Cutting-free application of CRISPR-mediated endogenous gene activation in human induced pluripotent stem cell derived cardiomyocytes and engineered human myocardium

Background Imbalanced transcriptional networks characterize cardiomyocyte stress and result in cardiac remodelling. We hypothesize that re-establishing homeostatic gene networks in cardiomyocytes will prevent further tissue damage. To tackle this challenge, we applied CRISPR-based endogenous gene activation (CRISPRa) in vivo and in vitro. Methods We employ precision transcriptome editing tools based on CRISPR/Cas9 with enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) to induce target gene expression by directing dCas9VPR to promoter regions by guide RNAs (gRNA). Results Homozygous CRISPRa hiPSC cell lines were generated by targeted integration of a CAG promoter driven dCas9VPR-T2A-tdTomato expression cassette into the AAVS1 locus by CRISPR/Cas9 editing and homology directed repair. Expression of dCas9VPR was evaluated by immunoblotting and co-expressed reporter fluorescence in spontaneously beating hiPSC-CM. We previously identified a crosstalk between WNT signalling and Krueppel-like factor 15 (KLF15) necessary for controlling cardiac homeostasis. We designed and tested 8 non-overlapping gRNAs in the –400 bp region upstream of the KLF15 transcriptional start site (TSS) and tested individual gRNA effectiveness for gene activation in HEK293T cells. Five gRNAs were identified inducing KLF15 transcript levels between 2- and 5-fold compared to non-targeted (NT) gRNA transfected cells (n=3 experiments). The single most effective gRNA was transduced by lentiviral particles into CRISPRa hiPSC-CM increasing KLF15 transcript levels to 1.5-fold compared to NT-gRNA control. Synergistic effects of 3 instead of single gRNA increased KLF15 transcript levels by 3-fold compared to controls (n≥3 experiments). We hypothesized that dCas9VPR expression could be harnessed as an additional option for gene dose titration and we generated hiPSC lines with enhanced dCas9VPR expression (v2.0). We observed up to 5-fold KLF15 gene activation when triple gRNA and v2.0 were combined (n≥4 experiments). Engineered human myocardium (EHM) was generated consisting of CRISPRa cardiomyocytes, fibroblasts and collagen and we observed similar contractility in 4-week cultured EHM suggesting innocuous dCas9VPR and gRNA expression. CRISPRa component expression was maintained over the entire culture period as evaluated by dCas9VPR immunoblotting and KLF15 transcriptional activation (1.4 fold, v1.0 CRISPRa hiPSC-CM, n≥8 tissues) indicating sustained gene activition. Conclusions Targeted gene activation with CRISPR/Cas9 is a precise and effective tool for transcriptional activation in hiPSC-CM. We observed titratability of gene activation by 1.) dCas9VPR expression levels and 2.) single versus multiple gRNA use. We furthermore elucidated general rules for effective gRNA targeting within the 5' TSS of genes of interest which confirmed a dependency of baseline gene activity as a limiting factor for endogenous gene activation. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG) - Collaborative Research Center 1002German Center for Cardiovascular Research (DZHK)

Abstract P494: Truncated Titin Protein In Dilated Cardiomyopathy

Truncating variations in the gene coding for titin (TTNtv) have been known to cause dilated cardiomyopathy for nearly 20 years. Efforts to detect direct evidence of either haploinsufficiency or dominant negative mechanisms have thus far failed, leaving the mechanism open to controversy. By analyzing a collection of 184 post-transplant human hearts, 22 of which bear TTNtv’s, we show evidence supporting both haploinsufficient (lack of sufficient full length titin to maintain normal cardiomyocyte contractility) and dominant-negative (toxic gain of function due to truncated titin) mechanisms. Using allele specific proteomics as well as epitope specific agarose gel immunoblotting we show that TTNtv are present in human myocardium at the expected molecular weight and bear only the epitopes expected to be present in TTNtv protein. TTNtv associate with the sarcomere bearing insoluble fraction of human myocardium but are more weakly attached to the sarcomere than full length titin, consistent with their lack of thick filament and M-line attachment sites. We further show that DCM hearts bearing TTNtv have less full length titin than non-TTNtv bearing DCM hearts, by both total protein and in ratio to sarcomeric proteins, indicating TTN haploinsufficiency is also present in TTNtv hearts. This unambiguous detection of TTNtv protein in the myocardium of DCM combined with a reduction in full length titin supports a combined dominant negative and haploinsufficient mechanism of pathogenesis of TTNtv induced DCM.

Multicellular Human Cardiac Organoids Transcriptomically Model Distinct Tissue-Level Features of Adult Myocardium

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for disease modeling and drug cardiotoxicity screening. To this end, we recently developed human cardiac organoids (hCOs) for modeling human myocardium. Here, we perform a transcriptomic analysis of various in vitro hiPSC-CM platforms (2D iPSC-CM, 3D iPSC-CM and hCOs) to deduce the strengths and limitations of these in vitro models. We further compared iPSC-CM models to human myocardium samples. Our data show that the 3D in vitro environment of 3D hiPSC-CMs and hCOs stimulates the expression of genes associated with tissue formation. The hCOs demonstrated diverse physiologically relevant cellular functions compared to the hiPSC-CM only models. Including other cardiac cell types within hCOs led to more transcriptomic similarities to adult myocardium. hCOs lack matured cardiomyocytes and immune cells, which limits a complete replication of human adult myocardium. In conclusion, 3D hCOs are transcriptomically similar to myocardium, and future developments of engineered 3D cardiac models would benefit from diversifying cell populations, especially immune cells.