CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials

Abstract

Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 KO mouse model. In humans, CAPN5 loss-of-function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.

Calpains as mechanistic drivers and therapeutic targets for ocular disease

“…In a proof-of-principle study, a CAPN5-specific, short hairpin RNA post-transcriptionally silenced target gene expression, effectively reduced CAPN5 expression in neuroblastoma SH-SY5Y cells [97], suggesting that a gene-silencing strategy could target CAPN5 specifically. Notably, the eyes of CAPN5 KO mice appear to function normally, as evaluated by retina histology and ERG [56], supporting the concept that eliminating CAPN5 expression in the eye would pose little risk for offtarget effects. To overcome gene-delivery hurdles (e.g., large molecular size, immense negative charge, and poor enzymatic stability), genetic material is often delivered by engineered nonviral or viral vectors.…”

“…Nevertheless, concern remains regarding off-target effects. Even with intraocular drug delivery, calpain inhibitors may need to be highly specific to avoid off-target inhibition of other proteases (e.g., cathepsins) [52][53][54][55][56]. Prior to clinical application, fine-tuning strategies to selectively target pathologic calpain activity while sparing normal calpain activity in healthy cells is possible.…”

Molecular Surgery: Proteomics of a Rare Genetic Disease Gives Insight into Common Causes of Blindness

“…Since the discovery of the first NIV-causing mutations, efforts have been taken to understand the structure, function, targets, and downstream signaling effectors of the CAPN5 protease ( Bassuk et al., 2015 ; Gakhar et al., 2016 ; Mahajan et al., 2012 ; Schaefer et al., 2016 ; Wert et al., 2014 , 2015 , 2019 ). Our group solved the structure of the CAPN5 protease core domain (termed CAPN5-PC), a finding that furthers the study of NIV disease mechanisms ( Velez et al., 2020 ).…”