New approaches to overcome the sputum barrier to gene delivery

基因载体穿透呼吸道中的CF痰屏障是基因成功转染的先决条件 用雾化基因载体治疗CF。然而，我们发现，主要的病毒和非病毒基因 迄今为止，我们测试的CF载体不能穿透高粘性和弹性的痰液 从CF患者的气道获得。这一发现可能有助于解释令人失望的临床结果， 基因疗法在气道，并指出迫切需要发现新的方法，促进病毒和 非病毒基因载体通过CF痰的渗透。我们的总体假设是， 病毒和非病毒基因载体的渗透可以通过以下方式实现：（i）调节生物物理性质 和/或（ii）用单克隆抗体掩蔽基因携带者或CF痰本身的粘附部分， 在人类中有安全使用历史的化学物质。该提案将利用强大的生物物理技术， 首先量化主要病毒和非病毒基因载体通过未稀释和未操作的 CF患者咳出的痰液，包括来自项目1和核心A的AAV（血清型1、2、5）， 由Project IV提供的重组AAV载体，目前在人类临床上使用的紧凑的DNA纳米颗粒， 试验，以及我们实验室设计的压实DNA纳米颗粒的非粘膜粘附变化（Aim 1A）。 我们还将研究限制基因载体快速渗透的机制， 研究CF痰液的微观结构和纳米级流变行为， 来自目标1A（Aim 1 B）的颗粒。随后，我们将测试潜在的协同方法， 降低痰液阻碍基因载体转运的屏障性质，包括：（i）以下的组合： 靶向痰液主要成分的粘液溶解剂（目标2A），（ii）给予牺牲性 在CF痰液中产生大开口的颗粒（Aim 2B），和（iii）使CF痰液中的大开口最小化的化学涂层， 基因携带者对痰液的粘附（Aim 2C）。如果成功，我们的工作将提供基本的见解， CF痰屏障，确定了新的策略，使有前途的基因载体，以克服可怕的 CF痰屏障，并导致基因递送效率的大幅提高 相关性（参见说明）： 基因载体穿透厚粘液分泌物是成功的基因治疗的必要条件， 囊性纤维化（CF）。我们已经发现，目前正在考虑的几种基因递送系统， CF潜在治愈剂不能穿透这些粘液分泌物。我们试图测试小说 策略，以提高有前途的病毒和非病毒基因载体的CF基因治疗的渗透率。

Gene carrier penetration through the CF sputum barrier in the airways is a prerequisite for successful gene therapy with aerosolized gene carriers for CF. However, we found that the leading viral and nonviral gene carriers for CF that we have tested to date are incapable of penetrating the highly viscous and elastic sputum obtained from the airways of CF patients. This finding may help explain the disappointing clinical results with gene therapy in the airways, and points to a dire need to discover new approaches that facilitate viral and non-viral gene carrier penetration through CF sputum. Our overall hypothesis is that improved sputum penetration by viral and non-viral gene carriers may be achieved by: (i) modulating the biophysical properties of CF sputum, and/or (ii) masking the adhesive portions of gene carriers or of CF sputum itself with simple chemicals with a history of safe use in humans. This proposal will utilize powerful biophysical techniques to first quantify the transport of leading viral and nonviral gene carriers through undiluted and unmanipulated sputum expectorated by CF patients, including AAV (serotypes 1,2,5) from Project 1 and Core A, recombinant AAV vectors provided by Project IV, compacted DNA nanoparticles currently in human clinical trials, and non-mucoadhesive variations of compacted DNA nanoparticles engineered by our lab (Aim 1 A). We will also investigate the mechanisms responsible for restricting rapid penetration of gene carriers by investigating the microstructure and nano-scale rheological behavior of CF sputum as probed by the various particles from Aim 1A (Aim1 B). Subsequently, we will test potentially synergistic methods by which to decrease the barrier properties of sputum that hinder gene carrier transport, including: (i) combinations of mucolytic agents that target major components of sputum (Aim 2A),(ii) pre-administration of sacrificial particles that create large openings in CF sputum (Aim 2B),and (iii) a chemical coating that minimizes adhesion of gene carriers to sputum (Aim 2C). If successful, our work will provide fundamental insights into the CF sputum barrier, identify new strategies that allow promising gene carriers to overcome theformidable CF sputum barrier, and lead to strongly improved gene delivery efficiency RELEVANCE (Seeinstructions): Penetration of gene carriers through thick mucus secretions is a requirement for successful gene therapy for Cystic Fibrosis (CF). We have discovered that several gene delivery systems currently being considered for the potential cure of CF are incapable of penetrating these mucus secretions. We seek to test novel strategies to improve the penetration rates of promising viral and nonviral gene carriers for CF gene therapy.