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）的非病毒基因载体的主要限制是对粘膜屏障对基因载体稳定性的影响以及对其进入和有效进入、在内部运输并将货物DNA递送到肺上皮细胞核的能力的影响的关注较少。黏液成分的吸收往往使基因载体不稳定，并显著改变影响基因传递效率的重要载体理化性质。然而，旨在克服一个障碍（如粘蛋白吸收）的修饰可能会产生其他重大障碍（如降低细胞进入率）。我们的总体假设是，一种更全面和定量的“系统”方法来识别CF基因成功传递的障碍，将使我们能够合理地合成新的基因载体，这些基因载体能够抵抗粘液吸收，并能够：(i)通过粘膜屏障快速运输；（ii）在CF粘液中孵育后容易进入人CF支气管上皮细胞；（iii）在细胞进入细胞后的几分钟内，在细胞核周围和细胞核内有效和活跃地积累；（iv）细胞培养和CF小鼠模型中基因载体在CF粘液中预孵育的基因表达显著提高。具体来说，从合成和表征新型高度致密（小直径<22 nm）聚合物基因载体（Aim 1）开始，本提案将利用许多强大的生物物理技术来识别和量化基因载体通过人类CF粘液（Aim 2）和通过细胞到细胞核（Aim 3）的有效运输的速率限制障碍。为了确保临床相关性，我们将在化脓性/感染的痰液和在空气界面培养的分化的原代人支气管上皮细胞中研究基因载体的转运，每一个细胞都是从CF患者新鲜获得的。有希望的携带者将在CF小鼠模型中测试其功效（Aim 4）。Aims 2-4中重要障碍的识别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Aims 1），以潜在地克服瓶颈。一个跨学科的团队，汇集了生物工程/生物物理学、气溶胶基因传递、细胞运输/生物学和CF方面的专业知识，以安全有效的CF基因治疗的长期目标来研究这一假设。

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.