CRISPR/CasRx-mediated RNA knockdown targeting β-catenin and Ihh signaling alleviates osteoarthritis

Area of Science:

• Biotechnology
• Molecular Biology
• Orthopedics

Background:

• Osteoarthritis (OA) is a chronic, incurable joint disease.
• Aberrant Wnt/β-catenin and Indian hedgehog (Ihh) signaling pathways contribute to OA development and progression.
• Novel therapeutic strategies are needed to manage OA.

Purpose of the Study:

• To investigate the therapeutic potential of targeting β-catenin (encoded by Ctnnb1) and Ihh signaling (via Smo) using CRISPR/CasRx for OA treatment.
• To evaluate the efficacy and specificity of CRISPR/CasRx-mediated gene knockdown in vitro and in vivo.

Main Methods:

• Screening of CRISPR-derived RNAs (crRNAs) targeting Ctnnb1 and Smo in vitro.
• Validation of CasRx-mediated knockdown efficiency and specificity.
• Intra-articular injection of adeno-associated virus-delivered crRNAs into an OA mouse model.
• Assessment of OA progression using Micro-CT, histological, and histomorphometric analyses.

Main Results:

• CasRx-mediated knockdown of Ctnnb1 and Smo demonstrated high efficiency and specificity in vitro with no significant off-target effects.
• Intra-articular delivery of crRNAs via adeno-associated virus successfully reduced OA pathological damage in the mouse model.
• Targeting both β-catenin and Ihh signaling pathways decelerated joint degeneration in experimental OA.

Conclusions:

• CRISPR/CasRx-mediated knockdown of Ctnnb1 and Smo is a viable strategy for decelerating osteoarthritis progression.
• This approach offers a potential new therapeutic avenue for managing osteoarthritis.
• Further research into CasRx-mediated gene therapy for OA is warranted.