HUMAN GENE THERAPY CLINICAL DEVELOPMENT 25:1–11 (June 2014) ª Mary Ann Liebert, Inc. DOI: 10.1089/humc.2014.019

Research Article

Aerosol Delivery of DNA/Liposomes to the Lung for Cystic Fibrosis Gene Therapy Lee A. Davies,1,2 Graciela A. Nunez-Alonso,1,2 Gerry McLachlan,2,3 Stephen C. Hyde,1,2 and Deborah R. Gill1,2

Abstract

Lung gene therapy is being evaluated for a range of acute and chronic diseases, including cystic fibrosis (CF). As these therapies approach clinical realization, it is becoming increasingly clear that the ability to efficiently deliver gene transfer agents (GTAs) to target cell populations within the lung may prove just as critical as the gene therapy formulation itself in terms of generating positive clinical outcomes. Key to the success of any aerosol gene therapy is the interaction between the GTA and nebulization device. We evaluated the effects of aerosolization on our preferred formulation, plasmid DNA (pDNA) complexed with the cationic liposome GL67A (pDNA/GL67A) using commercially available nebulizer devices. The relatively high viscosity (6.3 – 0.1 cP) and particulate nature of pDNA/GL67A formulations hindered stable aerosol generation in ultrasonic and vibrating mesh nebulizers but was not problematic in the jet nebulizers tested. Aerosol size characteristics varied significantly between devices, but the AeroEclipse II nebulizer operating at 50 psi generated stable pDNA/GL67A aerosols suitable for delivery to the CF lung (mass median aerodynamic diameter 3.4 – 0.1 lm). Importantly, biological function of pDNA/GL67A formulations was retained after nebulization, and although aerosol delivery rate was lower than that of other devices (0.17 – 0.01 ml/min), the breath-actuated AeroEclipse II nebulizer generated aerosol only during the inspiratory phase and as such was more efficient than other devices with 83 – 3% of generated aerosol available for patient inhalation. On the basis of these results, we have selected the AeroEclipse II nebulizer for the delivery of pDNA/GL67A formulations to the lungs of CF patients as part of phase IIa/b clinical studies.

therapy clinical trials (all in patients with CF) have incorporated aerosol delivery (Griesenbach and Alton, 2009). A key barrier to the development of viable aerosol gene therapies has been the susceptibility of many GTAs to hydrodynamic shear forces generated during aerosol production. With the exception of adeno-associated virus (Leung et al., 2007), the majority of viral vectors so far investigated have suffered a significant loss of viability after aerosolization (Katkin et al., 1995). This is exemplified by recombinant Sendai virus (SeV), a highly efficient vector for lung gene transfer that demonstrates less than 1% of initial viral activity after jet nebulization (Griesenbach et al., 2011). More encouraging results have been obtained after aerosol delivery of nonviral formulations consisting of plasmid DNA (pDNA) complexed with cationic lipids or polymers. While pDNA is shear sensitive and prone to rapid degradation during nebulization (Davies

Introduction

T

he preparation and delivery of gene transfer agents (GTAs) remains a key challenge to the realization of gene therapy in the clinic. In particular, the potential to treat a range of lung diseases, including cystic fibrosis (CF), emphysema, and lung cancer, requires the development of stable gene transfer formulations suitable for lung administration via aerosolization. The relative accessibility of the pulmonary epithelium makes aerosol delivery an attractive option for noninvasive application to target cells within the lung while minimizing potential risks associated with systemic delivery. However, despite promising preclinical studies using both viral and nonviral GTAs in the lung (Griesenbach and Alton, 2009), aerosolization of many GTAs has so far proven highly problematic and, to date, only eight gene

1 Gene Medicine Research Group, Nuffield Division of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom. 2 United Kingdom Cystic Fibrosis Gene Therapy Consortium, United Kingdom. 3 The Roslin Institute & R(D)SVS, University of Edinburgh, Roslin, EH25 9RG, United Kingdom.

