**7. Future needs**

*Genetic Engineering - A Glimpse of Techniques and Applications*

specificity of both gene deletion and gene insertion in genomes.

minimal number of steps, as discussed below, is highly valuable.

the feasibility of rapidly making animals with multiple TG.

variable TG copy numbers and expression levels.

porate larger DNAs into the genome.

**6.4 Site-specific recombination**

**6.3 Multigenic insertion**

repair machinery. Application of ZFN, TALEN and CRISPR technologies have shown that homologous recombination efficiencies are improved when one or more doublestrand DNA breaks are introduced into the genome at the desired site of insertion [67] with CRISPR exhibiting bi-allelic targeting rates as high as 90% [68]. The use of these more advanced genome engineering tools has greatly improved the rates and

Gene insertion by homologous recombination for xenotransplantation has not advanced as rapidly as other approaches, in part due to the challenges of using primary porcine cells, for which the efficiencies can be extremely low, particularly with large DNA constructs. Use of improved genome engineering tools with increased targeting efficiencies have already been applied in pig and will continue to grow in impact [69]. However, even with improved efficiencies of gene insertion, breeding pigs with multiple, independently segregating loci is challenging. The number of litters required to produce animals bearing all of the genetic modifications greatly increases with each additional locus, which can be impractical for large animals such as pigs. Therefore, the ability to insert the maximal amount of genetic information into the genome in the

The most straightforward example of multigenic targeting at a single site takes advantage of the observation that multiple DNA vectors co-transfected into cells will tend to insert together at a given genome site. This approach was used successfully to simultaneously introduce as many as five large transgenes in a single step into porcine cells [70]. Although difficulties in producing mature cloned pigs limited the study to animals with fewer integrated genes, the study demonstrated

Another, relatively less complicated, way to introduce multiple TG is to generate large DNA constructs bearing multiple TGs for integration at random into the genome [71]. This greatly reduces the complexity of screening, while increasing the efficiency of insertion, but still relies upon random integrants which can have

Multiple genomic regions have been defined, such as Rosa26, which allow expression of heterologous genes at similar levels regardless of cell type. These "safe harbor" regions are believed to have a chromatin structure that is more easily accessible to the gene expression machinery, regardless of cell type. Targeting at porcine homologs of murine safe harbor sites such as Rosa26 has been described and demonstrates the utility of this approach [70, 72]. On a practical level, the use of safe harbor sites yields more reproducible gene expression than random TG insertions, so fewer lineages are required to select animals with desired TG levels. Furthermore, the defined location and copy number of TG inserted at a safe harbor site makes breeding and genotyping more straightforward, and is expected to provide a less complicated regulatory pathway for clinical use. As DNA synthesis and assembly has improved, increasingly large DNA constructs encoding a variety of TG are possible, however, as the size of the DNA increases, the rate of insertion decreases. Considering the lower insertion rates observed for large animals such as pig, alternate approaches are necessary to incor-

Bacteriophage- and yeast-derived site-specific recombinases are, as the name suggests, proteins which catalyze recombination between two specific DNA recognition sites, small (<50 base pair) sequences that are unique to the recombinase being

**40**

The advancements in genome engineering, both in general and in their application to xenotransplantation, have been significant, but many needs remain to be addressed. As new genome engineering tools are identified and further refined, improvement of targeting efficiencies will allow more sophisticated modifications of the pig genome. Ideally, the pig genome will become as readily manipulated as the mouse, allowing researchers to further leverage approaches shown to be effective in murine models.

One major technological difference in the genetic modification of mice and pigs (and many other mammalian species) is the lack of ES cells possessing significant rates of homologous recombination that can be grown in culture for extended periods and subjected to multiple manipulations without losing the ability to produce viable pigs. Efforts to identify natural or induced pluripotent stem cells (iPSC) suitable for these purposes have been described, but have yet to demonstrate practical application for porcine genome engineering [74]. Ongoing work will be required to identify and validate cells which meet these needs.

The function of the TGs themselves can also be further improved. The majority of TG constructs used in pigs have used constitutive promoters to drive high level expression of the proteins encoded by the TGs. In some cases, such as CRPs, this approach may be useful, however, overexpression of TGs which inhibit critical immune processes may create risks of immune deficiency and infections. For this reason, use of expression control elements which can turn on and off TG activity is of increasing interest in xenotransplantation. There are multiple examples of inducible promoters employed in mice which can be controlled by exogenously applied small molecules (such as the tetracycline repressor system), or by endogenous signals (such as promoters for innate immune response genes) [75]. Advanced DNA synthesis and assembly methods also allow synthetic biology approaches to create novel signaling pathways and networks not present in nature.

Immune tolerance is another very active area in xenotransplantation research. As the molecular mechanisms controlling the balance between immunity and tolerance are further elucidated, manipulation of the human immune system itself to specifically reduce or eliminate responses to porcine targets, while leaving intact immunity to infectious diseases, will help overcome xenorejection. Multiple approaches are currently being tested and genome targets identified to encourage human immune tolerance of porcine cells and tissues [76, 77].
