Background:
Cardiac fibrosis, a hallmark of heart failure, is driven by a complex interplay between cell types, primarily the activation of cardiac fibroblasts. Among the critical regulators of profibrotic gene expression is the signal integrator Serum Response Factor (SRF), juxtaposed at the intersection of multiple pathways as a transcriptional on switch. To dissect the distinct roles of SRF in different cell populations, a vector system capable of cell specific gene delivery is necessitated. Here we show simultaneous modulation SRF expression in cardiomyocytes (CM) and fibroblasts (FB) in a large animal model.
Methods:
We have previously described nanoparticle coating of AAV9 to enable endothelial cell transduction. Here, we use modularity of that system to retarget fibroblasts. AAV9 particles were generated encoding for dCasPhi-VPR under transcriptional regulation of pPOSTN along with complementary AAVs encoding U6-crRNAs targeting promoter, proximal enhancer and distal enhancer regions of SRF. These AAVs were coated with modified polyamidoamines targeting FBs, with the aim of cardiac FB overexpression of SRF (SRF-FB-OE). On the other hand, AAV9 particles encoding for SpCas9 sgRNAs against SRF and a DNA repair factor were coated with modified nanoparticles with CM-affine peptides, for Cas9 mediated knockout of SRF in CMs (SRF-CM-KO). Both vectors were locally administered via catheterization into the LAD of a Cas9 knock-in pig. Animal was sacrificed at four weeks post transduction and LV was systematically sampled, including tissue dissociation for gravitational enrichment of CMs and flow sorting for FBs.
Results:
Initial analyses revealed successful CM specific knockout of SRF with up to 40 percent. Interestingly, this genetic intervention was accompanied by a drop of EF (39±2% vs. 48±3% in untreated Cas9-tg pigs, n=3). In contrast, levels of SRF transcripts were increased by 9±1.7 fold in FBs, along with expected dCasPhi-VPR transcripts. Western blot of bulk tissue reveals mild increase in overall SRF protein. A precursory hyaluronidase assay hints at increased fibrosis, which is to be confirmed with spatial resolution provided via picrosirius red stain.
Conclusion:
Through the “dual modulation” of SRF demonstrated in this study, we present a platform for investigation of cell specific effects of pleiotropic factors in tissue remodeling via utilization of multiple assets developed in our lab.
