Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a.
Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a.
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E2a 缺陷的重组腺病毒在棉鼠肺中延长转基因表达。
DOI：
10.1089/hum.1994.5.10-1217
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发表时间：
1994
影响因子：
4.2
通讯作者：
Wilson,JM
中科院分区：
文献类型：
--
作者：
Engelhardt,JF;Litzky,L;Wilson,JM
重组腺病毒在囊性纤维化肺病的基因治疗中具有巨大的潜力。第一代重组病毒，使复制缺陷删除E1，已与高水平的重组基因表达在气道上皮细胞直接给药时，肺。小鼠和非人灵长类动物的经验表明转基因表达是瞬时的（即，持续不到21天），并与炎症的发展有关。我们提出了一种假设来解释这些发现，这是基于病毒蛋白在转基因细胞中的表达，导致破坏性的细胞免疫反应和非转基因细胞的肺上皮细胞的再增殖。本研究使用棉鼠模型对这一假设进行了评估。将第一代LacZ病毒H5.010CBlacZ注入棉鼠气道，导致传导和呼吸气道中的高水平基因表达，该表达是短暂的，并与大量单核细胞、CD 8主导的浸润相关。用环孢霉素治疗动物减弱了炎症反应，延长了传导和呼吸道中重组基因的表达。在传导气道和肺泡的表达lacZ的上皮细胞亚群中检测到病毒早期和晚期基因的表达。将病毒滴注到在无胸腺/numice中生长的棉鼠气管异种移植物中导致在不存在病理的情况下有效且稳定的转基因表达，强调了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E2 a基因中引入温度敏感性突变以及缺失E1序列来使其复制能力丧失。将该病毒滴注到棉鼠气道中导致高水平的转基因表达，其比用第一代病毒实现的更稳定，并且与较少的早期和晚期基因表达以及传导气道上皮中的CD 8 +T细胞浸润减少相关。有趣的是，E2 a突变的引入对转基因表达的持续性、晚期病毒基因表达的模式以及肺泡细胞内的CD 8 +T细胞反应没有影响。这些数据表明，在重组腺病毒的细胞生物学细胞特异性变异存在于肺。目前在棉鼠中的研究证实了细胞免疫在腺病毒介导的肺基因治疗的生物学中的作用，并表明重组腺病毒设计中的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。
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-generationlacZvirus, 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 oflacZ-expressing epithelial cells of conducting airway and alveoli. Instillation of virus into cotton rat tracheal xenografts grown in athymicnu/numice 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.