686. Bone Formation Inhibitors in Animal Sera

GENE THERAPY FOR CONNECTIVE TISSUE 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 AAV2bFGF were significantly higher than those in the cells wthout 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.

685. Inhibitory Effect of Ribbon-type NF-kB Decoy Oligodeoxynucleotides on Osteoclast Induction and Activity Yasuo Kunugiza,1,2 Tetsuya Tomita,1 Naruya Tomita,2 Mariana Kiomy Osako,2 Takuji Kizawa,1 Keishi Sekiguchi,2 Ryuichi Morishita,2 Hideki Yoshikawa.1 1 Orthopaedic Surgery, Osaka University, Graduate School of Medicine, Osaka, Japan; 2Clinical Gene Therapy, Osaka University, Graduate School of Medicine, Osaka, Japan. Purpose: Osteoclasts, multinucleated giant cells that resorb bone, develop from hematopietic cells of the monocyte/macrophage lineage. We examined the effect of ribbon-type NF-kB decoy oligodeoxynucleotides ODN (RDODN) on induction and activity of osteoclasts. Method: We extracted bone marrow cells from the femur of rats and non-adherent cells were incubated with RANKL (100ng/ml) and M-CSF (20ng/ml). First, cells were incubated with FITC-labeled RDODN without reagent and 24 hours later transfer efficiency of RDODN was examined by counting the number of the fluorescencepositive cells. To examine the effect of RDODN on induction and activity of osteoclasts, cells were incubated with or without RDODN and TRAP staining was performed on day 7 and the number of TRAP-positive multinucleated cells was counted. To examine the Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

effect of RDODN on the bone resorbing activity of mature osteoclasts, we performed the bone-resorbing assay. Non-adherent bone marrow cells were cultured on Osteologic Bone Cell Culture System. On day 8 cells were incubated with RDODN and on day 10 calcified matrix resorption area on each disc was measured using the MacSCOPE image analyzer. Result: About 80% of cells could be transferred with RDODN. The total number of TRAP- positive multinucleated cells were significantly decreased in RDODN treated group compared to nonRDODN treated group or scrambled RDODN treated group (p<0.01). The average resorbed area of calcified matrix was significantly decreased in RDODN treated group compared to other groups (p<0.05). Conclusion: We confirmed the high transfer efficiency of RDODN to osteoclasts, the inhibitory effect of RDODN on induction and activity of osteoclasts. Taken together, these data may suggest the therapeutic potential of RDODN in joint destruction of arthritis.

686.

Bone Formation Inhibitors in Animal Sera

Jin Zhong Li,1 Ann-Shung Lieu,1 Gerald R. Hankins,1 Gregory A. Helm.1,2 1 Neurological Surgery, University of Virginia, Charlottesville, VA; 2 Biomedical Engineering, University of Virginia, Charlottesville, VA. Based on previous gene therapy studies in bone formation induced by BMP adenoviral vectors, the osteogenic potentials of different BMP adenoviral vectors (ADhBMP-2, -4, -6, -7, and -9) are very high in immunodeficient animals but vary significantly among immunocompetent animals. We have demonstrated that the amounts of bone formation in immunocompetent animals were not significantly affected by use of different generations of adenoviral vectors and different species of BMPs. The similar protein expression curves between immunodeficient and immunocompetent rats indicated that reduced amount of BMP expression was not the main factor limiting the bone formation. Nevertheless, some components of sera may specifically block the signal transduction pathways of particular BMPs, thus interrupting bone formation. We use the term “bone formation inhibitors (BFIs)” to describe these immune factors to distinguish them from “BMP antagonists”, which are mainly secreted by bone tissue. A mouse skeletal muscle myoblast cell line (C2C12) was used as a model. Reduction of alkaline phosphatase (AP) expression was used as an index to evaluate the inhibiting activity of serum. The distribution of BFIs among different animal species and strains was investigated. Sera from rabbit, rat, and mice were tested. Sera from two strains of rabbit included 14 normal control sera and five adenovirus-vaccinated sera. Most rabbit sera did not exhibit significant inhibiting activity on cell response to any BMP. Only one serum from adenovirus-vaccinated rabbit displayed inhibiting activity on cell response to BMP6. Two sera from NOD SCID mice did not show any inhibiting activity on cell response to BMP2, -6 and -9. Forty-four sera obtained from athymic nude, SD, and Wistar rats were tested. From athymic nude rats we obtained five normal and seven adenovirus-vaccinated sera. All the rat sera inhibited response of cells to BMP6. Two sera obtained from adenovirus (ADLUC)-vaccinated athymic nude rats 16 months after the vaccination inhibited cell response to BMP2, -6, and -9. Four sera from untreated Wistar rats demonstrated inhibiting activities to all three BMPs. Sera from13 normal, and 15 adenovirusvaccinated SD rats, all displayed inhibiting activity to BMP6. Some sera also showed inhibition of cell response to BMP2 and -9. The results indicate that BFIs are widely distributed among different animal species and strains. Adenovirus-vaccinated sera may have higher titers than normal sera. This finding will provide a new point of view to assess the relationship between the immune system and BMPs. S265