Gregg A. Duncan

The mucus barrier to inhaled gene therapy

Recent evidence suggests that the airway mucus gel layer may be impermeable to the viral and synthetic gene vectors used in past inhaled gene therapy clinical trials for diseases like cystic fibrosis. These findings support the logic that inhaled gene vectors that are incapable of penetrating the mucus barrier are unlikely to provide meaningful benefit to patients. In this review, we discuss the biochemical and biophysical features of mucus that contribute its barrier function, and how these barrier properties may be reinforced in patients with lung disease. We next review biophysical techniques used to assess the potential ability of gene vectors to penetrate airway mucus. Finally, we provide new data suggesting that fresh human airway mucus should be used to test the penetration rates of gene vectors. The physiological barrier properties of spontaneously expectorated CF sputum remained intact up to 24 hours after collection when refrigerated at 4 °C. Conversely, the barrier properties were significantly altered after freezing and thawing of sputum samples. Gene vectors capable of overcoming the airway mucus barrier hold promise as a means to provide the widespread gene transfer throughout the airway epithelium required to achieve meaningful patient outcomes in inhaled gene therapy clinical trials.

Barriers to inhaled gene therapy of obstructive lung diseases: A review

Knowledge of genetic origins of obstructive lung diseases has made inhaled gene therapy an attractive alternative to the current standards of care that are limited to managing disease symptoms. Initial lung gene therapy clinical trials occurred in the early 1990s following the discovery of the genetic defect responsible for cystic fibrosis (CF), a monogenic disorder. However, despite over two decades of intensive effort, gene therapy has yet to help patients with CF or any other obstructive lung disease. The slow progress is due in part to poor understanding of the biological barriers to inhaled gene therapy. Encouragingly, clinical trials have shown that inhaled gene therapy with various viral vectors and non-viral gene vectors is well tolerated by patients, and continued research has provided valuable lessons and resources that may lead to future success of this therapeutic strategy. In this review, we first introduce representative obstructive lung diseases and examine limitations of currently available therapeutic options. We then review key components for successful execution of inhaled gene therapy, including gene delivery systems, primary physiological barriers and strategies to overcome them, and advances in preclinical disease models with which the most promising systems may be identified for human clinical trials.

Microstructural alterations of sputum in cystic fibrosis lung disease

The stasis of mucus secretions in the lungs of cystic fibrosis (CF) patients leads to recurrent infections and pulmonary exacerbations, resulting in decreased survival. Prior studies have assessed the biochemical and biophysical features of airway mucus in individuals with CF. However, these measurements are unable to probe mucus structure on microscopic length scales relevant to key players in the progression of CF-related lung disease, namely, viruses, bacteria, and neutrophils. In this study, we quantitatively determined sputum microstructure based on the diffusion of muco-inert nanoparticle probes in CF sputum and found that a reduction in sputum mesh pore size is characteristic of CF patients with reduced lung function, as indicated by measured FEV1. We also discovered that the effect of ex vivo treatment of CF sputum with rhDNase I (Pulmozyme) on microstructure is dependent upon the time interval between the most recent inhaled rhDNase I treatment and the sample collection. Microstructure of mucus may serve as a marker for the extent of CF lung disease and as a parameter for assessing the effectiveness of mucus-altering agents.

