Human primary 2D culture as a tool to explore JAK1 pathway inhibition in the intestinal epithelium using siRNA technology

Abstract Background Oral inhibitors of JAK1 have become promising therapeutic agents for the treatment of inflammatory bowel diseases (IBD); however, concerns have been raised regarding their specificity and safety profiles. Currently, a local therapy based on specific JAK1 siRNA combined with lipid nanoparticle (LNP) technology is under investigation as a safer alternative to JAK inhibitors.The purpose of this study is to explore the inhibition of the JAK1 pathway in the intestinal epithelium mediated by siRNA/LNP technology, using human primary 2D culture. Methods Human primary 2D cultures were generated from colonic 3D organoids of non-IBD donors. The efficiency of JAK1 pathway inhibition was tested in IFN-y stimulated cultures using either filgotinib (a JAK1 inhibitor, used as a control) or the novel human JAK1 siRNA. JAK1 siRNA transfection was performed using Lipofectamine or LNPs. qPCR was performed on a panel of JAK1 target genes to evaluate the efficiency of JAK1 pathway inhibition. Results Incubation of the 2D culture with IFN-y induced the activation of the JAK1 pathway, as suggested by the significant up-regulation of JAK1-dependent genes (i.e., CXCL10, SOCS1, SOCS3 and PLA2G2A). The addition of filgotinib to the culture efficiently inhibited the JAK1 pathway by suppressing the expression of JAK1-target genes. JAK1 siRNA transfection using Lipofectamine reduced JAK1 mRNA expression by 50%, which was mirrored by the concomitant down-regulation (between 60 and 80%) of JAK1-dependent genes. Importantly, the silencing efficiency of the JAK1-dependent pathway by LNPs was comparable to that observed using Lipofectamine. Conclusion Organoid-derived 2D culture is a useful model for investigating the activation of the JAK1 pathway and its pharmacological inhibition in human intestinal epithelium. In particular, siRNA/LNP nanoplexes may be a promising technology for locally delivering highly specific siRNAs to the intestinal mucosa, which could pave the way for the development of more effective treatments for IBD patients.