Although gene therapy seems the best hope for treatment of cystic fibrosis (CF), many strategies tested so far have been disappointing. Some provoke deleterious host responses at doses below those required for genetic correction, or stimulate a host immune response which prevents effective subsequent dosing, when it is clear that repeat dosing will be necessary for long term therapeutic benefit no matter what the vector, if applied postnatally. In addition, death in CF results from relentless, progressive infection and inflammation, and the connection of this disease to the lack of CFTR chloride transport remains unclear. The ideal gene therapy vector for CF must not itself provoke disease nor initiate immune responses, and must correct the vulnerability to bacterial infection and the tendency toward excess inflammatory responses. Since excess inflammation is critical in disease progression, understanding the cellular origins of the excess inflammatory response is key. In the last grant period, we obtained data from both gene transfer and bone marrow transplant experiments that bone marrow-derived cells in addition to epithelial cells may contribute to the excess inflammatory response in the lung of CF mice, and that providing CFTR to macrophages can ameliorate the response. Moreover, in the last grant period, we developed a molecular conjugate vector that delivers both reporter genes and therapeutic genes to the airway epithelium of mice without provoking limiting inflammatory response or immune responses. This vector consists of a uniform polycation, a 30-mer of lysine with an N-terminal cysteine, conjugated to polyethylene glycol, which is used to condense plasmid DNA into particles with minor diameter 10-12 nm. These particles transfect nasal epithelium of CF mice in vivo, and give full electrophysiologic correction in one third the mice studied. A clinical trial in the nose of CF patients showed no detectable toxicity, but electrophysiologic improvement in two-thirds of subjects. In the next grant period we propose to pursue experiments to elucidate the cellular origins of the excess inflammatory response of the CF mice, to determine whether dosing strategies to increase the intensity and extent of gene transfer with this reagent confer limiting toxicity, and to determine whether this reagent, dosed in optimal fashion, can not only deliver electrophysiologic correction but also correct some of the important downstream consequences of Cftr lack in mice, including the downregulation of nitric oxide synthase and IL-10, expression, the upregulation of B7 expression on macrophages, and the excess cellular and cytokine responses to pulmonary challenge with pseudomonas, either alone or embedded in agar beads, or with pseudomonas lipopolysaccharide.