rAAV-delivered alpha-1-antitrypsin compositions and method for the treatment and prevention of diabetes
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example 1
In vitro Studies in Murine C2C12 Myoblasts
[0065]In order to determine the relative strength of a number of constitutively active promoters in the context of AAV-AAT vectors, packageable AAV-AAT expression vectors containing one of the CMV, EF1, U1a or U1b promoters (FIG. 1) were constructed. Each of these constructs were transfected in to the murine C2C12 myoblast cell line. Both the EF1 and the CMV promoter were active for AAT expression, with EF1 construct (AAV-E-AT) expressing 850 ng / 105 cells / day and the CMV construct (AAV-C-AT) expressing approximately 670 ng(105 cells / day, as measured by a human-specific ELISA assay for AAT (FIG. 2). This difference was not statistically significant. The levels of expression from the U1a and U1b constructs were undetectable.
[0066]In order to better characterize the level and duration of expression in the setting of vector transduction, cultures of C2C12 cells were transduced with either AAV-E-AT or AAV-C-AT at multiplicities of infection rangi...
example 2
In vivo Expression of hAAT from Murine Skeletal Muscle
[0067]In order to determine whether the AAV-AAT constructs would be active in vivo in skeletal muscle, doses of vector were injected into the quadriceps femoris muscle of mice. Circulating serum levels of hAAT were then measured for 11 to 15 weeks after the initial injection. Four saline-injected animals from each mouse strain served as controls. In the case of the C-AT vector (FIG. 5A), levels of expression were sufficient to achieve serum levels in excess of 800 μg / ml in SCD mice after a single injection of 1.4×1013 particles. A dose-effect relationship was observed, with expression levels in SCID being at least 20-fold lower at the 5×1011 particle dose. The levels of expression increased over the first several weeks after injection and were stable thereafter until the time of sacrifice. Since hAAT has a half-life of less than 1 week, this indicated continuous expression. Levels from C57B1 / 6 mice were comparable, and also achie...
example 3
Immunologic Studies
[0069]In studies in Balb / c mice, antibody levels against hAAT were high in 2 of 3 animals injected. The one which did not have circulating anti-hAAT was the only animal with levels of hAAT expression similar to those in the C57B1 / 6 and SCID groups. The high-dose C57-C-AT injection group had detectable levels of antibody directed against VP3, but not hAAT.
[0070]In order to determine whether any cell-mediated immune responses were mounted, lymphocyte proliferation assays were performed using either hAAT or AAV-VP3 for antigenic stimulation of primary splenic lymphocytes harvested at the time of animal sacrifice, 16 weeks post-vector injection. Using this method, no immune responses were detectable in any of the mice.
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