1:Centro de Investigacion Medica Aplicada (CIMA); 2:Universidad Francisco de Vitoria

CRISPR based technologies have offered novel opportunities for the development of new therapies that could overcome the traditional way to treat inherited monogenic diseases. Primary hyperoxaluria type 1 (PH1) is a rare inborn disorder where a mutated form of alanine glyoxylate aminotransferase (AGT) presents a reduction in its enzymatic activity that affects glyoxylate metabolism. AGT deficiency results in excessive hepatic oxalate production that accumulates in the kidney forming CaOx stones that lead to end-stage renal disease. A substrate reduction therapy (SRT) based on CRISPR-Cas9 editing of Hao1 gene, results in the reduction of glycolate oxidase (GO) expression, has been previously shown to be an attractive alternative therapy for PH1. However, potential side effects associated to off target effects remains a concern for this type of therapeutic approaches.

Here we have explored the use of CRISPR/CasRx, a programmable RNA-guided, RNA-targeting CRISPR system with nuclease activity that allows target gene knockdown without altering the genome. First, in vitro studies were performed to select the most efficient guide-RNA (gRNA) targeting GO mRNA. We showed that CRISPR/CasRx system resulted in the reduction of the expression of the targeted protein, although simultaneous reduction of non-targeted exogenous protein was observed in mammalian cells, while endogenous controls remained unaltered. Lastly, the most efficient gRNAs that demonstrated lower collateral cleavage will be tested in a PH1 mouse model using an adenoassociated vector serotype 8 (AAV8) carrying  CasRx and single or multiple targeting guides.

In summary, CasRx represents a very attractive system to reduce protein expression without altering the cellular genome.