Prolonged Transgene Expression in Cotton Rat Lung with Recombinant Adenoviruses Defective in E2a

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Abstract
Recombinant adenoviruses have tremendous potential for gene therapy of cystic fibrosis (CF) lung disease. First-generation recombinant viruses, rendered replication defective by deleting E1, have been associated with high-level recombinant gene expression in airway epithelial cells 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 inflammation. We suggest an hypothesis to explain these findings that is based on expression of viral proteins in genetically modified cells that leads to destructive cellular immune responses and repopulation of lung epithelia with non-transgene-containing cells. This hypothesis has been evaluated in the current study using the cotton rat model. Instillation of the first-generation lacZ virus, H5.010CBlacZ, into cotton rat airway led to high-level gene expression in conducting and respiratory airway that was transient and associated with a substantial mononuclear, CD8-dominated, infiltrates. Treatment of the animals with cyclosporine 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. Instillation of virus into cotton rat tracheal xenografts grown in athymic nu/nu mice led to efficient and stable transgene expression in the absence of pathology, underscoring the importance of T cell-mediated immunity. A recombinant adenovirus was constructed that is disabled in its capacity to replicate by the introduction of a temperature-sensitive mutation in the E2a gene as well deletion of E1 sequences. Instillation of this virus into cotton rat airway led to high-level transgene expression that was more stable than that achieved with the first-generation virus and was associated with less early and late gene expression as well as a diminished infiltration of CD8+ T cells in conducting airway epithelium. Interestingly, the introduction of the E2a mutation had no effect on the persistence of transgene expression, the pattern of late viral gene expression, nor the CD8+ T cell response within alveolar cells. These data suggest that cell-specific variation in the cell biology of recombinant adenoviruses exists in the lung. The present studies in cotton rats confirm the role of cellular immunity in the biology of adenovirus-mediated gene therapy to the lung and suggest that modifications in the design of recombinant adenoviruses to minimize or ablate transgene expression will be useful in improving the potential of this technology for gene therapy of CF. Recombinant E1-deleted adenoviral vectors have proven to be useful tools in transferring genes to numerous organs in vivo. As a result, the potential application of these vectors in the treatment of CF lung disease is extensively under investigation in several clinical trials. Despite the high efficiency in which these vectors can transfer genes to the respiratory epithelium, their application has been limited by lack of persistent gene expression and elicited immune response. Alterations in current E1-deleted adenoviral vectors which incorporate a temperature-sensitive mutation within the E2a gene, have led to the development of second-generation recombinant adenoviral vectors that have increased the length of transgene expression in vivo. This increased persistent in transgene expression is concordant with a decreased ability of recombinant virus to transcribe late genes as detected by in situ hybridization and immunocytochemical localization of hexon. Furthermore, the decreased level of hexon gene expression was directly correlated with decreased levels of CD8+ T cell infiltrates within bronchiolar epithelium. These studies present encouraging evidence that, by further manipulation of the adenoviral genome, recombinant vectors may be generated that reduce the frequency of repetitive dosing for gene therapy.