Engelhardt J F, Litzky L, Wilson J M
Institute for Human Gene Therapy, University of Pennsylvania Medical Center, Philadelphia 19104-4268.
Hum Gene Ther. 1994 Oct;5(10):1217-29. doi: 10.1089/hum.1994.5.10-1217.
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.
重组腺病毒在囊性纤维化（CF）肺部疾病的基因治疗中具有巨大潜力。第一代重组病毒通过缺失E1基因而导致复制缺陷，当直接给予肺部时，已显示出在气道上皮细胞中具有高水平的重组基因表达。在小鼠和非人灵长类动物中的经验表明，转基因表达是短暂的（即持续时间少于21天），并且与炎症的发展相关。我们提出一个假说来解释这些发现，该假说基于转基因细胞中病毒蛋白的表达，这种表达会导致破坏性的细胞免疫反应以及肺上皮细胞被不含转基因的细胞重新占据。在当前的研究中，使用棉鼠模型对这一假说进行了评估。将第一代lacZ病毒H5.010CBlacZ滴注到棉鼠气道中，导致在传导气道和呼吸气道中出现高水平的基因表达，这种表达是短暂的，并且伴有大量以CD8为主的单核细胞浸润。用环孢素治疗动物可减弱炎症反应，并延长传导气道和呼吸气道中的重组基因表达。在传导气道和肺泡的lacZ表达上皮细胞亚群中检测到病毒早期和晚期基因的表达。将病毒滴注到无胸腺裸鼠体内生长的棉鼠气管异种移植物中，可在无病理学改变的情况下实现高效且稳定的转基因表达，这突出了T细胞介导的免疫的重要性。构建了一种重组腺病毒，通过在E2a基因中引入温度敏感突变以及缺失E1序列，使其失去复制能力。将这种病毒滴注到棉鼠气道中，可导致高水平的转基因表达，这种表达比第一代病毒所实现的表达更稳定，并且与早期和晚期基因表达减少以及传导气道上皮中CD8 + T细胞浸润减少相关。有趣的是，E2a突变的引入对转基因表达的持续性、晚期病毒基因表达模式以及肺泡细胞内的CD8 + T细胞反应均无影响。这些数据表明，肺部存在重组腺病毒细胞生物学中的细胞特异性差异。目前在棉鼠中的研究证实了细胞免疫在腺病毒介导的肺部基因治疗生物学中的作用，并表明对重组腺病毒设计进行修改以最小化或消除转基因表达，将有助于提高该技术用于CF基因治疗的潜力。