Protein nanocages that penetrate airway mucus and tumor tissue

Significance In designing new nanoparticle drug delivery systems, it is critical to identify simple formulations that overcome multiple biological barriers while being safe, reproducible, and scalable. We modified human ferritin nanocages using a unique PEGylation strategy, which provides a highly uniform, stable, and compact nanocarrier platform capable of overcoming multiple biological barriers, specifically penetration of airway mucus and tumor tissue, selective uptake by cancer cells, and drug release triggered only upon cell uptake. Surprisingly, PEGylation of ferritin to overcome the mucus barrier did not interfere with the ability of the nanocages to form particles, penetrate tumor tissues, and enter cells. Proof-of-concept of the system is provided in the treatment of an aggressive orthotopic model of lung cancer. Reports on drug delivery systems capable of overcoming multiple biological barriers are rare. We introduce a nanoparticle-based drug delivery technology capable of rapidly penetrating both lung tumor tissue and the mucus layer that protects airway tissues from nanoscale objects. Specifically, human ferritin heavy-chain nanocages (FTn) were functionalized with polyethylene glycol (PEG) in a unique manner that allows robust control over PEG location (nanoparticle surface only) and surface density. We varied PEG surface density and molecular weight to discover PEGylated FTn that rapidly penetrated both mucus barriers and tumor tissues in vitro and in vivo. Upon inhalation in mice, PEGylated FTn with optimized PEGylation rapidly penetrated the mucus gel layer and thus provided a uniform distribution throughout the airways. Subsequently, PEGylated FTn preferentially penetrated and distributed within orthotopic lung tumor tissue, and selectively entered cancer cells, in a transferrin receptor 1-dependent manner, which is up-regulated in most cancers. To test the potential therapeutic benefits, doxorubicin (DOX) was conjugated to PEGylated FTn via an acid-labile linker to facilitate intracellular release of DOX after cell entry. Inhalation of DOX-loaded PEGylated FTn led to 60% survival, compared with 10% survival in the group that inhaled DOX in solution at the maximally tolerated dose, in a murine model of malignant airway lung cancer. This approach may provide benefits as an adjuvant therapy combined with systemic chemo- or immunotherapy or as a stand-alone therapy for patients with tumors confined to the airways.

National Heart, Lung, and Blood Institute Workshop Summary: Enhancing Opportunities for Training and Retention of a Diverse Biomedical Workforce.

RATIONALE Committed to its mission of conducting and supporting research that addresses the health needs of all sectors of the nations population, the Division of Lung Diseases, National Heart, Lung, and Blood Institute of the National Institutes of Health (NHLBI/NIH) seeks to identify issues that impact the training and retention of underrepresented individuals in the biomedical research workforce. OBJECTIVES Early-stage investigators who received grant support through the NIH Research Supplements to Promote Diversity in Health Related Research Program were invited to a workshop held in Bethesda, Maryland in June, 2015, in order to (1) assess the effectiveness of the current NHLBI diversity program, (2) improve its strategies towards achieving its goal, and (3) provide guidance to assist the transition of diversity supplement recipients to independent NIH grant support. METHODS Workshop participants participated in five independent focus groups to discuss specific topics affecting underrepresented individuals in the biomedical sciences: (1) Socioeconomic barriers to success for diverse research scientists; (2) role of the academic research community in promoting diversity; (3) life beyond a research project grant: non-primary investigator career paths in research; (4) facilitating career development of diverse independent research scientists through NHLBI diversity programs; and (5) effectiveness of current NHLBI programs for promoting diversity of the biomedical workforce. MEASUREMENTS AND MAIN RESULTS Several key issues experienced by young, underrepresented biomedical scientists were identified, and solutions were proposed to improve on training and career development for diverse students, from the high school to postdoctoral trainee level, and address limitations of currently available diversity programs. Although some of the challenges mentioned, such as cost of living, limited parental leave, and insecure extramural funding, are also likely faced by nonminority scientists, these issues are magnified among diversity scientists and are complicated by unique circumstances in this group, such as limited exposure to science at a young age, absence of role models and mentors from underrepresented backgrounds, and social norms that relegate their career endeavors, particularly among women, to being subordinate to their expected cultural role. CONCLUSIONS The factors influencing the participation of underrepresented minorities in the biomedical workforce are complex and span several continuous or overlapping stages in the professional development of scientists from these groups. Therefore, a multipronged approach is needed to enable the professional development and retention of underrepresented minorities in biomedical research. This approach should address both individual and social factors and should involve funding agencies, academic institutions, mentoring teams, professional societies, and peer collaboration. Implementation of some of the recommendations, such as access to child care, institutional support and health benefits for trainees, teaching and entrepreneurial opportunities, grant-writing webinars, and pre-NIH career development (Pre-K) pilot programs would not only benefit biomedical scientists from underrepresented groups but also improve the situation of nondiverse junior scientists. However, other issues, such as opportunities for early exposure to science of disadvantaged/minority groups, and identifying mentors/life coaches/peer mentors who come from similar cultural backgrounds and vantage points, are unique to this group.

