889. Relationship between the Amount of Bone Formation and Protein Expression in Two Rat Stains Using Direct Adenoviral Vector Injection and Ex Vivo Method
Authors: Jin Zhong Li, Hongwei Li, El Noh, Lindsey Jacobson, An-Hsiang Liu, Dwight Saulle, Gerald R. Hankins, Gregory A. Helm
Journal: Molecular Therapy (2004)

Although adenoviral vectors have a very broad range and high efficiency of cell transduction both in vitro and in vivo, the uses of adenoviral vectors have been significantly limited in immunocompetent animals. We tested whether the amount of protein expression was the major factor determining the foreign gene functions. The recombinant adenoviral vectors of human bone morphogentic protein 4 and luciferase were used to answer this question. Direct viral injection and ex vivo methods were chosen in immunodefective athymic nude (AN) and immunocompetent SD rats. On Day 0, the primary rat bone marrow cells were infected with either ADhBMP4 or ADLUC at 107 PFU/flask or PBS. At the same time, five AN and SD rats were given a direct injection of either ADhBMP4 or ADLUC (107 PFU/100 μl) into the right thigh. On Day 1, both control and virally infected cells were injected into the left thigh with 2 × 106 cells/100 μl/flask. The rats underwent imaging performed with a cooled CCD before and after an intraperitoneal luciferin injection on Days 1 and 7. On Day 1, the densities of luminescent signals (×107 photons/sec) were the following: 1.83 ± 0.56 for SD rats with direct ADLUC injection; 3.73 ± 1.15 for SD rats with ex vivo ADLUC injection; 5.01 ± 1.91 for AN rats with direct ADLUC injection; and 11.9 ± 3.35 for AN rats with ex vivo ADLUC injection. The luminescent signals in the control groups were the same as background. By Day 7, the percentages of luminescence densities in ADLUC groups (compared with the same site on Day 1) were the following: 14.1% for SD rats with direct ADLUC injection; 1.06% for SD rats with ex vivo ADLUC injection; 25.4% for AN rats with direct ADLUC injection; and 0.8% for nude rats with ex vivo ADLUC injection. The animals were scanned with CT 1 month after injection. The bone volumes (cm3) of ADhBMP4 induced were 0.85 ± 0.08 for direct injection and 0.15 ± 0.03 for ex vivo in AN rats, and no bone for direct injection and 0.12 ± 0.02 for ex vivo in SD rats. No bone was observed at sites receiving injections of control cells or ADLUC. These data demonstrate that AN and SD rats display similar expression profiles of foreign gene expression delivered with adenoviral vectors, regardless of whether direct viral injection or the ex vivo method is used. As we know the amount of new bone is dose dependent in AN rats, and the average amount of bone induced by hBMP4 is smaller when the ex vivo method is employed than when direct viral injection is used. This result reflects the lower level of hBMP4 expression using the ex vivo method than directly viral injection. However, in SD rats, although hBMP4 expression may be lower after the ex vivo injection than after directly viral injection, the ex vivo ADhBMP4 delivery induces bone formation. These results indicate that the amount of BMP expression is not the major factor affecting the amount of new bone in SD rats. The major factors affecting the osteogenic potentials of ADhBMPs in SD rats may be some immune factors, which may have higher titer in direct adenoviral injection comparing to the ex vivo delivery.