The complexity of the CF lung disease phenotype has, up to now, defied precise explanation. Primary aberrations within the airways of CF patients include deficiency of cAMP-activated chloride conductance, sodium hyperabsorption, and a marked propensity for infection with Pseudomonas aeruginosa resulting in purulent inflammation and an imbalance of protease activity over antiprotease activity. These effects ultimately result in partial or complete obstruction of the airway lumen, cytotoxicity, and destruction of the extracellular matrix. The exact mechanisms underlying the propensity to inflammatory airways disease are unknown, but may relate to an alteration in the glycosylation pattern of surface glycoproteins, increased binding of Pseudomonas aeruginosa, and an exaggerated pro-inflammatory cytokine response that might relate to the presence of a cell stress response. Recent microarray studies have indicated that the complex phenotype of CF lung disease is reflected by a complex pattern of altered gene regulation, both at baseline and in response to Pseudomonas exposure. While it remains axiomatic that wild-type CFTR gene transfer itself will be the ultimate primary prevention for CF lung disease, there may yet be many individuals with existing CF lung disease for whom a secondary amelioration strategy may be more feasible. The primary hypotheses to be tested in this proposal are the following: Aberrant down-regulation of certain anti-protease, anti-inflammatory, and gly?osylation-related genes contribute to CF lung disease, and augmentation of these substances will ameliorate the CF lung disease phenotype. There is also a secondary hypothesis related to the observation that two of the anti-proteases that are down-regulated in CF, squamous cell carcinoma antigen-1 (SCCA-1) and SCCA-2 appear to be primarily intracellular anti-proteases that are up-regulated in malignant lung tumors and down-regulated in CF. This might lead one to speculate as to whether these intracellular anti-proteases could also be anti-apoptotic (perhaps through caspase inhibition), thus contributing to the immortalization of tumor cells, and conversely to epithelial cell death in CF. The latter hypothesis will also be studied in the context of CF lung disease models.
Four aims are proposed: (1) The down-regulation of anti-proteases, anti-inflammatory cytokines and glycosylation enzymes will be examined in CF cell lines, the CFTR knock-out mouse, and clinical samples of CF lung tissue; (2) The effects of exogenous vector-mediated expression of a number of anti-proteases will be evaluated in the Pseudomonas-infected CFTR knock-out mouse model; (2a) Depending on the results of Aim 1, the potential value of augmenting other gene products, such as anti-inflammatory cytokines and glycosylation enzymes, will be evaluated; (3) The in vivo dynamic range of transcriptionally-regulated vector cassettes will be assessed; and (4) The anti-protease-rAAV constructs determined to biologically active and safe in the infected lung models outlined in Aim 2 will undergo formal preclinical toxicology testing will be performed as a prelude to clinical trials in CF patients. We anticipate that whether or not gene augmentation of these specific molecules becomes practical, that lessons learned in these models might serve as target validation for these molecules or for agents with similar mechanisms of action.
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