Tyrosine-Mutant AAV8 Vector Mediated Efficient and Safe Gene Transfer of Pigment Epithelium-Derived Factor to Mouse Lungs.

BACKGROUND/AIMS: Recombinant adeno-associated viruses (rAAV) are an important tool for lung targeted gene therapy. Substitution of tyrosine with phenylalanine residues (Y-F) in the capsid have been shown to protect the AAV vector from ubiquitin/proteasome degradation, increasing transduction efficiency. We tested the mutant Y733F-AAV8 vector for mucus diffusion, as well as the safety and efficacy of pigment epithelium-derived factor (PEDF) gene transfer to the lung. METHODS: For this purpose, Y733F-AAV8-PEDF (1010 viral genome) was administered intratracheally to C57BL/6 mice. Lung mechanics, morphometry, and inflammation were evaluated 7, 14, 21, and 28 days after injection. RESULTS: The tyrosine-mutant AAV8 vector was efficient at penetrating mucus in ex vivo assays and at transferring the gene to lung cells after in vivo instillation. Increased levels of transgene mRNA were observed 28 days after vector administration. Overexpression of PEDF did not affect in vivo lung parameters. CONCLUSION: These findings provide a basis for further development of Y733F-AAV8-based gene therapies for safe and effective delivery of PEDF, which has anti-angiogenic, anti-inflammatory and anti-fibrotic activities and might be a promising therapy for lung inflammatory disorders.

Inhaled gene therapy of preclinical muco-obstructive lung diseases by nanoparticles capable of breaching the airway mucus barrier.

INTRODUCTION: Inhaled gene therapy of muco-obstructive lung diseases requires a strategy to achieve therapeutically relevant gene transfer to airway epithelium covered by particularly dehydrated and condensed mucus gel layer. Here, we introduce a synthetic DNA-loaded mucus-penetrating particle (DNA-MPP) capable of providing safe, widespread and robust transgene expression in in vivo and in vitro models of muco-obstructive lung diseases. METHODS: We investigated the ability of DNA-MPP to mediate reporter and/or therapeutic transgene expression in lung airways of a transgenic mouse model of muco-obstructive lung diseases (ie, Scnn1b-Tg) and in air-liquid interface cultures of primary human bronchial epithelial cells harvested from an individual with cystic fibrosis. A plasmid designed to silence epithelial sodium channel (ENaC) hyperactivity, which causes airway surface dehydration and mucus stasis, was intratracheally administered via DNA-MPP to evaluate therapeutic effects in vivo with or without pretreatment with hypertonic saline, a clinically used mucus-rehydrating agent. RESULTS: DNA-MPP exhibited marked greater reporter transgene expression compared with a mucus-impermeable formulation in in vivo and in vitro models of muco-obstructive lung diseases. DNA-MPP carrying ENaC-silencing plasmids provided efficient downregulation of ENaC and reduction of mucus burden in the lungs of Scnn1b-Tg mice, and synergistic impacts on both gene transfer efficacy and therapeutic effects were achieved when DNA-MPP was adjuvanted with hypertonic saline. DISCUSSION: DNA-MPP constitutes one of the rare gene delivery systems providing therapeutically meaningful gene transfer efficacy in highly relevant in vivo and in vitro models of muco-obstructive lung diseases due to its unique ability to efficiently penetrate airway mucus.

Roles of mucus adhesion and cohesion in cough clearance.

