IMPROVING AAV VECTOR TRANSDUCTION IN AIRWAY CELLS

描述 (摘自申请者摘要)囊性纤维化(CF)是最常见的 广泛研究遗传和呼吸道疾病作为基因靶点 转移疗法的发展。它也可以作为一个重要的模式 其他疾病，特别是其他肺部疾病的基因转移治疗。 腺相关病毒的长期表达和缺乏致病性 囊性纤维化跨膜电导调节剂(AAV-CFTR) 基因转移载体用于检测慢性粒细胞白血病患者。虽然申请人 证明了AAV介导的CFTR基因转移是有效的和 在上颌窦持续进行的一项临床试验 患者，表情模棱两可。适当的表达方式对 有效而成功的基因转移治疗慢性萎缩性胃炎。许多因素会改变 野生型AAV和重组AAV载体的表达。转换为 AAV DNA单链转变为双链可能是主要因素 限制表达。腺病毒基因的表达具有良好的特征 在这一过程中发挥作用。对包括遗传毒性在内的其他因素知之甚少 也可以诱导细胞处于一种“允许”状态并增加 AAV的表达。这项提议侧重于与以下两个假设相关的 重组AAV载体的表达：(1)已知的基因毒剂和其他因素 为野生型AAV转录诱导允许的细胞状态 AAV载体在呼吸道上皮细胞中的高表达及(2) 感染增加rAAV载体的表达和降低感染的因素 炎症会降低呼吸道上皮细胞中载体的表达。 为了检验这些假设，我们提出了以下具体研究 目的：(1)表征遗传毒性物质、热休克、 紫外线和伦琴辐射对重组腺病毒载体转导的影响 转化和原代培养的CF型和非CF型呼吸道上皮细胞 来源；(2)确定细菌感染对rAAV载体的影响 呼吸道上皮细胞的转导；以及(3)确定其作用。 RAAV载体上的抗菌剂和抗炎药 感染和未感染的呼吸道上皮细胞的转导。渐增 重组腺相关病毒载体的表达是CF基因治疗成功的关键 以及潜在的其他遗传性疾病，并可能被证明是临床上重要的。 确定细菌感染的影响以及 抗微生物和消炎剂可能会直接影响 未来临床rAAV载体基因转移治疗方案。(末尾 摘要)

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)