Targeting autophagy for the treatment of osteoarthritis - Phase I: CRISPR-cas9 screen

Active: 2019.10.01 - 2020.12.30
UK SPINE Scientific Liaison: Monica Spisar

Autophagy is the main cellular bulk degradation pathway. Biological aging and specific diseases such as osteoarthritis are characterized in part by loss of autophagic efficacy.

TFEB is a master regulator of autophagy and lysosomal biogenesis. The researchers have previously identified a novel signalling pathway for inducing autophagy via post-translational donation of spermidine’s aminobutyl moiety to a specific lysine on eIF5A, known to regulate TFEB. Spermidine, however, is not a good candidate for development as an FDA approved therapeutic as it is too pleiotropic. This work aims to identify alternate targets, including in the eIF5A/TFEB pathway, for modulating autophagy via regulation of TFEB expression. Such targets will be identified via a genome wide pooled CRISPR screen, using an osteoarthritic model; top hits will be validated with in vitro assays.

The single plasmid system TKO V3 genome wide library targeting 18,053 protein-coding genes will be employed to transduce fibroblasts from OA patients with 500 sgRNAs/target to maintain library complexity throughout the screen. Post antibiotic selection, cells will be pooled for TFEB intracellular staining. The specificity of the staining has been validated using TFEB knock out cells. TFEBhi cells will be flow sorted and deep sequenced to identify the enriched genes in this population. Following the primary screen, hits will be reconfirmed in a repeat experiment under similar conditions.

The data from the screening activities will be fed into TargetHunter bioinformatics platform for annotation, and the top five hits will be validated in vitro in chondrocytes and fibroblasts of young, healthy old, and osteoarthritic donors via knock out and subsequent staining for TFEB by flow cytometry or lysed for TFEB, eIF5A hypusination and LC3 western blot. Effect on cartilage markers will be assessed by RT-qPCR and WB, and cytokine secretion detected by qPCR and ELISA. Post-validation, these hits will merit continued translational efforts to advance toward therapeutic candidates and funds will be sought to conduct a drug screen informed by the outputs of this work.