**6. Gene insertions**

Gene KO and editing techniques have been used for ablation of xenoantigen genes but do not address the need to express human or synthetic genes in pig cells and organs. Unlike gene deletions, gene insertions require heterologous DNA to be introduced into the genome in a manner that allows subsequent expression of the gene(s) encoded by the inserted DNA. Because they are being transferred into the genome from another source, these novel genes are referred to as "transgenes" (TG), whether they are derived from natural or synthetic sequences. The general approaches to introduce TG into the genome are detailed below.

## **6.1 Random integrant TG**

One of the earliest types of genetic modification described in mammals was insertion of DNA into the target genome by random integration. After transfection of DNA into nearly all mammalian cells, some portion of the heterologous DNA can be found incorporated at random sites in the genome [52]. The precise process for this is unclear, but presumably is a result of aberrant repair mechanisms. One hypothesis is that endogenous NHEJ machinery recognizes breaks in the genome and fortuitously utilizes the relatively higher concentration of the heterologous DNA vector sequences to repair the break [53], resulting in the insertion of the TG into the genome.

Agricultural species, including pig, were some of the earliest TG animals described, establishing the utility of this approach [54]. For xenotransplantation, several of the initial TG approaches focused on inhibiting human antibodymediated damage of porcine organs. The binding of human antibodies to porcine cells leads to complement pathway activation and subsequent cell ablation [55]. The complement function is controlled by several proteins, such as CD46, CD55 and CD59, referred to as complement regulatory proteins (CRPs). The CRPs are broadly expressed on many different cell types to prevent harm from complement activity by raising the threshold of antibody binding required for complement pathway

**39**

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

esis, instability or even lethality [65].

**6.2 Homologous recombination**

resistance to complement-mediated destruction [57].

induction [56]. The transgenic expression of human CRPs in pigs appears to overcome human complement activity, and may have the potential to reduce, or even eliminate, the need for xenoantigen KOs. By placing the human CRP TGs under the control of strong gene expression elements, the CRP protein levels on the porcine cells can be much higher than CRP levels on normal human cells, further increasing

Pig lineages developed by multiple labs have been engineered to express human CRPs, individually or in combination. In most cases, the porcine cells appeared to be more resistant to complement-mediated destruction, and organs from TG animals survived longer in xenotransplant experiments in non-human primates [58–60]. Because each of the CRPs control a different part of the complement pathway, the use of multiple human TGs was more effective in protecting cells from complement-mediated destruction than individual TGs [61]. Together with the removal of key xenoantigens, the expression of human CRPs by porcine cells has

Because random insertion of DNA does not require homologous recombination, it is relatively rapid and efficient to produce transgenic animals [62]. The process is so efficient that the selection methods that are critical for gene KO described above are often unnecessary for TG. Despite the speed and ease of generation, random integration of TGs has several drawbacks. Variabilities in genome structure can affect the expression level of genes inserted at distinct chromosomal regions, such that identical TGs may express at very different levels depending upon their specific location [63]. Furthermore, multiple copies of a given TG may be inserted into the genome at a single site, creating concatenated repeats which can be unstable and yield variable expression levels [64]. Random TG insertion may occur within or near endogenous genes and alter or inactivate their function, leading to tumorigen-

The development of techniques for precise gene KO by homologous recombination has been adapted for site-specific gene insertion or gene knock-in (KI). Similar to the KO vectors described above, the gene to be introduced is flanked by DNA sequences that are identical to regions of the genome to be targeted. After introduction of the heterologous DNA vector, the regions of DNA sequence identity are aligned with the target genome sequence, after which the homologous recombination machinery catalyzes reactions which swap the endogenous genomic DNA with the heterologous DNA within the construct. If the recombination event occurs with high fidelity, the gene of interest will functionally replace the gene that was removed [24]. Similar to gene KO, this approach is much less efficient than random TG integration. Therefore, vectors carrying the TG are often designed to incorporate selectable markers, similar to those used for gene KO, to allow the identification of cells carrying the desired TG in the genome. In this case, both targeted and randomly integrated TGs may be selected, requiring additional assays, such as PCR or Southern blotting, to distinguish between sequence-specific and random insertion events [30]. As described above for gene KO, specific targeting is more efficient in murine ES cells, which express the enzymatic machinery necessary for homologous recombination, than is currently possible for pig primary cells. Insertion of heterologous DNA into the mammalian genome is believed to be driven by endogenous DNA repair mechanisms, presumably in response to DNA breaks, whether randomly via NHEJ, or specifically via homologous recombination [66]. The deliberate introduction of double-stranded DNA breaks at the desired integration site should therefore improve the efficiency of heterologous DNA insertion by activating and recruiting the cellular

greatly reduced the effects of human serum antibodies on xenografts.

