Improved Tissue Repair in Articular Cartilage Defects in Vivo by rAAV-Mediated Overexpression of Human Fibroblast Growth Factor 2

Abstract

Therapeutic gene transfer into articular cartilage is a potential means to stimulate reparative activities in tissue lesions. We previously demonstrated that direct application of recombinant adeno-associated virus (rAAV) vectors to articular chondrocytes in their native matrix in situ as well as sites of tissue damage allowed for efficient and sustained reporter gene expression. Here we test the hypothesis that rAAV-mediated overexpression of fibroblast growth factor 2 (FGF-2), one candidate for enhancing the repair of cartilage lesions, would lead to the production of a biologically active factor that would facilitate the healing of articular cartilage defects. In vitro, FGF-2 production from an rAAV-delivered transgene was sufficient to stimulate chondrocyte proliferation over a prolonged period of time. In vivo, application of the therapeutic vector significantly improved the overall repair, filling, architecture, and cell morphology of osteochondral defects in rabbit knee joints. Differences in matrix synthesis were also observed, although not to the point of statistical significance. This process may further benefit from cosupplementation with other factors. These results provide a basis for rAAV application to sites of articular cartilage damage to deliver agents that promote tissue repair.

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

“…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.…”

“…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). The rAAV-LacZ plasmid is an AAV-2-based vector plasmid carrying the lacZ gene that encodes ␤-gal under the control of the cytomegalovirus immediate-early (CMV-IE) promoter (24,25,32). A human SOX9 (hSOX9) complementary DNA (cDNA; 1.7 kb) obtained from G. Scherer (Institute for Human Genetics and Anthropology, Albert-Ludwig University, Freiburg, Germany) (33) was modified to incorporate a FLAG tag on its 5Ј end.…”

“…The resulting plasmid vector is rAAV-FLAGhSOX9, where the presence of the FLAG-hSOX9 fragment was confirmed by sequencing. The rAAV vectors were packaged using adenovirus 5 to provide helper functions in combination with pAd8 (24,25,32). Vector preparations were purified by dialysis (24,25,32) and titered by real-time PCR (24,25,32), averaging 10 10 functional units/ml.…”

“…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.…”