890. Comparison of the Osteogenic Potential of AAVhBMP6 and ADhBMP6 between Two Rat Strains

Abstract

Adeno-associated virus (AAV) and adenovirus (AD) are the two most commonly viral vectors in gene therapy. Both vectors have their own advantages and limitations. In this study, a human bone morphogentic protein 6 (hBMP6) cDNA under control of an immediate CMV promoter was separately inserted into AAV type 5 (AAVhBMP6) and human adenovirus type 5 (ADhBMP6) vectors. Since no a reasonable viral unit could be used to give definite dose equivalence between these two viral vectors, the viral doses were chosen based on our previous experiments with these two vectors. The criterion to choose the viral dose was the amount of viral vector having the potential to induce bone formation in both athymic nude (AN) and Sprague-Dawley (SD) rats. Two-month-old AN and SD (15 of each strain) and 18-month-old SD (3 animals) rats were used in this experiment. Five young rats from each strain were directly injected with AAVhBMP6 (7 1012 particles/350 l) or ADhBMP6 (5 107 PFU/350 l) into the left thigh separately. Three old SD rats were injected with the above amount of AAVhBMP6 in the left thigh and ADhBMP6 in the right thigh. The percentages of CD4, CD8, CD11b, and CD45RA cells were measured 3 days later by using flow cytometry. The animals were scanned with CT on Days 15, 30, 60, and 90 after viral injection. The AN rats had significantly lower percentages of CD4+ (27.7%) and CD8+ (16%) than same age SD rats (CD4+ 43.4% and CD8+ 31.5%), but higher percentages of CD11b+ (38.7%) than SD rats (CD11b+ 15.8%). Most of the CD4+ and CD8+ cells in athymic nude rats were double stained with CD11b+. That indicates that these CD4+ and CD8+ cells in AN rats are macrophage cells, rather than true T cells. Two-month-old SD rats had the lower percentages of CD8+ (16%) and CD11b+ (15.8%) than 18-month-old SD rats (CD8+ 39.5% and CD11b+ 32.7%). The percentages of all cell types were not significantly different among different groups in the same age and strain of rats. The average amounts of bone, calculated only among rats that had the bone formation in each group, on Days 15, 30, 60, and 90 were the following: 0, 0.12, 0.25, and 0.23 cm3 for AAVhBMP6 (3/4 rats have bone, the same as following) and 2.52, 3.96, 3.65, and 3.98 cm3 for ADhBMP6 (5/5) in AN rats; 0, 0.15, 0.3, and 0.28 cm3 for AAVhBMP6 (5/5) and 0.07, 0.06, 0.07, and 0.08 cm3 for ADhBMP6 (1/5) in 2-month-old SD rats, and 0, 0.15, 0.20, and 0.18 cm3 for AAVhBMP6 (3/3) and 0 for ADhBMP6 (0/3) in 18-month-old SD rats. In addition, the viral concentration or volume may also affect the amount of bone formation because the same amount of ADhBMP6 in 50 l induced 1.2 cm3 in AN (5/5) and 0.12 cm3 in SD (4/5) rats. These results indicate that AAVhBMP6 has the potential to induce ectopic bone formation in both immunodefective and immunocompetent animals but takes longer time to form bone than ADhBMP6 in both rat strains. Interestingly, the amount of bone induced by AAVhBMP6 is smaller than ADhBMP6 in athymic nude rats, but is larger than ADhBMP6 in SD rats. Similar sizes of bone induced by AAVhBMP6 are observed in both rat strains. Eighteen-month-old rats still have the ability to form the ectopic bone.