title: Overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors.
abstract: Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Schuster
authors.affiliation: Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Current address: Departments of Neurosurgery and Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, Maryland, USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Kim
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Joshua C
authors.initials: JC
authors.lastname: Kays
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Mia M
authors.initials: MM
authors.lastname: Kanzawa
authors.affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: William B
authors.initials: WB
authors.lastname: Guggino
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Johns Hopkins Adult Cystic Fibrosis Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Boyle
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Rowe
authors.affiliation: Department of Molecular Genetics and Microbiology, and Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Muzyczka
authors.affiliation: Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jung Soo
authors.initials: JS
authors.lastname: Suk
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland, USA. Electronic address: hanes@jhmi.edu.
authors.forename: Justin
authors.initials: J
authors.lastname: Hanes
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D002460:Cell Line; D003550:Cystic Fibrosis / Q000628:therapy / Q000821:virology*; D000229:Dependovirus / Q000145:classification / Q000235:genetics / Q000502:physiology*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000627:therapeutic use*; D057809:HEK293 Cells; D006801:Humans; D008855:Microscopy, Electron, Scanning; D013183:Sputum / Q000187:drug effects / Q000821:virology*
chemical_list: D000111:Acetylcysteine