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P034 Human primary 2D culture as a tool to explore JAK1 pathway inhibition in the intestinal epithelium using siRNA technology

Dotti, I.(1);Sulpice, E.(2);Nougarède, A.(3);Jary, D.(3);Clément, F.(2);Gidrol, X.(2);Navarro, F.(3);Salas, A.(1);

(1)IDIBAPS, Department of Gastroenterology, Barcelona, Spain;(2)CEA-INSERM, Biomics, Grenoble, France;(3)CEA-Leti, Division for biology and healthcare technologies, Grenoble, France

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.

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