Alpha 1-antitrypsin deficiency is a genetic disorder caused by defective production of alpha 1-antitrypsin (AAT). Gene therapy approaches have been conducted in patients with AAT deficiency with successful AAT expression, but not to the therapeutic levels required to reduce the risk of emphysema. Codon optimization, a somewhat new and evolving technique, is used by many scientists to maximize protein expression in living organisms by altering translational and transcriptional efficiency as well as protein refolding. The purpose of this study was to develop single stranded and double stranded AAT gene constructs, test their protein expression in vitro, and compare with those levels expressed by the AAT construct that is currently in clinical trials. Three constructs were to be developed, yet only one construct was successfully cloned. This clone, optimized ds-CB-AAT, illustrated increased AAT protein expression as the transfection time increased. However, protein levels were appreciably lower in the optimized construct compared to the single stranded (long intron) AAT construct that is currently being administered in clinical trials. The data did not suggest that the optimized AAT construct does in fact express more AAT protein in vitro as expected. In order to achieve data that can be reproduced, the 2 remaining constructs need to be cloned and all of the isolated plasmid DNA should be prepared on the same scale to minimize any additional confounding variables.

Asthma and allergic rhinitis are almost invariable accompanied by elevated levels of immunoglobin E (IgE), and more importantly a genetic link between IgE levels and airway hyper-responsiveness has been established. We hypothesized that expression of soluble receptors directed against interleukin (IL)-13 and IL-17e would prevent the cytokines from engaging the cell-bound receptors and therefore help to attenuate allergic responses in a Cftr(-/-)-dependent mouse model of exaggerated-IgE responses. Cftr(-/-) mice were injected with recombinant adeno-associated virus 1 (rAAV1) intramuscularly expressing soluble receptors to IL-17e (IL-17Rh1fc) or IL-13 (IL-13Ralpha2Fc). Total IgE levels, in mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapy, were lower than in the control group. Interestingly Aspergillus fumigatus (Af)-specific IgE levels were undetectable in both the mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapies. Further flow cytometry analysis of intracellular gene expression suggests that blocking IL-17e may be interfering with signaling upstream of CD4(+) and CD11b(+) cells and reducing IgE levels by affecting signaling on these cell populations. In contrast it appears that IL-13 blockade acts downstream to reduce IgE levels probably by directly affecting B-cell maturation. These studies demonstrate the feasibility of targeting T helper 2 (Th2) cytokines with rAAV-delivered fusion proteins as a means to treat aberrant immune responses.