Genetically edited pigs comprising a modification in the RELA gene

#The present invention relates to a genetically edited non-human animal. In particular the invention relates to a genetically edited mammal, and particularly to a genetically edited pig. The animal is less affected by pathogen infection; in particular the animal is less likely to develop disease pathology upon virus infection. The genetically edited animal has altered RELA (p65) expression or activity. The altered RELA activity is preferably caused by a mutation affecting the transactivation domain encoding sequences of RELA. Pigs with altered RELA activity can be tolerant of viral infection, in particular infection by African Swine Fever Virus (ASFV).

#Infectious disease adversely affects livestock production and animal welfare, and often has major impacts upon human health and public perception of livestock production. For example, the costs of existing endemic diseases are estimated as 17% of turnover of livestock industries in the developed world and 35-50% in the developing world. Furthermore, epidemics, particularly in the developed countries, incur further major costs and profound impacts upon the rural economy and on public confidence in livestock production. Biomolecular approaches to enhance the underlying genetic ability of an animal to combat infectious disease will enhance animal production regimes. Such technologies, along with traditional disease control measures, will enable more effective and sustainable disease control.

#Central to the immune response a mammal activates up pathogen infection is the gene transcription pathway utilising the transcription factor NFkappaB, which is an obligate dimer protein. This transcription factor regulates, amongst other signalling pathways, the pathogen induced cytokine response that is central to the animal's immune response upon infection. The predominant components of the NFkappaB dimer are the RELA (p65) and NFKB1 proteins. The RELA protein is encoded by a gene which displays polymorphic variation within pig species. In particular African and Eurasian pigs display different allelic versions of RELA and functional studies indicate that the African RELA variant displays a reduced NFkappaB activity that is generally equivalent to the animal carrying only one copy of the ‘Eurasian’ gene. It is known from mouse studies that animals that carry a single copy of the RELA gene are viable.

#The invention will be further described by way of the following examples, which are in no way intended to limit the scope of the invention. The examples refer to the accompanying drawings, in which:

#FIG. 1 shows the Sus scrofa RELA cDNA sequence, GenBank accession number NM_001114281 (SEQ ID NO 1).

#FIG. 2 shows the Sus scrofa RELA amino acids sequence, GenBank accession number NP_001107753 (SEQ ID NO 2).

#Two types of editor were used: TALEN and ZFN. Both were designed to target the same region of porcine RELA gene.

#TALEN: All TALENs were designed using the TALE-NT software and assembled using methods described in Cermak et al. (2011)—Nucl. Acids Res. (2011) 39 (12): e82. Briefly, intermediary arrays were produced for each TALEN pair that were compatible for Golden Gate cloning into pC-+63-TAL modified vector (although other vectors such as pC-+231-TAL, RCIscript-+231-TAL, pC-GoldyTALEN or RCIscript-GoldyTALENetc. could be used). Arrays were joined in the above vectors as follows; 150 ng each pFUS_A, pFUS_B, pLR-X and the desired backbone were mixed in a 20 μl digestion/ligation reaction including 50 units T4 DNA ligase (New England Biolabs) and 10 units Esp3I (Fermentas) in 1×T4 ligase buffer (New England Biolabs). The reaction was incubated in a thermocycler for 10 cycles of 5 min at 37° C. and 10 min at 16° C., then heated to 50° C. for 5 min and then 80° C. for 5 min. Two microliters of each reaction was transformed into E. coli and plated on LB-carbenicillin plates. Plasmid DNA was purified and mRNA synthesized from SacI linearized RCIscript vectors using the mMessage Machine T3 Kit (Ambion).

#The target sequences for the TALENs used in this work are shown in FIGS. 4 and 5. It is of course possible that TALENs targeting other sequences within the RELA gene, or indeed elsewhere in the pig genome, could be used in the present invention. The person skilled in the art could readily construct TALENs adapted to target essentially any other desired sequence using the same techniques described above.