Clearance of intrapulmonary mucus by the high-velocity airflow generated by cough is the major rescue clearance mechanism in subjects with mucoobstructive diseases and failed cilial-dependent mucus clearance, e.g., subjects with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Previous studies have investigated the mechanical forces generated at airway surfaces by cough but have not considered the effects of mucus biophysical properties on cough efficacy. Theoretically, mucus can be cleared by cough from the lung by an adhesive failure, i.e., breaking mucus-cell surface adhesive bonds and/or by cohesive failure, i.e., directly fracturing mucus. Utilizing peel-testing technologies, mucus-epithelial surface adhesive and mucus cohesive strengths were measured. Because both mucus concentration and pH have been reported to alter mucus biophysical properties in disease, the effects of mucus concentration and pH on adhesion and cohesion were compared. Both adhesive and cohesive strengths depended on mucus concentration, but neither on physiologically relevant changes in pH nor bicarbonate concentration. Mucus from bronchial epithelial cultures and patient sputum samples exhibited similar adhesive and cohesive properties. Notably, the magnitudes of both adhesive and cohesive strength exhibited similar velocity and concentration dependencies, suggesting that viscous dissipation of energy within mucus during cough determines the efficiency of cough clearance of diseased, hyperconcentrated, mucus. Calculations of airflow-induced shear forces on airway mucus related to mucus concentration predicted substantially reduced cough clearance in small versus large airways. Studies designed to improve cough clearance in subjects with mucoobstructive diseases identified reductions of mucus concentration and viscous dissipation as key therapeutic strategies.

Nanoparticle diffusion in spontaneously expectorated sputum as a biophysical tool to probe disease severity in COPD.

Perturbations in airway mucus properties contribute to lung function decline in patients with chronic obstructive pulmonary disease (COPD). While alterations in bulk mucus rheology have been widely explored, microscopic mucus properties that directly impact on the dynamics of microorganisms and immune cells in the COPD lungs are yet to be investigated.We hypothesised that a tightened mesh structure of spontaneously expectorated mucus (i.e. sputum) would contribute to increased COPD disease severity. Here, we investigated whether the mesh size of COPD sputum, quantified by muco-inert nanoparticle (MIP) diffusion, correlated with sputum composition and lung function measurements.The microstructure of COPD sputum was assessed based on the mean squared displacement (MSD) of variously sized MIPs measured by multiple particle tracking. MSD values were correlated with sputum composition and spirometry. In total, 33 samples collected from COPD or non-COPD individuals were analysed.We found that 100 nm MIPs differentiated microstructural features of COPD sputum. The mobility of MIPs was more hindered in sputum samples from patients with severe COPD, suggesting a tighter mucus mesh size. Specifically, MSD values inversely correlated with lung function.These findings suggest that sputum microstructure may serve as a novel risk factor for COPD progression and severity.

Structure and properties of tethered bilayer lipid membranes with unsaturated anchor molecules.

The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [Z-20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol] and mixed HC18/ß-mercaptoethanol (ßME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection-absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/ßME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients relative to those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore-forming toxin α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicate that HC18 results in tBLMs with improved physical properties for the incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution are imperative.

Membrane association of the PTEN tumor suppressor: electrostatic interaction with phosphatidylserine-containing bilayers and regulatory role of the C-terminal tail.

The phosphatidylinositolphosphate phosphatase PTEN is the second most frequently mutated protein in human tumors. Its membrane association, allosteric activation and membrane dissociation are poorly understood. We recently reported PTEN binding affinities to membranes of different compositions (Shenoy et al., 2012, PLoS ONE 7, e32591) and a preliminary investigation of the protein-membrane complex with neutron reflectometry (NR). Here we use NR to validate molecular dynamics (MD) simulations of the protein and study conformational differences of the protein in solution and on anionic membranes. NR shows that full-length PTEN binds to such membranes roughly in the conformation and orientation suggested by the crystal structure of a truncated PTEN protein, in contrast with a recently presented model which suggested that membrane binding depends critically on the SUMOylation of the CBR3 loop of PTEN's C2 domain. Our MD simulations confirm that PTEN is peripherally bound to the bilayer surface and show slight differences of the protein structure in solution and in the membrane-bound state, where the protein body flattens against the bilayer surface. PTEN's C2 domain binds phosphatidylserine (PS) tightly through its CBR3 loop, and its phosphatase domain also forms electrostatic interactions with PS. NR and MD results show consistently that PTEN's unstructured, anionic C-terminal tail is repelled from the bilayer surface. In contrast, this tail is tightly tugged against the C2 domain in solution, partially obstructing the membrane-binding interface of the protein. Arresting the C-terminal tail in this conformation by phosphorylation may provide a control mechanism for PTEN's membrane binding and activity.