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

*Genetic Engineering - A Glimpse of Techniques and Applications*

tion for genome engineering in xenotransplantation.

are required as discussed below.

**6. Gene insertions**

**6.1 Random integrant TG**

into the genome.

The use of CRISPR for the rapid modification of the pig genome was recently

Gene deletion has been instrumental in the advancement of xenotransplantation, however, there are limitations to its application; the genes of interest must be non-essential to pig viability, development, fertility and, most importantly, organ function. The number of distinct loci to be targeted is also a serious consideration, since independently-assorting alleles will be challenging to breed together in a reasonable timeframe. To address these concerns, additional engineering strategies

Gene KO and editing techniques have been used for ablation of xenoantigen genes but do not address the need to express human or synthetic genes in pig cells and organs. Unlike gene deletions, gene insertions require heterologous DNA to be introduced into the genome in a manner that allows subsequent expression of the gene(s) encoded by the inserted DNA. Because they are being transferred into the genome from another source, these novel genes are referred to as "transgenes" (TG), whether they are derived from natural or synthetic sequences. The general

One of the earliest types of genetic modification described in mammals was insertion of DNA into the target genome by random integration. After transfection of DNA into nearly all mammalian cells, some portion of the heterologous DNA can be found incorporated at random sites in the genome [52]. The precise process for this is unclear, but presumably is a result of aberrant repair mechanisms. One hypothesis is that endogenous NHEJ machinery recognizes breaks in the genome and fortuitously utilizes the relatively higher concentration of the heterologous DNA vector sequences to repair the break [53], resulting in the insertion of the TG

Agricultural species, including pig, were some of the earliest TG animals described, establishing the utility of this approach [54]. For xenotransplantation, several of the initial TG approaches focused on inhibiting human antibodymediated damage of porcine organs. The binding of human antibodies to porcine cells leads to complement pathway activation and subsequent cell ablation [55]. The complement function is controlled by several proteins, such as CD46, CD55 and CD59, referred to as complement regulatory proteins (CRPs). The CRPs are broadly expressed on many different cell types to prevent harm from complement activity by raising the threshold of antibody binding required for complement pathway

approaches to introduce TG into the genome are detailed below.

demonstrated with the ablation of porcine endogenous retroviral (PERV) sequences. The pig genome carries 25 or more copies of these gamma retroviral sequences, which are transmitted from parent to offspring through inheritance. Application of CRISPR was able to eliminate the PERV sequences from the genome of porcine cells [50]. Although the potential risk of infectious disease from porcine organs caused by PERV sequences in xenotransplantation is debatable, the results show the ability of CRISPR to target multiple, homologous loci throughout the genome. A key question that has arisen regarding large scale CRISPR targeting at multiple genomic sites is whether significant numbers of off-target double-stranded breaks were introduced, which may create unexpected mutations in the resulting pigs [51]. Nonetheless, the ease of use of CRISPR has resulted in widespread adop-

**38**

induction [56]. The transgenic expression of human CRPs in pigs appears to overcome human complement activity, and may have the potential to reduce, or even eliminate, the need for xenoantigen KOs. By placing the human CRP TGs under the control of strong gene expression elements, the CRP protein levels on the porcine cells can be much higher than CRP levels on normal human cells, further increasing resistance to complement-mediated destruction [57].

Pig lineages developed by multiple labs have been engineered to express human CRPs, individually or in combination. In most cases, the porcine cells appeared to be more resistant to complement-mediated destruction, and organs from TG animals survived longer in xenotransplant experiments in non-human primates [58–60]. Because each of the CRPs control a different part of the complement pathway, the use of multiple human TGs was more effective in protecting cells from complement-mediated destruction than individual TGs [61]. Together with the removal of key xenoantigens, the expression of human CRPs by porcine cells has greatly reduced the effects of human serum antibodies on xenografts.

Because random insertion of DNA does not require homologous recombination, it is relatively rapid and efficient to produce transgenic animals [62]. The process is so efficient that the selection methods that are critical for gene KO described above are often unnecessary for TG. Despite the speed and ease of generation, random integration of TGs has several drawbacks. Variabilities in genome structure can affect the expression level of genes inserted at distinct chromosomal regions, such that identical TGs may express at very different levels depending upon their specific location [63]. Furthermore, multiple copies of a given TG may be inserted into the genome at a single site, creating concatenated repeats which can be unstable and yield variable expression levels [64]. Random TG insertion may occur within or near endogenous genes and alter or inactivate their function, leading to tumorigenesis, instability or even lethality [65].
