A Hybrid Viral/Nonviral Vector for CFTR Delivery to CF Pig Airways

项目摘要/摘要 基因治疗是近30年前首次提出的一种可行的遗传病治疗策略。 最近有许多值得注意的成功的多种疾病的基因治疗试验；然而，基因 囊性纤维化(CF)的治疗遇到了许多意想不到的障碍。例如，1)呼吸道细胞具有 进化出许多障碍来阻止吸收外来DNA，2)CFs的小鼠模型通常没有肺部疾病， 3)每一种标准的基因传递工具都有优缺点。我们有良好的业绩记录 利用多种类型的病毒和非病毒试剂将基因输送到呼吸道。基于病毒的 载体是将基因输送到呼吸道细胞的最有效的方式。我们的长期目标是设计一种试剂 对于将基因传递到细胞是治疗CF肺部疾病的有效方法，但迫切需要 改进了基因传递工具。在这里，我们开发了一种新型试剂。我们建议证明我们的 工程载体可以整合到呼吸道祖细胞的基因组中并表达转基因 在适当的表面细胞中。为此，我们结合了基于腺病毒的载体的效率 以DNA转座子为基础的非病毒载体的持续表达。此外，我们还测试了这种“混血”。 在相关的CF大型动物模型中携带载体。在这里，我们演示了该载体将：1)将CFTR传递到CF 细胞并在体外纠正阴离子转运体缺陷，使用一种新的策略来集中细胞整合和 表达，2)验证必要水平的基因转移在活体猪的呼吸道中是可以实现的，以及3) 纠正猪CF模型呼吸道阴离子通道缺陷和疾病进展。我们调查了一个 基因传递工具，可用于解决其他试剂无法解决的问题。有&gt；2000 引起cftr突变的已知疾病。我们的目标是提供一种终身基因替换策略， 将是有效的，无论是什么疾病导致突变。这项研究具有很强的创新性。 结合基于DNA转座子的载体和研究良好的腺病毒载体的新兴技术 提供了一个令人兴奋的新机会来提高治疗基因输送载体的效用，例如 CFTR。这些实验产生的试剂、方法和数据将为基因研究提供指导 其他单基因疾病的治疗，从而显著推进了基因治疗领域。

Project Summary/Abstract Gene therapy was first proposed as a viable therapeutic strategy for genetic disease nearly 30 years ago. There are many notable recent successful gene therapy trials for multiple diverse diseases; however, gene therapy for cystic fibrosis (CF) has presented many unexpected roadblocks. For example, 1) airway cells have evolved many barriers to prevent uptake of foreign DNA, 2) mouse models of CF generally lack lung disease, and 3) each of the standard gene delivery vehicles have pros and cons. We have a demonstrated track record of utilizing many categories of viral and non-viral based reagents for gene delivery to the airways. Viral based vectors are the most efficient way to deliver genes to airway cells. Our long-term goal is to engineer a reagent for gene delivery to cells that is an effective therapeutic for CF lung disease, yet there is a critical need for improved gene delivery tools. Here, we develop a novel reagent. We propose to demonstrate that our engineered vector can integrate into the genome of progenitor cells in the airways and express the transgene in the appropriate surface cells. To this end, we have combined the efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based non-viral vector. In addition, we test this `hybrid' vector in a relevant large animal model of CF. Here we demonstrate that this vector will: 1) deliver CFTR to CF cells and correct the anion transporter defect in vitro using a novel strategy to focus cellular integration and expression, 2) verify that the necessary levels of gene transfer are achievable in pigs airways in vivo, and 3) correct the anion channel defect and disease progression in the airways of a pig CF model. We investigate a gene delivery tool that can be used to address questions that no other reagent can address. There are >2000 known disease causing mutations in CFTR. Our goal is to provide a life-long gene replacement strategy that would be efficacious regardless of the disease causing mutation. This proposed research is highly innovative. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral delivery presents an exciting new opportunity to improve the utility of delivery vehicles for therapeutic genes such as CFTR. The reagents, methods, and data generated by these experiments will provide guidance for gene therapies for other monogenic disorders, thereby significantly advancing the gene therapy field.