Abstract P1145: All-in-one AAV CRISPR/dCas9VPR Vectors Enable Endogenous Gene Activation In Cardiomyocytes

The transcriptional landscape of cardiomyocytes harbors the cellular adaptation upon stress, but remains out of scope for therapies preventing heart failure progression. CRISPR/Cas9 mediated endogenous gene activation (CRISPRa) using enzymatically inactive Cas9 (dCas9) fused to transcription activation domains (VPR) allows for precise and fine-tuned gene activity control when targeted to promoter or enhancer regions of interest via guide RNAs (gRNAs). Transgenic mouse models and human induced pluripotent stem cell (hiPSC) lines were established for proof-of-concept (PoC) gene activation studies in cardiomyocytes. However, the size of the commonly used Streptococcus pyogenes Cas9 exceeds the packaging limits of gene therapy relevant adeno-associated virus (AAV), hampering the therapeutic use of the technology. We aimed to engineer AAV vectors based on smaller, enzymatically inactive Cas9 variants fused to a minimalized VPR-transactivation domain co-expressing a single gRNA for all-in-one gene activation strategies. We rendered mini-Cas9s enzymatically inactive by mutating the evolutionary conserved RUVC and HNH regions and C-terminally attached a minimal VPR domain as well as a HA-tag. As PoC, we expressed mini-dCas9VPR under the CMV promoter in HEK293T cells and confirmed expression of the Cas9 variants via immunoblotting. When we targeted mini-dCas9VPR to the anti-hypertrophic transcription factor KLF15 promoter region or the PoC target POU5F1 promoter, we could confirm significant gene activation compared to non-transfected and non-targeted gRNA controls (KLF15: 3-fold change, POU5F1: 4-fold change, n ≤ 4). We next exchanged the CMV promoter by the TNNT2 promoter to allow cardiomyocyte-specific expression of mini-dCas9VPR. Upon targeting the KLF15 promoter with these vectors transfected in hiPSC-cardiomyocytes, we observed significantly elevated KLF15 mRNA levels compared to controls (1.5-fold change, n ≤ 4) and robust expression of the mini-CRISPRa system, respectively. In conclusion, we present novel vectors for AAV mediated cardiomyocyte-specific endogenous gene regulation which are currently tested in hiPSC-based models, patient-derived cardiac slices and for in vivo gene activation.