Despite the progress made in AAV-based gene therapy targeting different organ systems, lung-targeted gene therapy using AAV vectors has not been effective, mostly due to the poor transduction and un-sustained gene expression in airway epithelium. Furthermore, concerns over possible harmful insertional mutagenesis seen in other cell types, particularly hepatocytes, raised a question about AAV safety. In this study, we evaluate the long-term persistence of this vector in mouse lungs and any possible harmful integration of these vectors into the host genome. AAV6 vectors expressing reporter gene (firefly luciferase) were delivered to the lungs of C57BL/6 mice through intra-tracheal intubation. Despite the large variation among individual animals, most animals had high and sustained luciferase activity with a peak from 2 to 3 weeks post-transduction before a significant decline between 15 and 19 weeks post-transduction. More importantly, even after its decline, most animals maintained detectable luciferase expression for 150 days or more, which was confirmed by post-necropsy qPCR analysis of luciferase gene expression. At the termination point of experiments, an average of one copy of AAV expression cassette per mouse genome was detected. We also found that partial overlaps between the AAV6 expression cassette and the mouse genome were distributed broadly with no apparent systematic preference in any mouse chromosomal map location. In summary, our data suggest that AAV6 mediated long-term gene expression in the lungs with no evidence of genomic integration, and thus, any insertional mutagenesis.

Objective: Patients' with cystic fibrosis respiratory systems become colonized with pathogens as early as the first year of life. Some organisms are associated with a decline in pulmonary function and shortened survival, such as a mucoid strain of Pseudomonas aeruginosa (PA). Methods:We conducted a four year retrospective chart review study examining 158 pediatric and adult cystic fibrosis patients treated at an academic, tertiary care institution to compare patients treated by different antimicrobial regimens.Results: A higher proportion of mucoid PA study patients received alternate-monthly tobramycin therapy (83.3%), compared to those with non-mucoid PA (62.5%); although a large percentage of patients who had never had a mucoid PA infection were also receiving alternate-monthly tobramycin. Patients who had a mucoid PA infection had more hospitalizations and for longer periods of time than patients who had a non-mucoid PA infection; both mucoid and non-mucoid PA patients were hospitalized more often than patients who had never had a PA infection. We additionally found patients with mucoid PA infections to have strains that were either resistant or had intermediate resistance to tobramycin. Similar trends in resistance were not seen in patients who only received intermittent treatments with tobramycin. ConclusionsOur findings on increased rates of infection with both Aspergillus (in the alternate-monthly inhaled tobramycin group) and NTM (in the overall patient population) comparative to international averages were also interesting, and open the doors to future research regarding CF patients with these infections. J Microbiol Infect Dis 2017; 7(4):178-185