Gene function & annotation

Gene-Edited BMPR2 Heterozygous Sheep: A Novel Large Animal Model for Heritable Pulmonary Arterial Hypertension.

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Abstract

Pulmonary arterial hypertension (PAH) is a disease characterized by elevated pulmonary arterial resistance that leads to right heart failure. Approximately 20% of adult PAH cases and up to 50% of pediatric PAH cases are associated with mutations in the Bone Morphogenetic Protein Receptor Type 2 (BMPR2) gene. BMPR2-associated PAH is an autosomal dominant disease, but penetrance is highly variable with pathologic expression occurring in ~42% of females but only ~14% of males. Additionally, homozygous BMPR2(-/-) mutations are embryonic lethal as the fetus fails to form organized structures or mesoderm, and heterozygotes are haploinsufficient with a 50% reduction in functional receptor resulting in increased vascular cell proliferation and reduced apoptosis. Investigations using rodent models have advanced our understanding of BMPR2 signaling. However, the small size of rodents prevents them from adequately modelling human cardiopulmonary responses, presenting an essential need for clinically relevant large animal preclinical genetic models. To address this, we produced BMPR2(+/-) heterozygous gene-edited sheep with a targeted modification in exon 3 of the ovine BMPR2 gene by electroporating embryos with CRISPR/Cas9 gene editing reagents alongside a PAM-disrupting synonymous ssODN to generate eight gene-edited term lambs, all of which had at least one allele where the ssODN repair template has been used to reconstitute a wildtype allele. Four lambs that were liveborn (3♀,1♂), and all carried at least one BMPR2 knockout allele. The one BMPR2(+/-) F0 ram produced viable semen at maturity, transmitting frameshift mutant alleles -49 (58%) and -7 bp (17%), in addition to wildtype (26%) alleles. He was bred with nine wild-type ewes which resulted in eight pregnancies and 19 liveborn F1 offspring. Of these, 12 were heterozygous for BMPR2. One of these F1 heterozygous males was reared to maturity and his sperm was collected and analyzed. The result showed he transmitted both the -49bp deletion and wildtype alleles in the germline. His sperm was further used for in vitro fertilization and the resulting blastocysts were genotyped. Eleven of twenty-three blastocysts inherited the -49bp (48%) allele, and twelve the wildtype allele, which is not different from the expected Mendelian 1:1 segregation ratio. Expression of BMPR2 in the ear, lung, and heart of the BMPR2(+/-) F0 male #4 was analyzed by qPCR normalized to expression of a reference gene (ACTB) and was found to be significantly lower as compared to wildtype animals (P< 0.05) except in lungs. Moreover, in the heart and lung of F1 lambs, BMPR2(+/-) heterozygous animals had significantly lower BMPR2 expression in both tissues as compared to BMPR2(+/+) wildtype controls (P< 0.05). Edited BMPR2(+/-) heterozygous sheep may serve as a valuable large-animal preclinical biomedical model for heritable PAH.

Keywords: 2026

How to Cite:

Falade, O., Datar, S., Brown, A., Fitzpatrick, D., Trott, J., Hutchings, R., Amin, E., Johnson, E., Morgan, J., Nawaytou, H., Gonzales Viera, O., Urbano, T., McNabb, B., Austin, E., Fineman, J. & Van Eenennaam, A., (2026) “Gene-Edited BMPR2 Heterozygous Sheep: A Novel Large Animal Model for Heritable Pulmonary Arterial Hypertension.”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2285536. doi: https://doi.org/10.31274/wcgalp.23733

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