IMPROVING AAV VECTOR TRANSDUCTION IN AIRWAY CELLS

改善气道细胞中的 AAV 载体转导

• 批准号: 2600702
• 负责人: JOHN A WAGNER
• 金额: $ 8.39万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-16 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2600702
• 关键词: X ray; adeno associated virus group; antibacterial agents; antiinflammatory agents; cell transformation; chloride channels; cystic fibrosis; gene expression; genetic transduction; inflammation; method development; mutagens; respiratory epithelium; tissue /cell culture; transfection /expression vector; ultraviolet radiation

DESCRIPTION (Adapted from applicants' abstract) Cystic fibrosis (CF) is one of the most extensively researched genetic and respiratory diseases as a target for gene transfer therapy development. It may also serve as an important model for gene transfer therapy of other diseases, especially other lung diseases. Long-term expression and lack of pathogenicity make adeno-associated virus cystic fibrosis transmembrane conductance regulator (AAV-CFTR) an attractive gene transfer vector for testing in CF patients. Although the applicant demonstrated that AAV mediated gene transfer of CFTR was efficient and persistent in a phase I clinical trial in the maxillary sinus of CF patients, expression was equivocal. Adequate expression is critical to effective and successful gene transfer therapy for CF. Many factors alter expression of wild type AAV and recombinant AAV vectors. Conversion of single stranded AAV DNA to double stranded forms is likely the main factor limiting expression. Adenoviral gene expression has a well characterized role in this process. Less is known about other factors including genotoxic agents that can also induce a 'permissive' cellular state and increase expression of AAV. This proposal focuses on two hypotheses related to expression of rAAV vectors: (1) Genotoxic agents and other factors known to induce a permissive cellular state for wild type AAV transcription will increase expression of AAV vectors in respiratory epithelial cells and (2) infection increases rAAV vector expression and factors reducing infection and inflammation decrease vector expression in respiratory epithelial cells. To test these hypotheses, the following studies are proposed as Specific Aims: (1) To characterize the effects of genotoxic agents, heat shock, ultraviolet radiation, and roentgen radiation on rAAV vector transduction of transformed and primary cultured airway epithelial cells of CF and non-CF origin; (2) To determine the effects of bacterial infection on rAAV vector transduction of airway epithelial cells; and (3) To determine the effects of anti-microbial agents and anti-inflammatory agents on rAAV vector transduction of infected and uninfected airway epithelial cells. Increasing expression of rAAV vectors is critical to the success of gene therapy for CF and potentially other genetic disorders, and may prove clinically important. Determining the effects of bacterial infection as well as the role of anti-microbial and anti-inflammatory agents will likely directly impact future clinical rAAV vector gene transfer therapy protocols. (End of Abstract)

描述 （改编自申请人摘要）囊性纤维化（CF）是最常见的 广泛研究的遗传和呼吸系统疾病作为基因治疗的目标， 转移疗法的发展。 它也可以作为一个重要的模式， 基因转移治疗其他疾病，特别是其他肺部疾病。 长期表达和缺乏致病性使腺相关病毒 囊性纤维化跨膜传导调节因子（AAV-CFTR）是一种有吸引力的 基因转移载体用于CF患者的测试。 虽然申请人 证明了AAV介导的CFTR基因转移是有效的， 在CF上颌窦的I期临床试验中持续存在 患者，表达模棱两可。 充分的表达对于 有效和成功的基因转移治疗CF。 许多因素改变 野生型AAV和重组AAV载体的表达。 转化 单链AAV DNA到双链形式可能是主要因素 限制表达。 腺病毒基因表达具有良好的特征性 在这个过程中的作用。 对包括遗传毒性在内的其他因素知之甚少 也可以诱导“许可”细胞状态并增加 AAV的表达。 该提案侧重于两个假设， rAAV载体的表达：（1）已知的遗传毒性剂和其他因子， 诱导允许野生型AAV转录细胞状态将 AAV载体在呼吸道上皮细胞中表达增加，和（2） 感染增加rAAV载体表达和减少感染的因子 和炎症降低呼吸道上皮细胞中的载体表达。 为了验证这些假设，提出以下研究作为具体的 目的：（1）研究遗传毒性物质、热休克、 紫外线和伦琴辐射对rAAV载体转导的影响， 转化和原代培养的CF和非CF气道上皮细胞 （2）确定细菌感染对rAAV载体的影响 气道上皮细胞的转导;和（3）确定 rAAV载体上的抗微生物剂和抗炎剂 感染和未感染的气道上皮细胞的转导。 增加 rAAV载体的表达是CF基因治疗成功的关键 和潜在的其他遗传疾病，并可能证明临床重要性。 确定细菌感染的影响以及 抗微生物和抗炎剂可能会直接影响 未来的临床rAAV载体基因转移治疗方案。 (End的 摘要）