Recombinant Adeno-Associated Virus Vectors Efficiently and Persistently Transduce Chondrocytes in Normal and Osteoarthritic Human Articular Cartilage

Abstract

Successful gene transfer into articular cartilage is a prerequisite for gene therapy of articular joint disorders. In the present study we tested the hypothesis that recombinant adeno-associated virus (rAAV) vectors are capable of effecting gene transfer in isolated articular chondrocytes in vitro, articular cartilage tissue in vitro, and sites of articular damage in vivo. Using an rAAV vector carrying the Escherichia coli beta-galactosidase gene (lacZ) under the control of the cytomegalovirus (CMV) immediate-early promoter/enhancer (rAAV-lacZ), transduction efficiency exceeded 70% for isolated normal human adult articular chondrocytes, and osteoarthritic human articular chondrocytes. These were comparable to the transduction efficiency obtained with neonatal bovine articular chondrocytes. Transduction of explant cultures of articular cartilage resulted in reporter gene expression within the tissue of all three cartilage types to a depth exceeding 450 microm, which remained present until 150 days. When rAAV-lacZ vectors were applied to femoral chondral defects and osteochondral defects in vivo in a rat knee model, reporter gene expression was achieved for at least 10 days after transduction. These data suggest that AAV-based vectors can efficiently transduce and stably express foreign genes in articular chondrocytes, including chondrocytes of normal and osteoarthritic human articular cartilage. The data further suggest that the same rAAV vectors are capable of transducing chondrocytes in situ within their native matrix to a depth sufficient to be of potential clinical significance. Finally, the data demonstrate that these rAAV vectors are capable of effectively delivering recombinant genes to chondral and osteochondral defects in vivo.

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

“…All patients provided informed consent prior to inclusion in the study. Explant cultures and chondrocytes were prepared as previously described (11,24).…”

“…The activity of ␤-gal was detected by X-Gal staining to determine the transduction efficiencies (24,25,32,34). Transgene expression was also monitored by indirect immunostaining (25).…”

“…Spheres and explant cultures were processed, and paraffinembedded sections (5 m) were stained with Safranin O to detect proteoglycans and with hematoxylin and eosin (H&E) to detect cells (11,24,25). Type II collagen was detected with a specific antibody, a biotinylated secondary antibody (Vector), and the avidinϪbiotinϪperoxidase method (Vector) using diaminobenzidine as the chromogen.…”

“…All patients provided informed consent prior to inclusion in the study. Explant cultures and chondrocytes were prepared as previously described (11,24).Plasmids and rAAV vectors. Plasmid pACP is an AAV-2-derived vector plasmid from the genomic clone pSSV9, and pAd8 contains the AAV-2 replication and encapsidation functions (31).…”

“…Recombinant adeno-associated virus (rAAV) vectors are particularly well suited for this purpose because they can transfer genes into human OA chondrocytes in vitro and in situ with high efficiencies and for extended periods of time (23)(24)(25). The rAAV vectors are derived from a replication-defective human parvovirus that is nonpathogenic in humans.…”