Technical Review: Circular RNA-based protein replacement therapy mitigates osteoarthritis in male mice

Authors: Jinlong Suo, Ling Li, Wuyuan Tan, Xubin Yin, Jinghui Wang, Rui Shao, Shaokun Sun, Si-Kun Guo, Jingyi Feng, Bao-Qing Gao, Ying Wang, Meng-Yuan Wei, Lijun Wang, Heng Feng, Xiang Gao, Ping Hu, Xianyou Zheng, Ling-Ling Chen, Guanghua Lei, Youkui Huang, and Weiguo Zou

Affiliations: Shanghai Jiao Tong University, Shanghai, China

Background Introduction

Osteoarthritis (OA) is the most prevalent degenerative joint disorder worldwide, characterized by progressive cartilage destruction and the absence of disease-modifying treatments. While mRNA therapeutics have shown remarkable success, particularly in COVID-19 vaccines, they face challenges including limited stability, transient expression, immunogenicity, and potential off-target effects. Circular RNAs offer distinct advantages over linear mRNAs, including superior stability, reduced immunogenicity, and enhanced safety profiles due to their covalently closed loop structure that confers resistance to nuclease degradation.

This research addresses a critical gap in understanding how RNA-binding proteins (RBPs) contribute to osteoarthritis pathogenesis and explores the therapeutic potential of protein replacement therapy using innovative circular RNA delivery systems. This offers new perspectives on treating degenerative joint diseases through precision molecular interventions.

Fig 1. Schematic illustration of intra-articular injection of ivcRNA-LNPs encoding SOX5 to treatment DMM mice. ivcRNA-based therapy treated 10-week-old mice of sham and DMM. Then, the therapeutic phenotypes of OA were evaluated in sham and DMM mice after 7-week-treatment by micro-CT, SO&FG staining and immunohistochemistry.

Materials and Methodology

The researchers first conducted RNA-sequencing analysis to identify differentially expressed RBPs in osteoarthritis progression, where Musashi2 (MSI2) in articular chondrocytes was identified as the primary candidate. In vitro-transcribed circular RNA (ivcRNA) was then optimized for translation efficiency and encapsulated in lipid nanoparticles (LNPs). After initial in vitro testing with chondrocytes, an in vivo study was conducted on a murine model with intra-articular injection of RNA-LNPs into mice affected with experimental osteoarthritis.

Results

MSI2 Identification and Validation

The systematic screening revealed MSI2 as significantly downregulated in both mouse OA models and human OA cartilage samples, with expression levels correlating inversely with disease severity. Functional validation demonstrated that MSI2 deficiency in chondrocytes led to decreased expression of anabolic markers (aggrecan, COL2) and increased catabolic markers (MMP13, ADAMTS5). These findings establish MSI2 as a critical regulator of chondrocyte homeostasis and a promising therapeutic target, representing the first identification of this RNA-binding protein's role in osteoarthritis pathogenesis.

Circular RNA Platform Development

The researchers successfully developed a highly efficient ivcRNA synthesis platform, achieving circularization efficiencies exceeding 80% through systematic IRES optimization. Comparative analysis demonstrated that ivcRNAs exhibited superior stability and prolonged protein expression compared to linear mRNAs, with therapeutic proteins detectable for up to five days versus three days for conventional mRNA. This represents a significant technical advancement in RNA therapeutic design, demonstrating that circular architecture can substantially extend therapeutic protein expression duration.

Therapeutic Efficacy Studies

In the DMM osteoarthritis model, intra-articular delivery of ivcRNA encoding MSI2 significantly reduced cartilage degradation, decreased OARSI histopathological scores, and prevented subchondral bone thickening compared to controls. Micro-CT analysis revealed marked reduction in meniscal calcification and osteophyte formation. Importantly, therapeutic supplementation of SOX5, identified as a downstream effector of MSI2, achieved similar protective effects, validating the mechanistic pathway. These results demonstrate that ivcRNA-based protein replacement can effectively modify disease progression in a clinically relevant osteoarthritis model, achieving therapeutic outcomes superior to conventional mRNA approaches.

Fig 2. Schematic representation of ivcRNA-based therapy compensating SOX5, a downstream target of MSI2, to alleviate the phenotypes of OA in DMM mice. MSI2 can bind to the 3’UTR of Sox5 transcripts to promote SOX5 translation, and DMM surgery resulted in a significant decrease in SOX5 protein expression. Injecting ivcRNA-LNPs into the joint cavity ofmice after DMM surgery to effectively express SOX5 protein in chondrocytes.

Safety and Delivery Optimization

The SM-102 LNP formulation achieved joint-specific targeting with minimal systemic distribution, as confirmed by bioluminescence imaging and fluorescent tracking studies. Immunogenicity assessment revealed significantly lower inflammatory responses compared to linear mRNA, supporting the enhanced safety profile of circular RNA constructs. The therapeutic dosing (100-300 ng per injection) was substantially lower than previously reported mRNA therapies for OA (5-50 µg), indicating improved therapeutic efficiency. This optimization demonstrates the potential for safer, more targeted RNA therapeutic delivery with reduced off-target effects and lower immunogenicity risks.

Conclusion

Suo and colleagues established circular RNA-based protein replacement therapy as a potentially transformative approach for osteoarthritis treatment. The identification of MSI2 deficiency as a key pathogenic mechanism opens new therapeutic avenues, while the innovative ivcRNA delivery platform addresses fundamental limitations of current RNA therapeutics. The superior stability, reduced immunogenicity, and enhanced targeting achieved through this approach represent significant advances in the field of molecular medicine.

This paper utilized the PreciGenome NanoGenerator Flex-M for LNP synthesis. Uniform synthesis conditions are essential for quality RNA-LNPs, and the pressure-based microfluidics used by this instrument are optimized for production on the pre-clinical scale.

For further details, check out the link to the article here:

https://www.nature.com/articles/s41467-025-63343-z