dCas9-KRAB 매개 Runx2의 억제를 통한 골관절염 유전자치료 전략 = dCas9-KRAB Mediated Repression of Runx2 as a Gene Therapy Strategy for Osteoarthritis

다국어 초록 (Multilingual Abstract)

Osteoarthritis (OA), also known as degenerative joint disease (DJD), is a chronic, degenerative disease in various joints. The pathogenesis of OA involves changes in all tissues of the synovial joint. Current therapeutic interventions for osteoarthritis (OA) are primarily focused on symptomatic relief, particularly pain management, rather than targeting the underlying mechanisms for cartilage degradation. This limitation stems from an incomplete understanding of the precise pathophysiology involved in OA. The absence of disease-modifying treatments highlights the need for innovative approaches that specifically target the molecular pathways driving cartilage degeneration.
The present study is predicated on the hypothesis that the transcription factor Runx2, a pivotal regulator of cartilage catabolism during endochondral ossification, is aberrantly reactivated in OA, thereby accelerating joint deterioration. To safely and persistently repress the pathological expression of Runx2, a targeted-gene therapy approach using the dCas9-KRAB system is developed. This system utilizes a catalytically inactive dCas9 fused to the Krüppel-associated box (KRAB) transcriptional repression domain. For efficient and stable delivery of the dCas9-KRAB system, recombinant lentiviral vector system is applied. Recombinant lentiviruses are advantageous because they are able to transduce both dividing and non-dividing cells. Also they can carry a large-sized cargo insert, and ensure long-term, stable gene expression through genomic integration. Additionally they are known for its low immunogenicity. These features make lentiviral vector system suitable for in vivo delivery.
This study demonstrates that the dCas9-KRAB system effectively repressed Runx2 gene expression in murine primary chondrocytes. This targeted repression led to a subsequent upregulation of the genes for cartilage matrix substances, and a significant decrease in the expression of key catabolic enzymes, such as MMPs and ADAMTs. These molecular changes translated to a cellular-level effect, successfully exhibiting increased level of type II collagen matrix.
In conclusion, this study validates Runx2 as a key driver of cartilage degeneration in OA and provides compelling evidence that its regulation using the dCas9-KRAB system can be an effective therapeutic strategy. These findings may contribute to developing a novel, disease-modifying targeted-gene therapy that has the potential to halt or even reverse the progression of OA.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 6
• 2-1. Primary murine chondrocyte isolation and cell culture 6
• 2-2. Production of guide RNAs 7
• 2-3. Transfection of sgRNA/dCas9-KRAB by Lipofection 7
• 2-4. Lentiviral vector construction and virus production 7
• 2-5. Lentiviral transduction of chondrocytes 8
• 2-6. Inflammatory stimulation 8
• 2-7. Quantitative real-time PCR 9
• 2-8. SDS-PAGE and Immunoblot analysis 9
• 2-9. Safranin O staining and ECM analysis 10
• 2-10. Immunocytochemistry, Immunofluorescence 10
• 3. RESULTS 12
• 3-1. Design and validation of sgRNAs targeting Runx2 12
• 3-2. Lentiviral delivery of CRISPRi system targeting Runx2 15
• 3-3. Validation of off-target effects of CRISPRi system 15
• 3-4. On-target effect of lentiviral delivery of dCas9-KRAB system on primary chondrocytes 18
• 3-5. Runx2 inhibition by CRISPRi shifts chondrocytes into a more anabolic state 18
• 3-6. Runx2 inhibition by CRISPRi reverses inflamed chondrocytes from osteoarthritic phenotype 21
• 4. DISCUSSION 29
• 5. CONCLUSION 33
• 6. REFERENCES 34
• 국 문 초 록 41