The lung remains an attractive target for the gene therapy of monogenetic diseases such as cystic fibrosis (CF). Despite over 27 clinical trials, there are still very few gene therapy vectors that have shown any improvement in lung function; highlighting the need to develop formulations with improved gene transfer potency and the desirable physiochemical characteristics for efficacious therapy. Herein, we introduce a novel cell penetrating peptide (CPP)-based non-viral vector that utilises glycosaminoglycan (GAG)-binding enhanced transduction (GET) for highly efficient gene transfer. GET peptides couple directly with DNA through electrostatic interactions to form nanoparticles (NPs). In order to adapt the GET peptide for efficient in vivo delivery, we engineered PEGylated versions of the peptide and employed a strategy to form DNA NPs with different densities of PEG coatings. We were able to identify candidate formulations (PEGylation rates ≥40%) that shielded the positively charged surface of particles, maintained colloidal stability in bronchoalveolar lavage fluid (BALF) and retained gene transfer activity in human bronchial epithelial cell lines and precision cut lung slices (PCLS) in vitro. Using multiple particle tracking (MPT) technology, we demonstrated that PEG-GET complexes were able to navigate the mucus mesh and diffuse rapidly through patient CF sputum samples ex vivo. When tested in mouse lung models in vivo, PEGylated particles demonstrated superior biodistribution, improved safety profiles and efficient gene transfer of a reporter luciferase plasmid compared to non-PEGylated complexes. Furthermore, gene expression was significantly enhanced in comparison to polyethylenimine (PEI), a non-viral gene carrier that has been widely tested in pre-clinical settings. This work describes an innovative approach that combines novel GET peptides for enhanced transfection with a tuneable PEG coating for efficacious lung gene therapy.

Diffusion of the viral vectors evaluated in inhaled gene therapy clinical trials to date are largely hindered within airway mucus, which limits their access to, and transduction of, the underlying airway epithelium prior to clearance from the lung. Here, we discovered that adeno-associated virus (AAV) serotype 6 was able to rapidly diffuse through mucus collected from cystic fibrosis (CF) patients, unlike previously tested AAV serotypes. A point mutation of the AAV6 capsid suggests a potential mechanism by which AAV6 avoids adhesion to the mucus mesh. Significantly greater transgene expression was achieved with AAV6 compared to a mucoadhesive serotype, AAV1, in air-liquid interface cultures of human CF bronchial epithelium with naturally secreted mucus or induced mucus hypersecretion. In addition, AAV6 achieved superior distribution and overall level of transgene expression compared to AAV1 in the airways and whole lungs, respectively, of transgenic mice with airway mucus obstruction. Our findings motivate further evaluation and clinical development of AAV6 for inhaled gene therapy.

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE (DD-MM-YYYY) 2. REPORT TYPE 3. DATES COVERED (From -To) 00-10-2014 Technical15 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S)DTRA/J9CBI SPONSOR/MONITOR'S REPORT NUMBER(S)DTRA-TR-15-1 DISTRIBUTION / AVAILABILITY STATEMENTDisitribution Statement A: Approved for public release; distribution is unlimited. SUPPLEMENTARY NOTESThis work was sponsored by the Defense Threat Reduction Agency. ABSTRACT Introduction and PurposeAccurate modeling of medical countermeasure efficacy against chemical, biological and radiological agents (CBR agents) is essential to understanding the vulnerability of our warfighters on the modern battlefield. In helping to calculate the benefit of countermeasures, modeling can inform data-driven purchasing decisions and logistical tradeoffs. In this study, Gryphon Scientific and Applied Research Associates, Inc. (ARA) have developed models to predict the efficacy of medical countermeasures in preventing casualties and reducing the severity and duration of illness caused by CBR agents. This report (prepared by ARA) is one of five, describing the medical countermeasure models constructed for this project. This volume focuses exclusively on the modeling approach, parameters, and calculations used for the medical countermeasure model (MCM) for cesium-137 (Cs-137) exposure. The composite model includes the appropriate parameters necessary for calculating the impact of Prussian Blue (PB) countermeasure treatment on Cs-137 exposure.This paper presents an inhalation exposure model for calculating the absorbed fraction of cesium-137 from a given air concentration. The paper also presents a biokinetic model for calculating the distribution of cesium-137 within the body, a Prussian Blue model for binding and removal of cesium-137 circulating through the gastrointestinal tract, and a radiation dose model for calculating the absorbed radiation dose in the whole body and to the critical organ, the red bone marrow. Each model approach is described and justified along with the assumptions and key parameters that are implemented in each model. The composite m...

Here, a new paradigm is introduced to promote pulmonary DNA vaccination. Specifically, it is demonstrated that nanoparticles designed to rapidly penetrate airway mucus (mucus-penetrating particle or MPP) enhance the delivery of an inhaled model DNA vaccine (i.e., ovalbumin-expressing plasmids) to pulmonary dendritic cells (DC), leading to robust and durable local and trans-mucosal immunity. In contrast, mucus-impermeable particles are poorly taken up by pulmonary DC following inhalation, despite their superior ability to mediate DC uptake in vitro compared to MPP. In addition to the enhanced immunity achieved in mucosal surfaces, inhaled MPP unexpectedly provides significantly greater systemic immune responses compared to gold-standard approaches applied in the clinic for systemic vaccination, including intradermal injection and intramuscular electroporation. It is also shown here that inhaled MPP significantly enhances the survival of an orthotopic mouse model of aggressive lung cancer compared to the gold-standard approaches. Importantly, it is discovered that MPP-mediated pulmonary DNA vaccination induces memory T-cell immunity, particularly the ready-to-act effector memory-biased phenotype, both locally and systemically. The findings here underscore the importance of breaching the airway mucus barrier to facilitate DNA vaccine uptake by pulmonary DC and thus to initiate full-blown immune responses.