Disulfide disruption reverses mucus dysfunction in allergic airway disease.

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma.

In-plane homogeneity and lipid dynamics in tethered bilayer lipid membranes (tBLMs).

Tethered bilayer lipid membranes (tBLMs) were prepared by the self-assembly of thiolated lipidic anchor molecules on gold, followed by phospholipid precipitation via rapid solvent exchange. They were characterized by their in-plane structure, dynamics and dielectric properties. We find that the in-plane homogeneity and resistivity of the tBLMs depend critically on a well-controlled sample environment during the rapid solvent-exchange procedure. The in-plane dynamics of the systems, assessed by fluorescence correlation spectroscopy (FCS) as the diffusivity of free, labeled phospholipid dissolved in the membrane, depend on the density of the lipidic anchors in the bilayer leaflet proximal to the substrate as well as on details of the molecular structure of the anchor lipid. In DOPC tBLMs in which tethers are laterally dilute (sparsely tethered bilayer lipid membranes, stBLMs), measured diffusivities, D ≈ 4 µm(2) s(-1), are only slightly greater than those reported in physisorbed bilayers (M. Przybylo, J. Sykora, J. Humpolíckova, A. Benda, A. Zan and M. Hof, Langmuir, 2006, 22, 9096-9099). However, when we distinguish label diffusion in the proximal and in the distal bilayer leaflets, we observe distinct diffusivities, D ≈ 2 µm(2) s(-1) and 7 µm(2) s(-1), respectively. The value observed in the distal leaflet is identical to that in free membranes. stBLMs completed with phytanoyl lipids (DPhyPC) show consistently lower label diffusivity than those completed with unsaturated chains (DOPC). As the length of the tether chain increases, a reduction in the apparent diffusivity is observed, which we interpret as an increased propensity of the proximal bilayer leaflet to host free lipid. We also investigated preparation conditions that control whether the tBLMs are laterally homogeneous, as assessed by optical microscopy. In laterally heterogeneous bilayers, the label diffusivity varies only by a factor of ~2 to 4, indicating that the regions in the bilayers with different label solubilities do not correspond to distinct phases, such as a fluid phase coexisting with a gel phase.

Validation of hyaluronic acid-agar-based hydrogels as vitreous humor mimetics for in vitro drug and particle migration evaluations.

Artificial vitreous humor holds immense potential for use in in vitro intravitreal drug delivery assays. In this study, we investigated rheological properties and drug or nanoparticle migration in hyaluronic acid (HA) - agar based hydrogels and compared these characteristics with bovine vitreous humor. Gel compositions identified in literature containing HA (0.7-5.0 mg/ml) and agar (0.95-4.0 mg/ml) were classified as either high (VH), medium (VM) or low (VL) polymer load. Viscoelastic behavior was evaluated using oscillatory rheology, and migration of differently sized and charged polystyrene nanoparticles (NPs) through the different gels was determined via multiple particle tracking. Comparable rheological behaviour was observed between VL and bovine vitreous. Tracking evaluations revealed that increasing particle size and gel viscosity slowed NP migration. Additionally, 100 nm anionic NPs migrated slower than neutral NPs in VL and VM, while cationic NPs were immobile in all gels. Finally, distribution and clearance of sodium fluorescein was used to model drug mobility through the gels using a custom-built eye model. Flow and angular movement only influenced drug migration in VL and VM, but not VH. Finally, VL and VM demonstrated to have the most similar sodium fluorescein clearance to that of bovine vitreous humor. Together, these evaluations demonstrate that low viscosity HA-agar gels can be used to approximate nanoparticle and drug migration through biological vitreous humor.

Adhesive and Cohesive Peel Force Measurement of Human Airway Mucus.

