Description:
<jats:p>
Transcriptional changes in cardiomyocytes drive heart failure progression, however, precise control over endogenous gene expression remains challenging. The expression of Krueppel-like factor 15 (
<jats:italic>KLF15</jats:italic>
), an evolutionary conserved nuclear and cardiomyocyte specific inhibitor of WNT/CTNNB1 signalling in the heart, is lost upon cardiac remodelling, and accompanied by aberrantly active WNT/CTNNB1 resulting in heart failure progression. We investigated
<jats:italic>KLF15</jats:italic>
expression dynamics employing CRISPR/Cas9-based tools in mouse cardiomyocytes in vivo and in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) under the hypothesis that re-establishment of
<jats:italic>KLF15</jats:italic>
levels in myocardial stress conditions prevents heart failure progression. Using a mouse model expressing enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) under
<jats:italic>Myh6</jats:italic>
-promoter control, we activated
<jats:italic>Klf15</jats:italic>
in a murine pressure overload model by transverse aortic constriction. Delivery of Klf15 gRNAs targeted to the
<jats:italic>Klf15</jats:italic>
promoter region via AAV9 induced
<jats:italic>Klf15</jats:italic>
expression sufficiently to re-normalize
<jats:italic>Klf15</jats:italic>
expression to transcript levels comparable to sham surgery hearts. This was accompanied by reduced decrease of fractional shortening as well as reduced cardiomyocyte hypertrophy in stressed
<jats:italic>Klf15</jats:italic>
re-activated hearts compared to non-trageted (NT) gRNA hearts (n=3-8 per group, echo data from 4 and 8 weeks post-surgery). We achieved titratable
<jats:italic>KLF15</jats:italic>
activation in dCas9VPR transgenic hiPSC-CM by selection of single and multiple gRNAs (n=3-4 replicates) and used these cells to generate human engineered myocardium by combining hiPSC-CM and fibroblasts which we subjected to isometric contractions in order to induce mechanical stress, which resulted in KLF15 expressional decrease in line with our
<jats:italic>in vivo</jats:italic>
data. This transcriptional loss was rescued in CRISPR/dCas9VPR hiPSC-CM targeted to the
<jats:italic>KLF15</jats:italic>
locus compared to controls (n=6-9/2/4 tissues per group/casting sessions/differentiations). Additionally, TGFB1 induced cardiomyocyte stress resulted in decreased
<jats:italic>KLF15</jats:italic>
expression levels in 2D hiPSC-CM cultures which were rescued by dCas9VPR-
<jats:italic>KLF15</jats:italic>
targeting (n=3 experiments). In conclusion, we report controllable gene activity by CRISPR/dCas9VPR to restore the loss of
<jats:italic>KLF15</jats:italic>
in stressed mouse and human cardiomyocytes. We furthermore evaluate the potential to gain full control over gene dose titratability with these models to validate and define novel therapeutic targets for the prevention of heart failure progression.
</jats:p>