AIRWAY EPITHELIAL BIOLOGY OF AAV INTEGRATION

描述（直接取自应用程序） 囊性纤维化的基因治疗方法的有效性只会 一旦基本了解了载体与载体的相互作用， 了解气道的靶细胞。 最终，最好的治疗方法 CF的基因治疗方法将利用单剂量递送 在疾病发作之前整合载体。 到目前为止，只有两个矢量 包括重组逆转录病毒和AAV的系统具有以下能力： 将重组转基因整合到宿主基因组中。 Retroviruses 尽管在它们的整合过程中非常有效，但受到以下限制： 用于体内递送的可达到的滴度。 相比之下，AAV载体提供了 更有吸引力的替代品，具有更高的可实现滴度，但 最近一直困扰的集成效率低，研究细胞 线 然而，AAV整合过程的效率似乎 受受体细胞类型和细胞因子的影响， 融合过程。 因此，由于AAV用于 对气道的基因治疗将取决于受体细胞类型， 一个明显的问题仍然是， 靶向人气道的祖细胞。 这个问题太 与成年动物气道中AAV转导的效率不同 因此必须使用替代实验方法来研究这一点 问题 此外，野生型AAV的研究基础表明， AAV重组基因组整合位点特异性地在 19号染色体。 虽然目前的研究评估网站的具体 重组AAV在细胞系中整合表明， 随机的，关于这种整合的序列特异性知之甚少 过程 这些信息可用于潜在地改善 AAV整合的效率和靶向。 使用逆转录病毒标记 研究，我们的实验室已经定义了多种人气道祖细胞 具有不同分化能力的基因治疗靶点。 之间 这些祖细胞包括具有多能性的惩罚性干细胞， 气道表面上皮和粘膜下层的发育。 的 用于基因治疗的大多数功能相关的细胞靶点包括 干细胞或替代地产生CFTR那些祖细胞 在气道中表达细胞。 为了评估AAV载体用于 在气道和粘膜下腺体的子宫内基因治疗中，我们建议 使用支气管导管评估祖细胞亚群中的MV整合 异种移植模型系统。 评价细胞系中AAV整合的研究 在整合效率方面表现出高度的可变性。 这些差异可能是由于表达中的细胞特异性变异性 影响这种整合过程的细胞因子。 为此，委员会建议， 我们假设气道祖细胞的整合效率 也可以基于这些靶细胞的生物学差异而变化， 这是由它们不同的分化和再生能力所指示的。 此外，我们建议开发一种Alu PCR方法， 可以从体内显微切割的祖细胞克隆AAV的位点 克隆 这些信息将允许直接评估整合 序列特异性，并可能最终提供一个更好的理解， 影响整合过程的细胞差异。 更好的 了解AAV载体与适当气道的相互作用 靶向祖细胞可能最终导致合理方法来改变 载体整合效率和位点特异性。

DESCRIPTION (Taken directly from the application) The effectiveness of gene therapy approaches for cystic fibrosis will only be fully realized once a basic understanding of vector interactions with the target cell of the airway are understood. Ultimately, the best therapeutic approaches for gene therapy of CF will utilize single dose delivery of integrating vectors prior to the onset of disease. To date, only two vector systems including recombinant retroviruses and AAV have the ability to integrate recombinant transgenes within the host genomes. Retroviruses although extremely efficient in their integrating process are limited by achievable titers for in vivo delivery. In contrast AAV vectors provide a more attractive alternative with much higher achievable titers but have recently been troubled by the inefficiency of integration as studied in cell lines. However, the efficiency of the AAV; integrating process appears to be effected by the recipient cell type and cellular factors which regulate the integration process. Therefore, since the relative utility of AAV for gene therapy to the airway will be dependent on the recipient cell type, an obvious question remains as to the efficiency of integration in appropriate target progenitor cells of the human airway. This question is much different than the efficiency of AAV transduction in adult animal airways and hence must use alternative experimental approaches to investigate this question. Furthermore, the foundation of studies on wild type AAV suggests that the AAV recombinant genome integrates site specifically within chromosome 19. Although present studies evaluating the site specific integration of recombinant AAV in cell lines suggest that integration is random, little is known about the sequence specificity of this integration process. Such information could be used to potentially improve the efficiency and targeting of AAV integration. Using retroviral marking studies, our laboratory have defined multiple human airway progenitor cell targets for gene therapy with varying capacities for differentiation. Among these progenitors is included a punitive stem cell which has pluripotent development for surface airway epithelium as well as submucosal liands. The most functionally relevant cellular targets for gene therapy involve this stem cell or alternatively those progenitor cells which give rise to CFTR expressing cells in the airway. To evaluate the utility of AAV vectors for in utero gene therapy in the airway and submucosal glands, we propose to evaluate MV integration in progenitor cell subsets using a bronchial xenograft model system. Studies evaluating AAV integration in cell lines demonstrate a high level of variability in the efficiency of integration. These differences may be due to cell specific variability in the expression of cellular factors which influence this integration process. To this end, we hypothesize that the efficiency of integration in airway progenitor cells may also vary based on the differences in biology of these target cells as indicated by their varying capacity for differentiation and regeneration. Furthermore, we propose to developed an Alu PCR method by which integration sites of AAV can be cloned from in vivo microdissected progenitor cell clones. This information will allow for direct evaluation of integration sequence specificity and may ultimately provide a better understand of the cellular differences which effect the integration process. A better understanding of the interactions of AAV vectors with the appropriate airway target progenitor cells may ultimately lead to rational methods to alter vector integration efficiencies and site specificities.