In health, the high-speed airflow associated with cough represents a vital backup mechanism for clearing accumulated mucus from our airways. However, alterations in the mucus layer in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) leads to the mucus layer adhered to the airway surfaces, representing the nidus of chronic lung infection. To understand what is different about diseased mucus and why cough clearance is defective, there is a need for techniques to quantify the strength of the interactions limiting the ability of airflow to strip mucus from the airway surface (i.e., adhesive strength) or tear mucus apart (i.e., cohesive strength). To overcome the issues with measuring these properties in a soft (i.e., low elastic modulus) mucus layer, we present here novel peel-testing technologies capable of quantifying the mucus adhesive strength on cultured airway cells and cohesive strength of excised mucus samples. While this protocol focuses on measurements of airway mucus, this approach can easily be adapted to measuring adhesive/cohesive properties of other soft biological materials.

Widespread gene transfer to malignant gliomas with In vitro-to-In vivo correlation.

Gene therapy of malignant gliomas has shown a lack of clinical success to date due in part to inability of conventional gene vectors to achieve widespread gene transfer throughout highly disseminated tumor areas within the brain. Here, we demonstrate that newly engineered polymer-based DNA-loaded nanoparticles (DNA-NP) possessing small particle diameters (~50 nm) and non-adhesive surface polyethylene glycol (PEG) coatings efficiently penetrate brain tumor tissue as well as healthy brain parenchyma. Specifically, this brain-penetrating nanoparticle (BPN), following intracranial administration via convection enhanced delivery (CED), provides widespread transgene expression in heathy rodent striatum and an aggressive brain tumor tissue established orthotopically in rats. The ability of BPN to efficiently traverse both tissues is of great importance as the highly invasive glioma cells infiltrated into normal brain tissue are responsible for tumor recurrence. Of note, the transgene expression within the orthotopic tumor tissue occurred preferentially in glioma cells over microglial cells. We also show that three-dimensional (3D) multicellular spheroids established with malignant glioma cells, unlike conventional two-dimensional (2D) cell cultures, serve as an excellent in vitro model reliably predicting gene vector behaviors in vivo. Briefly, DNA-NP possessing greater surface PEG coverage exhibited more uniform and higher-level transgene expression both in the 3D model and in vivo, whereas the trend was opposite in 2D culture. The finding here alerts that gene transfer studies based primarily on 2D cultures should be interpreted with caution and underscores the relevance of 3D models for screening newly engineered gene vectors prior to their in vivo evaluation.

Interfacial binding and aggregation of lamin A tail domains associated with Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome is a premature aging disorder associated with the expression of ∆50 lamin A (∆50LA), a mutant form of the nuclear structural protein lamin A (LA). ∆50LA is missing 50 amino acids from the tail domain and retains a C-terminal farnesyl group that is cleaved from the wild-type LA. Many of the cellular pathologies of HGPS are thought to be a consequence of protein-membrane association mediated by the retained farnesyl group. To better characterize the protein-membrane interface, we quantified binding of purified recombinant ∆50LA tail domain (∆50LA-TD) to tethered bilayer membranes composed of phosphatidylserine and phosphocholine using surface plasmon resonance. Farnesylated ∆50LA-TD binds to the membrane interface only in the presence of Ca(2+) or Mg(2+) at physiological ionic strength. At extremely low ionic strength, both the farnesylated and non-farnesylated forms of ∆50LA-TD bind to the membrane surface in amounts that exceed those expected for a densely packed protein monolayer. Interestingly, the wild-type LA-TD with no farnesylation also associates with membranes at low ionic strength but forms only a single layer. We suggest that electrostatic interactions are mediated by charge clusters with a net positive charge that we calculate on the surface of the LA-TDs. These studies suggest that the accumulation of ∆50LA at the inner nuclear membrane observed in cells is due to a combination of aggregation and membrane association rather than simple membrane binding; electrostatics plays an important role in mediating this association.

Membrane association of the PTEN tumor suppressor: molecular details of the protein-membrane complex from SPR binding studies and neutron reflection.

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P(2)) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P(2) were determined to be K(d)∼12 µM and 0.4 µM, respectively, and K(d)∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P(2). The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P(2) and an increased apparent affinity to PI(3,4,5)P(3), due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P(2) and no synergy in its binding with PS and PI(4,5)P(2). Neutron reflection measurements show that the PTEN phosphatase "scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.