First gene-edited calf with reduced susceptibility to a major viral pathogen

Authors: Workman, Aspen; Heaton, Michael - Mike; VANDER LEY, BRIAN - University Of Nebraska; WEBSTER, DENNIS - Recombinetics, Inc; SHERRY, LUKE - Recombinetics, Inc; BOSTROM, JONATHAN - Recombinetics, Inc; LARSON, SABREENA - Acceligen Inc; KALBFLEISCH, THEODORE - University Of Kentucky; Harhay, Gregory; JOBMAN, ERIN - University Of Nebraska; CARLSON, DANIEL - Recombinetics, Inc; SONSTEGARD, TAD - Acceligen Inc

Submitted to: Proceedings of the National Academy of Sciences-Nexus
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2023
Publication Date: 5/9/2023
Citation: Workman, A.M., Heaton, M.P., Vander Ley, B., Webster, D., Sherry, L., Bostrom, J.R., Larson, S., Kalbfleisch, T., Harhay, G.P., Jobman, E., Carlson, D., Sonstegard, T.S. 2023. First gene-edited calf with reduced susceptibility to a major viral pathogen. Proceedings of the National Academy of Sciences-Nexus. 2(5). Article pgad125. https://doi.org/10.1093/pnasnexus/pgad125.
DOI: https://doi.org/10.1093/pnasnexus/pgad125

Interpretive Summary: Bovine viral diarrhea virus (BVDV) is a widespread and economically important viral infection in cattle, causing respiratory and gastrointestinal diseases, and reproductive failure. While vaccines for BVDV have been available for more than 50 years, BVDV remains a significant global pathogen. Modifying the bovine receptor for BVDV via CRISPR/Cas9 gene-editing technology offers a novel way to potentially control infections in cattle. The main bovine receptor for BVDV is CD46, which we modified by replacing six amino acids in the domain that binds to the virus. This six amino acid substitution dramatically reduced susceptibility to BVDV infection when tested in the laboratory. The same CD46-edit was then made in skin cells taken from an adult Gir breed cow and used to produce reproductively cloned, gene-edited animals for study. At 100-days gestation, primary cells were isolated from multiple organs of a gene-edited fetus and compared those from an unedited fetal clone. When infected in the laboratory, cells from all organs of the gene-edited fetus had a significant reduction in BVDV susceptibility. In 2021, a normal and healthy Gir heifer calf was born with the CD46 edit and has continued to thrive with no observed adverse effects from the CD46 edit in the first year of life. To test whether the gene-edited Gir calf could resist BVDV infection, she was co-housed with a Holstein calf that was naturally and persistently infected with BVDV. Upon this natural exposure BVDV challenge, the CD46-edited Gir calf had minimal signs of infection and her white blood cells were not infected. Together, these results provide proof-of-concept for using intentional genome alterations in CD46 to reduce the burden of BVDV infections in cattle. This represents the first demonstration of editing a bovine gene to reduce susceptibility to a viral pathogen.

Technical Abstract: Background: Bovine viral diarrhea virus (BVDV) is one of the most important viruses causing respiratory and gastrointestinal diseases, and reproductive failure in cattle throughout the world. The main BVDV cellular receptor in cattle is CD46 and residues G82QVLAL87 are among those essential for binding and entry.Substituting residues A82LPTFS87 in CD46 was previously shown to reduce BVDV susceptibility in vitro. Our goal was to produce calves with homozygous CD46 A82LPTFS87 substitutions to evaluate the impact on BVDV susceptibility and calf viability. Methods: CRISPR-mediated homology-directed repair was used to make the CD46 A82LPTFS87 substitution in a bovine kidney cell line (MDBK) and in a primary fibroblast cell line derived from a Gir breed female. Reproductive cloning based on somatic cell nuclear transfer was used to produce fetal clones for isolating primary cells and an edited calf for BVDV challenge. Primary cell lines and calf white blood cells were tested ex vivo while the 10-month old CD46-edited calf was exposed to BVDV by cohabitation with a persistently infected calf. Results: BVDV susceptibility in MDBK cells with the CD46 A82LPTFS87 substitution was equivalent to a CD46 knockout, indicating the edit eliminated the virus' ability to use this entry receptor. In primary cell lines from fetal calves, the same CD46 substitution caused a similar dramatic reduction in BVDV infection. A CD46-edited calf was born and appears normal and healthy at one year of age with no obvious adverse effects from the on-target edit. Upon BVDV challenge, the edited calf had minimal signs of infection and her peripheral blood mononuclear cells (PBMCs) were not infected. Conclusion: Stepwise experiments showed that substituting A82LPTFS87 in CD46 dramatically reduced BVDV susceptibility in vitro, ex vivo, and in a natural challenge study. The results provide proof-of-concept for using intentional genome alterations in CD46 to reduce the burden of BVDV-associated diseases in cattle.