title: 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: BACKGROUND
  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-hSOX9 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-hSOX9, 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-hSOX9 delivery reduced the levels of markers of hypertrophy, terminal and osteogenic/adipogenic differentiation in hMSCs (type I and type X collagen, alkaline phosphatase (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.
authors: Center of Experimental Orthopaedics, Saarland University Medical CenterHomburg/Saar, Germany.
authors: Jagadeesh K
authors: JK
authors: Venkatesan
authors: Myriam
authors: M
authors: Ekici
authors: Henning
authors: H
authors: Madry
authors: Gertrud
authors: G
authors: Schmitt
authors: Dieter
authors: D
authors: Kohn
authors: Magali
authors: M
authors: Cucchiarini
mesh_terms: D050156:Adipogenesis; D000469:Alkaline Phosphatase / Q000378:metabolism; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D019070:Cell Lineage; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism; D020219:Chondrogenesis; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D010012:Osteogenesis; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; C493709:RUNX2 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D000469:Alkaline Phosphatase; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13