Friday October 11, 2019 from 08:00 to 08:30
Removal of PERV-C proviruses from xenotransplanation donor pigs by marker-assisted breeding or Gene Editing
Ingrid Kola1, Stefanie Egerer1, Andrea Bähr1, Antonia Godehardt3, Ralf R. Tönjes3, David Ayares2, Eckhard Wolf1, Nikolai Klymiuk1.
1Molecular Animal Breeding and Biotechnology, LMU Munich, Munich, Germany; 2Revivicor Inc, United Therapeutics, Blacksburgh, VA, United States; 3Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany
Concerns about xenotransplantation safety are dominated by the potential infection of recipients by porcine endogenous retroviruses (PERV). Three different subfamilies A, B and C have been identified with PERV-C being the least abundant. The potential of this subfamily to form highly infectious recombinant with PERV-A, however, suggested that PERV-C proviruses should be extincted from the genome of xenotransplantation donors. Therefore, we have identified PERV-C integration sites in the LMU Munich xenotransplantation breeding herd, with proven infectious potential in standard in vitro assays.
As none of the PERV-C proviruses was abundant in all animals, we started to select breeding animals with low copy numbers and achieved PERV-C-free animals by marker assisted breeding. Attempts to completely eradicate PERV-C from the breeding herd are ongoing, but they are evidently time-consuming. Therefore, we explored the option to delete proviral integration sites by Gene Editing. We chose a GTKO.hCD46.bLEA triple modified individual that contained a single PERV-C provirus at chr1:41Mb in a large intergenic region and cultivated primary cells. gRNA binding in the up- and downstream flanking regions of the provirus were designed and applied to the primary cells together with a Cas9 encoding protein. By examination of single cell clones we revealed that 20 out of 100 examined cell clones underwent deletion of PERV-C. The precise status of the former provirus-containing allele as well as the sister allele was determined by Sanger sequencing. Finally, we confirmed that such cell clones did not contain any further PERV-C elements. Attempts to produce GTKO.hCD46.bLEA.PERVC-KO animals from these cell clones are ongoing.
Conclusively, we here illustrate that both, marker-assisted breeding as well as Gene Editing are sufficient tools to eliminate the load of PERV-C in donor pigs for xenotransplantation.
Transregional Collaborative Research Center 127, German Research Foundation.