New Gene Vectors for Cystic Fibrosis (CF): Extra- & Intracellular Barriers

描述（由申请人提供）：开发囊性纤维化（CF）非病毒基因载体的主要限制是很少关注粘膜屏障对基因载体稳定性的影响以及对它们进入和有效进入肺上皮细胞核、在其内运输和将货物DNA递送至肺上皮细胞核的能力的影响。粘液成分的吸收通常使基因载体不稳定，并显著改变影响基因递送效率的重要载体理化性质。然而，旨在克服一个障碍（例如粘蛋白吸收）的修饰可能会产生其他显著的障碍（例如降低细胞进入率）。我们的总体假设是，一个更全面和定量的“系统”方法来鉴定成功的CF基因递送的障碍，将允许我们合理合成新的基因载体，其抵抗粘液吸收，并且能够：（i）快速转运通过粘膜屏障;（ii）在CF粘液中孵育后容易进入人CF支气管上皮细胞;（iii）在细胞进入数分钟内在细胞核周围和细胞核中有效和主动的积累;和（iv）在细胞培养物中和在具有已在CF粘液中预孵育的基因载体的CF小鼠模型中显著改善的基因表达。具体而言，从新型高度致密（小于22 nm的小直径）聚合物基因载体的合成和表征（目标1）开始，该提议将利用许多强大的生物物理技术来鉴定和量化基因载体通过人CF粘液（目标2）和通过细胞到细胞核（目标3）的有效转运的限速障碍。为了确保临床相关性，将在化脓/感染痰和在空气界面生长的分化的原代人支气管上皮细胞中研究基因载体转运，每种细胞均新鲜获得自CF患者。将在CF小鼠模型中测试有前景的载体的功效（目标4）。目标2-4中重要障碍的鉴定将指导基因载体物理化学性质和表面化学的合理修饰（再次目标1）以潜在地克服瓶颈。一个具有生物工程/生物物理学、气溶胶基因递送、细胞贩运/生物学和CF专业知识的跨学科团队已经组装起来调查这一假设，其长期目标是安全有效的CF基因治疗。

DESCRIPTION (provided by applicant): A major limitation in the development of non-viral gene carriers for Cystic Fibrosis (CF) has been the sparse attention paid to effects of the mucosal barrier on stability of gene carriers and on their ability to access and efficiently enter, traffic within, and deliver cargo DNA to the nucleus of lung epithelial cells. Absorption of mucus components often destabilizes gene carriers and significantly changes important carrier physicochemical properties that affect gene delivery efficiency. However, modifications aimed at overcoming one barrier (e.g. mucin absorption) may create other significant barriers (e.g. reduced cell entry rate). Our overall hypothesis is that a more comprehensive and quantitative "systems" approach to the identification of barriers to successful gene delivery for CF will allow our rational synthesis of novel gene carriers that resist mucus absorption and are capable of: (i) rapid transport through the mucosal barrier; (ii) facile entry into human CF bronchial epithelial cells following incubation in CF mucus; (iii) efficient and active accumulation around and in the cell nucleus within minutes of cell entry; and (iv) significantly improved gene expression in cell culture and in CF mouse models with gene carriers that have been pre-incubated in CF mucus. Specifically, starting with the synthesis and characterization of novel highly compacted (<22 nm in minor diameter) polymeric gene carriers (Aim 1), this proposal will utilize a number of powerful biophysical techniques to identify and quantify the rate limiting barriers to efficient gene carrier transport through human CF mucus (Aim 2) and through the cell to the nucleus (Aim 3). To ensure clinical relevance, gene carrier transport will be investigated in purulent/infected sputum and differentiated primary human bronchial epithelial cells grown at an air-interface, each freshly obtained from CF patients. Promising carriers will be tested for efficacy in a CF mouse model (Aim 4).The identification of important barriers in Aims 2-4 will guide the rational modification of the gene carrier physicochemical properties and surface chemistries (Aim 1 again) to potentially overcome the bottleneck. An interdisciplinary team, with expertise in bioengineering/biophysics, aerosol gene delivery, cellular trafficking/biology, and CF, has been assembled to investigate the hypothesis, with a long-term goal of safe and effective CF gene therapy.