B08: In vivo manipulation of non-coding RNAs in pig hearts

Meg3, an imprinted gene of the Dlk1-Meg3 locus, is transcribed as a long non-coding RNA and exhibits antiangiogenic and profibrotic functions, which have been shown to contribute to pathologic hypertrophy in mice. In this project, we set out to identify the cell-type responsible for the Meg3 increase in hypertrophic porcine hearts. Concomitantly, we will devise strategies to delete Meg3 in postnatal pigs in a cell-specific way and assess functional effects in a model of hypertrophic heart failure. To this end, we will make use of two developments of the previous funding period: first, we will utilize hearts of Cas12-transgenic pigs devised during the first funding period, since its targeting gRNAs are compatible with gRNAs expressed under the control of cell-type specific promoters. Second, we will apply a novel adenoassociated virus (AAV) platform, which by virtue of targeting peptides and specific promoters allow for cell- type specific expression of Meg3-gRNA. Cas12-transgenic pig hearts will be subjected to AAVs carrying Meg3- gRNA transduction to delete the MEG3 cluster postnatally. After hypertrophy-induction via percutaneous aortic reduction stent implantation, we will assess the role of Meg3 expression in cardiomyocytes, fibroblasts, endothelial cells and pericytes. We will further develop transgenic animals that allow targeting of non-coding RNAs more precisely in defined cell subtypes within the heart.