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et al., 2005; Lentz et al., 2006; Arulmuthu et al., 2007), condensation of the pDNA with a nonviral GTA can confer some protection, although the level of protection, and hence the efficacy, of aerosolized formulations varies significantly between GTAs and between nebulizer devices (Schwarz et al., 1996; Eastman et al., 1997b). Recent advances in nebulizer design have resulted in a variety of clinically approved devices that utilize compressed air, ultrasonic waves, or a vibrating mesh to generate aerosols (Kesser and Geller, 2009). Such devices have proven highly successful for the aerosol delivery of aqueous, small-molecule pharmaceuticals, but little is yet known regarding the viability of these devices for the generation of clinical gene therapy formulations. Development of any viable aerosol gene therapy system will be dependent upon the identification of both a suitable GTA and a compatible nebulizer device to deliver the chosen formulation. Where such compatibility has been achieved, the results have been promising and robust gene expression has been observed in vivo following preclinical aerosol studies using polyethylenimine (PEI) (Densmore et al., 2000; Davies et al., 2008), nanoparticles (McLachlan et al., 2011), and lipidbased complexes (Densmore et al., 1999). Cationic liposome GL67A complexed with pDNA is a nonviral formulation that has been aerosolized to the lungs of CF patients with encouraging results. Data from two clinical trials showed successful aerosol delivery of a single dose of GL67A complexes incorporating the CF transmembrane conductance regulator (CFTR) gene (Alton et al., 1999; Ruiz et al., 2001). In one study, approximately 25% correction of CF ion-transport defects was observed in the lung (Alton et al., 1999), although the effects were only short-lived probably because of the use of a viral promoter for CFTR expression. Subsequently, the performance of the pDNA/GL67A formulation was improved by constructing a new generation of plasmids devoid of potentially inflammatory CG-dinucleotides with a promoter capable of directing persistent transgene expression (pGM169) (Hyde et al., 2008). When aerosolized to the mouse lung, the new pGM169/GL67A formulation was capable of directing persistent CFTR transgene expression for at least 2 months, with minimal inflammation. We aimed to translate the improvements seen with the new pGM169/GL67A formulation in mouse models to clinical studies in CF patients. As the aerosol equipment used in earlier GL67A clinical trials (Alton et al., 1999; Ruiz et al., 2001) is now obsolete, a new delivery system was required, which not only retained the formulation’s efficacy after aerosolization, but also could generate droplets with physical characteristics suitable for delivery to the target areas of the CF lung. Furthermore, the device needed to be clinically approved, mechanically robust, easy to use, and capable of delivering suitable volumes of formulation to the lungs of patients in a clinically acceptable timeframe. Finally, to maximize the potential clinical benefits of aerosol delivery and simultaneously minimize wastage of expensive gene therapy formulations, it is vital that the aerosol delivery device is also as efficient as possible. We therefore assessed the stability and efficacy of the pDNA/GL67A formulation after aerosolization using several commercially available nebulizers to take forward into clinical studies for the treatment of patients with CF lung disease.

DAVIES ET AL. Materials and Methods Animal studies

Female BALB/c mice, aged 6–12 weeks at the time of the procedure, were purchased from the Biomedical Services Unit (University of Oxford, Oxford, United Kingdom). Mice were housed in accordance with UK Home Office ethics and welfare guidelines and fed on standard chow and water ad libitum. All procedures were carried out under UK Home Office–approved project and personal licenses under the terms of the Animals (Scientific Procedures) Act 1986. Chemicals and plasmids

