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

描述(申请人提供)：囊性纤维化(CF)的非病毒基因载体发展的一个主要限制是对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、运输和运送货物DNA到肺上皮细胞核的能力的影响的关注很少。粘液成分的吸收通常会破坏基因载体的稳定性，并显著改变影响基因传递效率的重要载体的物理化学性质。然而，旨在克服一种障碍(例如，粘蛋白吸收)的修改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，用更全面和更定量的“系统”方法来确定成功传递CF基因的障碍将使我们能够合理地合成新型基因载体，这种基因载体可以抵抗黏液吸收，并且能够：(I)快速穿透黏膜屏障；(Ii)在CF黏液中孵育后轻松进入人CF支气管上皮细胞；(Iii)在细胞进入几分钟内在细胞核周围和细胞核内高效而活跃地积聚；(Iv)在细胞培养和CF小鼠模型中显著提高细胞培养和预先在CF黏液中孵育的基因载体的基因表达。具体地说，从新型高度致密(小直径22 nm)聚合物基因载体的合成和表征(目标1)开始，这项建议将利用一些强大的生物物理技术来鉴定和量化有效的基因载体通过人CF粘液(目标2)和通过细胞到细胞核(目标3)传输的限速障碍。为了确保临床相关性，将研究在空气界面上生长的化脓性/感染性痰和分化的原代人支气管上皮细胞中的基因载体运输，每种细胞都是新鲜从CF患者获得的。有希望的载体将在CF小鼠模型(AIM 4)上进行有效性测试。AIMS 2-4中重要障碍的识别将指导对基因载体的物理化学性质和表面化学的合理修饰(AIM 1)，以潜在地克服瓶颈。一个具有生物工程/生物物理学、气溶胶基因传递、细胞运输/生物学和CF的专业知识的跨学科团队已经被组织起来调查这一假说，其长期目标是安全和有效的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.