Development of conditional-siRNA programmable riboswitch for targeting adverse cardiac remodeling

Abstract

Heart Failure (HF) remains a global epidemic and a significant healthcare burden, with an unmet need for novel therapies to target the preceding pathological hypertrophy in vulnerable patients. Here we report the development of novel conditional-siRNA (Cond- siRNA) constructs that are selectively activated by disease-specific RNA biomarkers to enable cell-specific inhibition of a target disease-causing RNA. We designed a Cond- siRNA that can be activated by nppa mRNA, upregulated specifically in CMs under pathological stress, to silence the key pro-hypertrophic gene calcineurin by the effector siRNA. In cellular models including neonatal rat ventricular myocyte (NRVM) and rat cardiomyocyte cell-line (H9C2), Cond-siRNA exhibited low baseline activity in the absence of the disease biomarker but achieved targeted calcineurin silencing upon nppa mRNA induction by phenylephrine (PE)-induced stress in a two-dimensional (2D) cell culture system and pressure overload in three-dimensional (3D) heart-on a chip system. NRVM transfection with the Cond-siRNA resulted in a decreased expression of calcineurin mRNA specifically after PE or pressure-overload treatment, but not after vehicle treatment, proving nppa mRNA-specific activation of the effector siRNA against calcineurin. Specificity was confirmed as Cond-siRNA did not significantly silence calcineurin in cardiac fibroblasts and T cells, lacking nppa expression. Reduced calcineurin protein levels and NFATc1 nuclear translocation correlated with decreased NRVM hypertrophy after PE treatment, confirming Cond-siRNAs efficacy. This study offers proof-of-concept for Cond-siRNA as a targeted therapy to mitigate hypertrophic progression, paving the way for novel HF treatments. One sentence summaryConditional-siRNA targeting adverse cardiac remodeling GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/633434v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1b6532borg.highwire.dtl.DTLVardef@10e3836org.highwire.dtl.DTLVardef@8c839eorg.highwire.dtl.DTLVardef@1152883_HPS_FORMAT_FIGEXP M_FIG C_FIG