¹Gene Therapy, Department of Surgery, Roger Williams Medical Center, Boston University School of Medicine, Providence, RI

Abstract

Purpose: Transfer of exogenous growth factor genes to injured tendons is a new and promising method for strengthening tendon repairs. Genes have been delivered to tenocytes through plasmid or adenoviral vectors, but gene transfer through adeno-associated viral (AAV) vectors has not been reported. It is not known whether AAVs can effectively transduce tenocytes and whether transduction rate is different for different AAV serotypes. We explored the efficiency of transduction of intrasynovial tenocytes with 7 serotypes of AAV and the persistency of its expression of a growth factor transgene.

Methods: Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed to 82 wells in 8 culture plates and to 24 culture dishes. The tenocytes in the wells were treated with AAV1, 2, 3, 4, 5, 7, and 8 vectors containing LacZ gene, and a plasmid vector (pCMVb-LacZ). Cultured COS-7 cells known to be highly permissive to AAVs served as positive controls. The tenocytes were stained with in situ beta-galactosidase (X-gal) 5 days later. The basic fibroblast growth factor (bFGF) gene was cloned to the AAV2 vector to construct the AAV2-bFGF vector, which transduced tenocytes in culture dishes. Expression of the transgene and concentration of bFGF were measured over 3 weeks and statistically analyzed.

Results: AAV2 effectively delivered exogenous genes to proliferating intrasynovial tenocytes. In contrast, other tested AAVs transduced tenocytes minimally or not at all. The transduction rate by AAV2, indicated by percentage of cells with positive beta-galactosidase staining, was significantly greater than that by a plasmid vector (p=0.001). Gene expression and concentration of the bFGF in tenocytes transduced with the AAV2-bFGF were significantly higher than those in the cells without gene transfer over the 3-week period (p<0.01).

Discussion: Tendons usually have low growth factor activities and healing capacity of injured tendons is inherently weak. Delivery of growth factor genes to healing tendons is a new field of application of gene therapy. Little is known about appropriate vectors to deliver genes in tendon healing. Our study showed great differences in transduction rates of 7 serotypes of AAVs. AAV2 appears to transduce the tenocytes most efficiently and thus can be a vector for gene therapy in tenocytes. The trandcution rate was higher with AAV2 than with a plasmid vector, which indicates that AAV2-growth factor gene constructs may be more effective than plasmid-growth factor constructs in promoting tendon healing.

Conclusions: The bFGF gene can be delivered to intrasynovial tenocytes by the AAV2 vector effectively and the gene transfer significantly increases expression of bFGF over 3 weeks, but other AAV serotypes cannot effectively transduce tenocytes.