Background/Purpose: Long non-coding RNAs (lncRNAs) participate in the rheumatoid arthritis (RA) pathogenesis. The aim of this study was to examine the lncRNA H19 and HOTAIR expression in RA patients, and whether delivery of CRISPR interference (CRISPRi) with catalytically dead Cas9 (dCas9) targeting H19 can ameliorate the experimental rheumatoid joint.

Methods: Synovial tissue (ST) and venous blood samples were obtained from RA and osteoarthritis (OA) patients. Synovial fibroblasts (SFs) were purified from ST, and normal SFs were acquired commercially. To generate CRISPRi-H19 and CRISPRi-HOTAIR, guide RNA spacer sequences targeting H19 or HOTAIR were cloned into the CRISPRi vector containing the dCas9/Krüppel-associated box repressor domain with an efficient transcription repression ability. SFs and 293T cells were transduced with CRISPRi-H19 and CRISPRi-HOTAIR, respectively, to create stable transfectants. Therapeutic effects of intra-articular CRISPRi-H19 injection were assessed in a rat model of collagen-induced arthritis. LncRNAs expression were examined by quantitative real-time PCR. Cell invasion was assayed by Transwells with membrane coated with Matrigel.

Results: Increased synovial and peripheral H19 levels were found in RA patients with decreased H19 expression in venous mononuclear cells (MNCs) after a TNF  blockade (adalimumab) therapy as compared with OA counterparts. There were higher HOTAIR levels in MNCs from RA patients, but no differences after the anti-TNF therapy. H19 levels in SFs were enhanced by the TNF stimulation. Reduced H19, pGSK-3β (Wnt signaling), EZH2 and Snail expression, cell invasion and IL-6 production with increased Nkd1 (Wnt inhibitor) expression were identified in H19-silenced SF transfectants. Decreased Tri-Methyl-H₃K₂₇ expression were found in HOTAIR-silenced 293T transfectants. Lower arthritis indexes and histological scores with less erosion on cartilage/bone were demonstrated in the CRISPRi-H19-injected joints with enhanced NKd1 and decreased H19, pGSK-3β, EZH2, Snail and IL-6 expression, suggesting silencing H19 to ameliorate experimental rheumatoid joint through inactivating the Wnt signaling.

Conclusion: Our results demonstrate that intra-articular delivery of CRISPRi targeting lncRNA can alleviate the experimental rheumatoid joint. Such findings might contribute to the development of lncRNAs-related therapeutics in RA patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/experimental-rheumatoid-joint-ameliorated-by-crispr-interference-targeting-long-non-coding-rna-h19-through-wnt-signaling-inactivation/