Cystic fibrosis (CF) gene therapy can potentially benefit all CF patients, regardless of the specific gene mutation leading to the disease state. However, gene therapy trials, using viral and non-viral vectors, have had disappointing outcomes to date due to inability to overcome biological barriers, including the purulent sputum gel layer, periciliary layer and epithelial surface. In particular, we previously found that several clinically and preclinically teted viral and non-viral gene vectors are trapped in human CF sputum, which prevents them from reaching and delivering their therapeutic gene cargo to the underlying airway epithelium. We recently developed two delivery strategies: (i) ultra-small DNA-loaded nanoparticles, called mucus-penetrating DNA nanoparticles (DNA-MPP) that efficiently penetrate human CF sputum and (ii) mildly hypotonic aqueous vehicles, which further improves DNA-MPP penetration through the sputum gel layer and periciliary layer via osmosis-driven fluid absorption, and enhances DNA-MPP uptake by the airway epithelium via the regulatory volume decrease mechanism. We found that the combination of these two approaches led to efficient gene transfer to the airway epithelium of normal mice in vivo and primary human airway cells in vitro. Here we propose to further develop and validate our combined approach in relevant in vitro and in vivo settings. To this end, we will evaluate CFTR gene transfer to CF human primary airway epithelium grown at air-liquid interface and in the lung airways of CF mouse model thoroughly characterized for its CF-like lung diseases. We will also determine whether our delivery strategies allow persistent transgene expression upon a single and repeated dosing without eliciting acute and chronic toxicity. If we are successful, we will test our strategies in macaques and potentially clinical studies in the future.

Public Health Relevance

The gene that could cure cystic fibrosis (CF) has been available since 1989, yet no one has been cured; damage to the lungs is the primary source of morbidity and mortality in CF patients. We found that none of the gene delivery systems used in CF gene therapy clinical trials to date, including various viruses, are capable of overcoming the three foremost barriers to successful gene therapy in the airways: mucus gel barrier, periciliary layer barrier, and airway cell uptake barrier. We seek to test novel gene vectors in combination with a novel aqueous vehicle that we hypothesize will greatly enhance the effectiveness of gene transfer in the lungs.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Sheridan, John T
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Johns Hopkins University
Schools of Medicine
United States
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