GENE THERAPY STRATEGIES FOR TREATMENT OF MUCO-OBSTRUCTIVE LUNG DISEASES

Abstract

Knowledge of genetic origins and associations of muco-obstructive lung diseases has made inhaled gene therapy an attractive alternative to the current standards of care that are limited to managing disease symptoms. However, despite over two decades of intensive research and development, gene therapy has yet to help patients with cystic fibrosis (CF) or any other muco-obstructive lung diseases. The slow progress is due in part to poor understanding of the biological barriers to inhaled gene therapy. In this dissertation, I first introduce the pathobiology of representative muco-obstructive lung diseases and examine pitfalls of clinically investigated gene vectors of the past and of current options. I then review key components for successful execution of inhaled gene therapy, including gene delivery systems, physiological barriers and strategies to overcome them, and advances in preclinical models with which the most promising systems may be vetted for clinical trials. Secondly, I demonstrate that adeno-associated vectors (AAV), which are more commonly used gene vectors for clinical settings, differ in their ability to diffuse through the CF sputum barrier and mediate various levels of transduction depending on the surface chemistry. Specifically, I compared three AAV vectors in their ability to i) diffuse in CF sputum, ii) provide transgene expression in ALI culture of primary human CF bronchial epithelial cells, and iii) provide transgene expression in a mouse model of muco-obstructive lung diseases. Thirdly, I present the application of a synthetic biodegradable gene vector in ex vivo, in vitro, and in vivo models relevant to muco-obstructive diseases. This gene vector is composed of engineered poly(β-amino ester) polymers and nucleic acid that encodes reporter or therapeutically relevant genes. I found that this gene vector, compared to conventional gene vector, is able to i) efficiently diffuse through CF sputum, ii) safely mediate higher magnitude of and widespread transgene expression in healthy and muco-obstructive lung mouse model, and iii) apically transfer reporter gene to mucus-covered air-liquid interface (ALI) culture of primary human CF bronchial epithelial cells harvested from CF patient lungs with F508del homozygous mutation, the most common form of mutation in the CF patient population.