Rational design of hammerhead ribozymes as CD93 silencing tools for vascular diseases

While targeting angiogenesis represents a key therapeutic strategy in several pathological contexts, it comes with significant challenges associated with therapeutic efficacy and drug resistance. Therefore, developing novel, more effective and durable therapeutic modalities is paramount. We have previously reported that antibody-mediated inhibition of CD93, a vascular endothelial cell surface glycoprotein, can inhibit angiogenesis. Here, we describe a novel strategy to efficiently inhibit CD93 expression in cells, based on the targeted degradation of CD93 mRNA using trans-acting hammerhead ribozymes. To pinpoint single-stranded regions in CD93 mRNA that are amenable to ribozyme targeting in living cells, we performed RNA secondary structure mapping via targeted DMS-MaPseq analysis. Next, since exogenous hammerhead ribozymes are easily degraded and lose catalytic activity when expressed in living cells, we developed a novel scaffold RNA based on short stems from the 3' UTR of histone mRNAs to stabilize the active ribozyme structure and promote the CD93 cleavage under physiological conditions. Ectopic expression of these engineered ribozymes in primary endothelial cells resulted in efficient inhibition of CD93 expression, cell migration, and formation of tube-like structures in functional assays. Collectively, our data provides the proof-of-concept for the use of ribozyme-based therapeutics for the treatment of neovascular pathologies.