PROLONGED TRANSGENE EXPRESSION IN COTTON RAT LUNG WITH RECOMBINANT ADENOVIRUSES DEFECTIVE IN E2A

Recombinant adenoviruses have tremendous potential for gene therapy of cystic fibrosis (CF) lung disease. First-generation recombinant virus es, rendered replication defective by deleting El, have been associate d with high-level recombinant gene expression in airway epithelial cel ls when administered directly to the lung. Experience in mice and non- human primates indicates that transgene expression is transient (i.e., lasting less than 21 days) and associated with the development of inf lammation. We suggest an hypothesis to explain these findings that is based on expression of viral proteins in genetically modified cells th at leads to destructive cellular immune responses and repopulation of lung epithelia with non-transgene-containing cells. This hypothesis ha s been evaluated in the current study using the cotton rat model. Inst illation of the first-generation lacZ virus, H5.010CBlacZ, into cotton rat airway led to high-level gene expression in conducting and respir atory airway that was transient and associated with a substantial mono nuclear, CD8-dominated, infiltrates. Treatment of the animals with cyc losporine blunted the inflammatory response and prolonged recombinant gene expression in both conducting and respiratory airways. Expression of viral early and late genes was detected in a subpopulation of lacZ -expressing epithelial cells of conducting airway and alveoli. Instill ation of virus into cotton rat tracheal xenografts grown in athymic nu /nu mice led to efficient and stable transgene expression in the absen ce of pathology, underscoring the importance of T cell-mediated immuni ty. A recombinant adenovirus was constructed that is disabled in its c apacity to replicate by the introduction of a temperature-sensitive mu tation in the E2a gene as well deletion of E1 sequences. Instillation of this virus into cotton rat airway led to high-level transgene expre ssion that was more stable than that achieved with the first-generatio n virus and was associated with less early and late gene expression as well as a diminished infiltration of CD8(+) T cells in conducting air way epithelium. Interestingly, the introduction of the E2a mutation ha d no effect on the persistence of transgene expression, the pattern of late viral gene expression, nor the CD8(+) T cell response within alv eolar cells. These data suggest that cell-specific variation in the ce ll biology of recombinant adenoviruses exists in the lung. The present studies in cotton rats confirm the role of cellular immunity in the b iology of adenovirus-mediated gene therapy to the lung and suggest tha t modifications in the design of recombinant adenoviruses to minimize or ablate transgene expression will be useful in improving the potenti al of this technology for gene therapy of CF.