Purpose: We recently showed that the transcriptional co-factor Yesassociated protein (Yap) is a key modulator of function in mesenchymal stromal/stem cells (MSCs), and is upregulated in the hyperplastic synovium of mice in response to cartilage injury and of patients with intra-articular fracture or OA. We also demonstrated that ablation of Yap in Gdf5-lineage cells, which include fibroblast-like synoviocytes in the adult synovial lining, was sufficient to prevent synovial lining
hyperplasia in an articular cartilage injury mouse model in vivo. The aims of this study were to investigate the in vitro effects of Yap depletion and to develop RNA interference (RNAi) systems to achieve Yap silencing in synovial MSCs, with the ultimate goal of intra-articular therapeutic delivery.
Methods: To investigate the in vitro effects of Yap knockout (KO) on proliferation, synovial MSCs were isolated from the knee joints of Yapfl/fl mice additionally carrying a Cre-inducible TdTomato (Tom) fluorescent reporter (Yapfl/fl;Tom mice), or Tom-only mice as controls, and transduced with Cre-encoding lentivirus. The percentage of Tom-positive cells was analysed by flow cytometry and Yap expression levels were determined by quantitative RT-PCR (qPCR). For cell proliferation studies, the percentage of cells incorporating EdU into newly synthetized DNA was quantified by flow cytometry. Two Yap knockdown RNAi strategies, based on the use of 20 -O methylated DsiRNA/chitosan nanoparticles or Gapmers, were used. DsiRNA/chitosan nanoparticles, made at different ratios, were transfected in mouse and human synovial MSCs. Cytotoxicity and Yap expression were assessed by Alamar Blue assay and qPCR, respectively. Cellular internalisation of Cy5-labeled DsiRNA/chitosan nanoparticles was analysed by confocal microscopy.
Gapmers were transfected at different concentrations and incubation times either naked or in combination with Lipofectamine or TransITTKO in mouse primary synovial MSCs. Yap mRNA silencing and cellular uptake of Cy5-labeled Gapmers were assessed by qPCR and confocal
Results: An in vitro Yap knockout system was generated transducing Yapfl/fl;Tom synovial MSCs with a lentivirus-Cre, leading to Tom expression in 88 ± 7% of cells, and Yap mRNA reduction of 93 ± 4% compared with the untransduced control. Accordingly, there was a reduced EdU incorporation in Cre-transduced Yapfl/fl; Tom cells compared to untransduced control and Cre-transduced Tom cells. For the development of Yap RNAi strategies, synovial MSCs were transfected with DsiRNA/chitosan nanoparticles, showing no cytotoxic effects and leading to a 55 ± 6% and 74 ± 11% decrease in Yap mRNA levels in mouse and human synovial MSCs, respectively. Internalisation of Cy5-labeled
DsiRNA/chitosan nanoparticles into cells was confirmed. In parallel, of the three Yap Gapmer sequences designed, two resulted in Yap mRNA decreases of 77 ± 2% and 86 ± 8% when transfected with Lipofectamine or TransIT-TKO, respectively. The efficiency of naked transfection of
Gapmers was investigated and, despite the confirmation of cellular internalisation of Cy5-labeled Gapmers, Yap mRNA reduction was not achieved in any of the tested conditions.
Conclusions: These findings support Yap as a potential therapeutic target for pathological synovial hyperplasia. For Yap targeting, chitosan/ DsiRNA nanoparticles proved to be a safe and effective RNAi system in primary synovial MSCs in vitro. A further screening of new Gapmer formulations would be required. However, Gapmers could be used in combination with biocompatible delivery methods used for siRNA, such us chitosan nanoparticles.