232 Protecting pigs from viral infectivity with gene editing

Abstract

Abstract Pig models were created that were fully resistant to Porcine Reproductive and Respiratory Syndrome (PRRS) and the alphacoronaviruses, transmissible gastroenteritis virus (TGEV) by DNA editing of the cluster of differentiation (CD163) gene and aminopeptidase N (ANPEP) gene via CRISPR/Cas, respectively. Litters of pigs were bred with a gene edit in exon 7 of CD163 that resulted no CD163 protein expression (CD163-/-). The pigs were either challenged with multiple PRRSV isolates at 3 weeks of age or bred at maturity for a challenge with pregnant sows. The challenges demonstrated that the pigs were completely resistant to infectivity to both Type 1 and 2 isolates as measured by clinical signs, viremia, antibody response and lung histopathology. In a follow-up study, pregnant CD163 edited pigs were challenged with PRRSV to determine if absence of CD163 in the dam should be sufficient to protect the fetuses that have functional CD163 protein. The CD163-/- sows carrying both the CD163-/- and CD163+/- fetuses were all negative for PRRSV nucleic acid and showed no sign of fetal or placental failure clearly demonstrating that the absence of CD163 in the sow is sufficient to protect a PRRSV-susceptible CD163+/- fetus. Gene editing of CD163 in pigs, via CRISPR/Cas9, successfully blocked PRRSV infectivity in young growing pigs and pregnant sows and their fetuses. Similar methods were used to determine if TGEV infectivity could be prevented by editing the ANPEP gene. Exon 2 of ANPEP was edited and resulted an ANPEP -/- genotype. ANPEP -/- pigs and age matched wild type pigs were challenged with TGEV. The presence of virus nucleic acid was determined by PCR in fecal samples. ANPEP -/- pigs were fully resistant to TGEV infectivity. These studies clearly show that precise gene editing can be successfully used in pigs to prevent viral infectivity of both PRRSV and TGEV.