Immune modulation in pigs through editing of the RELA locus
Ballantyne, Maeve Kellett
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Livestock animals are an ancient, vital renewable natural resource. Many livestock species have the ability to convert inedible crops and waste food into food fit for human consumption, in the form of meat, eggs and dairy products. As the global demand for high value animal protein is ever increasing, the livestock market continues to play a major role in worldwide economics. Animal disease has the potential to be a huge burden on the livestock industry, impacting both welfare and production. Major outbreaks of transboundary diseases, such as foot and mouth disease, rinderpest and classical swine disease, have resulted in devastating global economic losses. As a result, scientific research is engaged in lowering this impact by generating effective preventative measures and treatments. One way to reduce livestock disease is to select animals that are genetically resistant, traditionally carried out through selective animal breeding programs; however, this is a time-consuming process and requires that appropriate genetic variation exists within the population. Advances in genome engineering technologies offer us an alternative approach, with the capability to make genetic improvements in livestock within a single generation. It is hypothesised that resilience to a disease, known as African swine fever (ASF), could be genetically engineered into the domestic pig. ASF is a highly contagious disease of domestic pigs and is a re-emerging global threat to the swine industry. It is a lethal haemorrhagic disease caused by a virus, known as the African swine fever virus (ASFV). At present, there is no vaccine or treatment for ASF, and disease control relies on rapid diagnosis, quarantine and the mass slaughter of animals. Unlike the domestic pig, swine indigenous to Sub-Saharan Africa, such as the warthog, show no clinical signs of disease following infection with ASFV. A comparative study was carried out to identify host genetic variation that could underlie the difference in response to ASFV, with candidate genes selected based on their potential involvement with the viral protein A238L, involved in immune evasion. Functional polymorphisms where identified in the porcine RELA gene, encoding RelA, a subunit of the NF-κB transcription factor family. This evolutionary conserved protein family plays a vital role in mediating inflammatory and immune responses. The specific RELA polymorphisms identified alter potential phosphorylation sites within the C-terminal transactivation domain of RelA which have been found to modulate NF-κB transcriptional activity in vitro. We set out to investigate whether genome editing tools could be employed to engineer the RELA sequence of domestic pigs. Initial attempts targeted the final exon of RELA, producing animals with a truncated RelA protein; modified animals lack the final 60 amino acids of the C-terminal transactivation domain. The aim of this thesis was to genotype and characterise the effects of this RELA modification at a molecular, cellular, morphological and whole organism level. The ultimate goal of this project was to investigate whether this RELA modification altered the domestic pig’s response to ASFV in vitro and in vivo. Unlike rela-/- mice which have an embryonic lethal phenotype, these RELA-edited pigs were born healthy and were fully viable when housed in a typical farm environment. Phenotypic analysis of lymphoid tissues from the RELA-edited pigs demonstrated no significant anatomical or histological changes compared to unmodified counterparts. Pigs homozygous for the RELA mutation had a significantly lower body weight compared to wild-type pigs. Molecular studies of samples from these pigs have shown that the modified RelA has an altered activity; however, the RELA modified pigs do develop the characteristic disease phenotype when challenged with ASFV. Finally, genome editors have been developed to introduce a specific warthog allele into the domestic pig RELA locus, these editors are currently being taken forward to produce a novel pig line.