Figure 4

Transduction of rabbit joint cells with rAAV1 and rAAV2 vectors in vitro and in vivo . (A) In vitro transduction of rabbit primary synoviocytes and chondrocytes with rAAV1/mCysC and rAAV2/mCysC. Transductions with adenovirus ts149 helper virus alone (‘Ad only’) and AAV2/EV were run as negative controls. Culture media was analyzed by Western blot 48 h postinfection. Protein molecular weight markers (MW) and purified mcysC protein were run as controls. Multiple protein sizes likely represent different degrees of glycosylation. (B-C) AAV capsid serotype comparison and identification of transduced cell types in rabbit joints in vivo. rAAV2/EV, rAAV1/LacZ and rAAV2/LacZ were administered by IA route (n = 3/treatment) and vector transduction sites were located by staining for β-gal activity 4 weeks post delivery. Panel B. Macroscopic analysis of rabbit joints after β-gal staining. The distal portion of the patella ligament was transected transversely to create a flap that was lifted and dissected proximally toward the body to expose the front and sides of the joint. Abbreviations: s, synovium; p, patella; fc, femoral condyle; sp, suprapatella pouch; m, muscle. Panel C. Histological analysis of synovium for β-gal positive cells. Representative sections of synovium from fat pad and patellar region counterstained with NFR are shown. Arrows indicate localization of rAAV-transduced (blue) cells. Magnification x400. EV, empty vector; IA, intra-articular; mCysC, mouse cystatin C; MW, molecular weight; NFR, nuclear fast red; rAAV, recombinant adeno-associated virus.