RNAi BASED APPROACH FOR MYOC-ASSOCIATED GLAUCOMA

Abstract

Purpose : Mutations in Myocilin (MYOC) are responsible for the most common genetic cause of glaucoma, accounting for 8-10% of autosomal dominant familial Juvenile Open Angle Glaucoma cases, as well as 2-3% of POAG cases. MYOC toxic gain of function mutant proteins aggregate intracellularly, leading to trabecular meshwork (TM) stress, elevated IOP, and glaucoma. In this study, we tested the hypothesis that silencing expression of disease-causing MYOC variants could lead to decreased TM stress and lowered IOP.

Methods : First, we made humanized MYOC mice to screen different siRNA duplexes in vivo. Next, we developed humanized MYOC.Y437H mice over-expressing human mutant MYOC using a CRISPRa approach (SAM-MYOC mice). We also validated the model using IOP lowering drugs (timolol and Rhopressa). We then tested several lead MYOC siRNA duplexes in the SAM-MYOC model to assess the IOP lowering efficacy. In addition, we also used ex vivo human eye perfusion culture model to screen this lead siRNA duplexes

Results : First, we found the several best siRNAs duplexes to lower both MYOC mRNA and protein. To develop animal model, out of several CRISPR guides delivered via AAV or Lentivirus in mouse eye, one guide led to significant upregulation (15-20-fold) of mutant MYOC and high IOP (5-6 mmHg from baseline). We showed that both timolol and Rhopressa lowered IOP in these mice, further validating the model. Next, 5 weeks after virally inducing high IOP in SAM-MYOC, we injected either control or MYOC siRNA intravitreally. While the control siRNA had no effect on IOP, in the MYOC siRNA group IOP was lowered to baseline within a week and this effect persisted for more than 5.5 months, when IOP elevated again gradually. Second, we used the ex vivo human eye perfusion culture system to explore the efficacy of MYOC siRNA to knockdown MYOC mRNA and protein in the TM and MYOC protein levels in the perfusate. Over 90% knockdown of both RNA and protein was achieved.

Conclusions : These results suggest that siRNA based knockdown of MYOC is a feasible therapeutic approach to treat human MYOC based glaucoma.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.