SOX9 gene transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cells

Abstract

Introduction

Transplantation of genetically modified human bone marrow-derived mesenchymal stem cells (hMSCs) with an accurate potential for chondrogenic differentiation may be a powerful means to enhance the healing of articular cartilage lesions in patients. Here, we evaluated the benefits of delivering SOX9 (a key regulator of chondrocyte differentiation and cartilage formation) via safe, maintained, replication-defective recombinant adeno-associated virus (rAAV) vector on the capability of hMSCs to commit to an adequate chondrocyte phenotype compared with other mesenchymal lineages.

Methods

The rAAV-FLAG-h SOX9 vector was provided to both undifferentiated and lineage-induced MSCs freshly isolated from patients to determine the effects of the candidate construct on the viability, biosynthetic activities, and ability of the cells to enter chondrogenic, osteogenic, and adipogenic differentiation programs compared with control treatments (rAAV- lacZ or absence of vector administration).

Results

Marked, prolonged expression of the transcription factor was noted in undifferentiated and chondrogenically differentiated cells transduced with rAAV-FLAG-h SOX9, leading to increased synthesis of major extracellular matrix components compared with control treatments, but without effect on proliferative activities. Chondrogenic differentiation ( SOX9, type II collagen, proteoglycan expression) was successfully achieved in all types of cells but strongly enhanced when the SOX9 vector was provided. Remarkably, rAAV-FLAG-h SOX9 delivery reduced the levels of markers of hypertrophy, terminal and osteogenic/adipogenic differentiation in hMSCs (type I and type X collagen, alkaline phosphatise (ALP), matrix metalloproteinase 13 (MMP13), and osteopontin (OP) with diminished expression of the osteoblast-related transcription factor runt-related transcription factor 2 (RUNX2); lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma 2 (PPARG2)), as well as their ability to undergo proper osteo-/adipogenic differentiation. These effects were accompanied with decreased levels of β-catenin (a mediator of the Wnt signaling pathway for osteoblast lineage differentiation) and enhanced parathyroid hormone-related protein (PTHrP) expression (an inhibitor of hypertrophic maturation, calcification, and bone formation) via SOX9 treatment.

Conclusions

This study shows the potential benefits of rAAV-mediated SOX9 gene transfer to propagate hMSCs with an advantageous chondrocyte differentiation potential for future, indirect therapeutic approaches that aim at restoring articular cartilage defects in the human population.