverse and individual patients will have a variable level of cross-reactive antibodies in their serum for a given set of SLA genes [41]. Although typing of patients and porcine donors to find the best HLA-SLA matches would be similar to the current system used for determining allotransplant cross-reactivity [42], use of gene targeting or editing technologies could easily eliminate the genes encoding SLA entirely. However, unlike the glycan epitopes described above, the SLA have a critical role in antigen presentation as part of the immune response, and thus the deletion of SLA could create risks of immune deficiencies that outweigh their risks as xenoantigens. Instead, alternate approaches seek to create engineered SLA proteins lacking the epitopes responsible for the immunogenicity while maintaining their antigen presentation functions [43].

The ease and efficiency of creating gene KO has improved recently through the use of engineered molecules to create genome disruptions in a process referred to as "gene editing". These novel molecules can be designed to generate double-strand DNA breaks at virtually any chosen genomic site *in situ*. Cellular machinery closely surveils the genome for double-strand breaks which are then recognized and often repaired by non-homologous end joining (NHEJ). Because NHEJ relies upon small single-strand overlaps at the ends of a break, the repair may be imprecise and, if within a coding

The most prominent of these novel tools for gene editing are Zinc Finger Nucleases

region, can lead to frame shift mutations which inactivate the gene [44].

(ZFN), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), each of which consists of two regions: a sequence-specific DNA binding domain and an enzymatic function that creates a double-strand break in the target DNA [45–47]. For ZFN and TALEN, the synthetic DNA binding domain is created by repetitive protein modules which can be joined combinatorially to recognize a particular DNA sequence. Both approaches, while successful, require a significant investment of time and resources to identify functional molecules. CRISPR, like ZFN and TALEN, has the ability to generate double-strand DNA breaks, however, the DNA binding domain relies upon RNA base-pairing with target DNA for its precision. The use of an RNA to guide specificity greatly improves the speed and efficiency in identifying optimal molecules at a much lower cost, which has led to its rapid adoption in genome engineering [48, 49].

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

*Genetic Engineering - A Glimpse of Techniques and Applications*

cloning, further limiting availability [28].

**5. Gene deletions**

**5.1 Gene knockout**

between the flanking sequences.

"Dolly". In this approach, pig oocyte nuclei are replaced with nuclei from the modified primary pig cells, and embryonic development stimulated electrochemically. The embryos are transferred to female surrogates and allowed to develop. The level of complexity and effort involved leads to lower efficiencies and higher costs for porcine SCNT relative to mouse ES cell cloning. Additionally, the size and scale of the facilities required for pig cloning is significantly greater compared with mouse

In the following sections, different types of gene modifications are described with examples of their application in porcine genome engineering for xenotransplantation.

As mentioned above, porcine cells produce molecules which are rapidly recognized by the human immune system and rejected. One straightforward approach to engineering the pig would be to simply eliminate the genes encoding reactive genes by either disrupting or removing the coding sequence. Several of the technical

Gene knockout (KO) approaches developed for use in mouse ES cells generally rely upon homologous recombination to replace a region of genomic DNA with a heterologous DNA sequence, which interrupts the function of the target gene [29]. To accomplish this, a DNA vector is generated with the heterologous DNA flanked on either side by sequences identical to regions flanking the genomic

sequences of the DNA vector first align with the cognate regions of the genome on either side of the target gene, after which the homologous recombination machinery replaces the genomic target with the heterologous DNA vector sequence found

Because homologous recombination occurs at a relatively low rate, in order to identify properly targeted cells within the larger cell population, it is common to include a gene in the heterologous DNA to be inserted into the genome, which, once properly inserted, allows selection of the desired cells. For example, genes which confer resistance to drugs which kill mammalian cells (neomycin hygromycin or puromycin resistance), or genes encoding molecules that enable cells to be isolated via flow cytometry (green fluorescent protein or novel cell surface markers), allow isolation of even extremely rare targeted cells from a large mixed population [30]. The use of gene KO approaches was one of the earliest successes in pig genetic modification for xenotransplantation [31]. The porcine genome encodes proteins that can be substantially different from their human counterparts, or that carry additional modifications which are not present in humans and can induce immune responses. These molecules are collectively referred to as "xenoantigens" [32]. Some of the most reactive of these targets are carbohydrate molecules found as post-translational modifications to proteins observed in pig, but not human, cells. Human serum can contain high levels of pre-existing antibodies specific for these porcine-specific glycan epitopes, leading to the destruction of pig cells expressing these molecules through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) mechanisms [33]. It is not entirely clear why human serum carries antibodies to these particular carbohydrates; one proposal is that the xenoantigens are related to glycans found in the cell walls of pathogens, others suggest that the human dietary consumption of pork causes

region to be eliminated. When introduced into mouse ES cells, the flanking

routes which can be employed to accomplish this are discussed below.

**36**

antibody generation to the porcine-specific molecules [34]. Because the novel carbohydrate structures are created by specific glycosylation enzymes, it is possible to eliminate the gene responsible for the enzymatic activity and prevent the xenoantigen from being expressed by pig cells.

The GGTA1 gene encodes the enzyme responsible for creating the highly reactive glycan Gal alpha (1,3) Gal epitope in pigs [35]. The KO of the GGTA1 gene is one of the earliest genetic modifications of pigs for application in xenotransplantation, and resulted in greatly reduced human antibody recognition of porcine cells [36, 37]. However, ablation of the GGTA1 gene alone did not completely eliminate porcine cell recognition by human serum antibodies. The enzymes responsible for other xenoantigens, such as CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase critical for Neu5Gc biosynthesis) and B4GALNT2 (beta 1,4 N-acetylgalactosaminyltran sferase), have been identified as sources of porcine-specific epitopes bound by antibodies found in human serum. In each case, the deletion of the gene responsible for creating the specific glycan leads to greatly decreased recognition of porcine cells by antibodies in human serum, and reduction in complement-mediated destruction [38, 39].

Another subset of xenoantigens is the swine leukocyte antigens (SLA), the physical and functional equivalent of the human leukocyte antigens (HLA) [40]. Much like the case for human HLA, the SLA genes are highly diverse and individual patients will have a variable level of cross-reactive antibodies in their serum for a given set of SLA genes [41]. Although typing of patients and porcine donors to find the best HLA-SLA matches would be similar to the current system used for determining allotransplant cross-reactivity [42], use of gene targeting or editing technologies could easily eliminate the genes encoding SLA entirely. However, unlike the glycan epitopes described above, the SLA have a critical role in antigen presentation as part of the immune response, and thus the deletion of SLA could create risks of immune deficiencies that outweigh their risks as xenoantigens. Instead, alternate approaches seek to create engineered SLA proteins lacking the epitopes responsible for the immunogenicity while maintaining their antigen presentation functions [43].