Tunable Aggregation by Competing Biomolecular Interactions

Measurements and models are reported for Concanavalin A (ConA) mediated aggregation of dextran coated colloids that is tunable via a competing ConA-glucose interaction. Video and confocal scanning laser microscopy were used to characterize ConA adsorption to dextran colloids and quasi-2D dextran coated colloid aggregation kinetics vs [ConA] and [glucose]. ConA adsorption to, and aggregation rates of, dextran coated colloids increased from negligible values to high coverage and rapid rates for increasing [ConA] in the range 0.1-10 mM and decreasing [glucose] in the range 1-100 mM, consistent with dissociation constant estimates. Analysis of colloidal aggregation kinetics indicates ConA bridge formation is the rate-limiting step controlling the transition from slow to rapid aggregation. Our findings reveal a mechanism for tuning colloidal interactions and aggregation kinetics through specific, competitive biomolecular interactions, which lends insights into aggregation phenomena in mixed synthetic-biomaterial and biological systems.

An Adeno-Associated Viral Vector Capable of Penetrating the Mucus Barrier to Inhaled 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.

PEGylated enhanced cell penetrating peptide nanoparticles for lung gene therapy

ABSTRACT 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.

Fate of PEGylated antibody fragments following delivery to the lungs: Influence of delivery site, PEG size and lung inflammation.

ABSTRACT Pulmonary administration of anti‐cytokine antibodies offers a targeted therapy in asthma. However, the rapid elimination of proteins from the lungs limits the efficacy of inhaled medications. PEGylation has been shown to increase the residence time of anti‐interleukin (IL)‐17A and anti‐IL‐13 antibody fragments in the lungs and to improve their therapeutic efficacy. Yet, little is known about the factors that affect the residence time of PEGylated antibody fragments in the lungs following pulmonary delivery. In this study, we showed that the molecular weight of polyethylene glycol (PEG), 20 kDa or 40 kDa, had a moderate effect on the residence time of an anti‐IL‐17A Fab′ fragment in the lungs of mice. By contrast, the site of delivery of the anti‐IL‐17A and anti‐IL‐13 Fab′ fragments within the lungs had a major impact on their residence time, with the deeper the delivery, the more prolonged the residence time. The nature of the Fab′ fragment had an influence on its residence time as well and the anti‐IL‐17A Fab′ benefited more from PEGylation than the anti‐IL‐13 Fab′ did. Acute lung inflammation slightly shortened the residence time of the anti‐IL‐17A and anti‐IL‐13 Fab′ fragments in the lungs but PEGylation was able to prolong their presence in both the healthy and inflamed lungs. Antibody fragments were predominately located within the airway lumen rather than the lung parenchyma. Transport experiments on monolayers of Calu‐3 cells and studies of fluorescence recovery after photobleaching in respiratory mucus showed that mechanisms involved in the prolonged presence of PEGylated Fab′ in the airway lumen might include binding to the mucus, reduced uptake by respiratory cells and reduced transport across lung epithelia. Finally, using I125‐labeled anti‐IL‐17A Fab′, we showed that the protein fragment hardly penetrated into the lungs following subcutaneous injection, as opposed to pulmonary delivery. Graphical abstract Figure. No Caption available.

Pseudomonas aeruginosa pyocyanin production reduced by quorum-sensing inhibiting nanocarriers

Graphical abstract Figure. No Caption available. Abstract Pseudomonas aeruginosa is an opportunistic gram‐negative pathogen that causes a wide range of infections; it is becoming increasingly difficult to treat due to antibiotic resistance. Quorum‐sensing (QS) based therapeutics, which function by disabling pathogen virulence without killing pathogens, are a promising class of drugs that may be used to treat bacterial infections without eliciting resistance development. The use of QS drugs to treat pulmonary P. aeruginosa infections, however, has been greatly limited due to the inability to deliver QS drugs at sufficiently high concentrations past physiological barriers such as pulmonary mucus. Here we apply a block copolymer‐directed self‐assembly process, Flash NanoPrecipitation, to develop a series of QS‐active formulations that are fully water dispersible, stable, and mucus‐penetrating. These formulations inhibit P. aeruginosa virulence without inhibiting cell growth. Particle size (70 nm–400 nm) and release rate (1 h–14 days) can be tuned by altering constructs’ physical properties and formulation excipients. We also demonstrate, to the best of our knowledge, the first instance of a QS nanocarrier platform technology that can penetrate through human cystic fibrosis pulmonary mucus. This work highlights the need to incorporate nanoformulation strategies into the development of next‐generation antimicrobial therapeutics.