Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.

Mucins are high molecular-weight glycoproteins involved in the protection and lubrication of respiratory, gastrointestinal, and reproductive tracts. Hypersecretory diseases such as cystic fibrosis (CF), chronic bronchitis, and asthma result in dysregulated levels of mucin production stemming from increased abundance of mucin-secreting cell types in the surface airway epithelium and submucosal glands. The isolation of at least nine mucin genes has prompted studies to characterize the cellular expression patterns of these mucins in normal and diseased tissues. In the present study, in situ hybridization and immunocytochemical methods were used to determine the cellular distribution of MUC5B and MUC7 expression in CF and non-CF human bronchus. Our findings indicate that MUC5B and MUC7 have expression patterns in human bronchial airways that are limited exclusively to submucosal glands. Specifically, MUC5B expression was confined to all mucous tubules, whereas MUC7 expression was seen in a subset of lysozyme expressing serous tubules of submucosal glands. Interestingly, heterogeneity of MUC7 expression between glands of the same bronchus ranged from 0 to 93% of serous tubules, suggesting that functional diversity may exist between glands within the same bronchial sample. No remarkable differences were observed in the expression patterns of MUC5B or MUC7 between CF (n ϭ 7) and non-CF (n ϭ 10) bronchial samples. In conclusion, MUC5B and MUC7 expressions define different cellular compartments within submucosal glands of human bronchus and lend insight into the heterogeneity of mucin production in the lung.

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