The plasmid pCIKLux (5.6 kb) containing the firefly luciferase gene under the control of the human cytomegalovirus immediate/early promoter/enhancer (Gill et al., 2001) was used in all studies. pDNA was prepared by Bayou Biolabs (Harahan, LA). Lipid GL67 (a gift from Genzyme Corporation, Cambridge, MA) was formulated together with DOPE (Avanti Polar Lipids, Alabaster, AL) and DMPE-PEG5000 (Avanti Polar Lipids) at a molar ratio of 1:2:0.05 (GL67:DOPE:DMPEPEG5000) to form GL67A (Eastman et al., 1997a) by OctoPlus (Leiden, The Netherlands). pDNA/GL67A complexes were prepared at a molar ratio of 8 mM:6 mM (McLachlan et al., 2007). Complexes of 25 kDa branched PEI (Sigma-Aldrich Company Ltd., Poole, United Kingdom) with pDNA (pDNA/ PEI) were prepared at a final pDNA concentration of 0.2 mg/ml and a PEI nitrogen (N)-to-pDNA phosphate (P) ratio of 10:1 as described previously (Davies et al., 2012). In vivo aerosol delivery

Aerosol delivery of pDNA/GL67A formulations was performed using a continuous, unrestrained whole-body exposure protocol. Mice (n = 6–24) were placed into an 8-liter Perspex exposure chamber and exposed to aerosols for the indicated period. Total lung luciferase was assayed in all animals 24 hr after aerosol administration as described previously (Davies et al., 2008). Nebulizer operation

The following nebulizers were investigated for aerosolization of pDNA/GL67A complexes: Euroneb (Medikare, Mainz, Germany), eFlow (Pari GmbH, Starnberg, Germany), I-neb (Philips Respironics UK, Chichester, United Kingdom), Sidestream (Philips Respironics), Aerotech II (Biodex Medical Systems Inc, New York, NY), LC+ (Pari), Sprint (Pari), Junior (Pari), Star (Pari), and AeroEclipse II (Trudell Medical International, London, Canada). Ultrasonic and vibrating mesh nebulizers were operated in continuous output mode, and pneumatic nebulizers were operated using medical air from a compressed air cylinder or using air from two commercially available nebulizer compressors, the TurboBoy S (Pari) or the Boy SX (Pari). Operating pressure was measured via an in-line Digitron 2023P digital pressure meter (Sifam Instruments Ltd., Torquay, United Kingdom) connected between gas source and nebulizer. Physical characterization of pDNA/GL67A formulations

Samples were prepared for electron microscopy using a routine negative staining technique. Carbon/formvar-coated

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copper grids were floated on drops of the various suspensions for 30 sec. The excess fluid was removed and the grids floated on drops of 1% methyl tungstenate, dried, and examined in a Jeol 1200EX transmission electron microscope. Particle size and zeta potential of pDNA/GL67A complexes was measured by laser light scattering using a Malvern Zetasizer Nano (Malvern Instruments, Malvern, United Kingdom). Five measurements per sample were performed at a fixed temperature of 25C and using a viscosity of water value of 0.89 cP. Viscosity measurements were performed using a falling ball viscometer (Gilmont Instruments, Barrington, IL) according to manufacturer’s instructions. A minimum of five measurements were performed for each sample at a temperature of 21C. Surface tension measurements were performed at 20C using a Du Nouy ring balance and 10 ml of starting sample. All measurements were performed in triplicate.

amount of lipid deposited on each filter was quantified using a modified fluorescamine assay (Ferrari et al., 1998). Briefly, lipid was extracted by vortexing collection filters for 30 sec in 20 ml of RLT buffer (Qiagen Ltd., Crawley, United Kingdom) before appropriate dilutions were prepared in sterile water. A volume of 100 ll of lipid sample was mixed with 900 ll of a 150 lM fluorescamine solution and fluorescence at 480 nm measured after excitation at 392 nm using a Gemini XPS fluorescent plate reader (Molecular Devices, Sunnyvale, CA). Collected samples were quantified relative to standard curves prepared from the initial aerosol formulation. Inhalation efficiency was defined as the proportion of total collected aerosol (inhalation plus expiration filters) isolated from the inhalation filter.

Aerosol size characterization

To facilitate measurement of pDNA and lipid concentration in nebulizer reservoirs and aerosol, each component was fluorescently labeled before complexation. GL67A was supplemented with carboxyfluorescein-labeled DOPE (Ext 488 nm, Ems 515 nm; Avanti) at 0.1% of standard DOPE levels and pDNA was labeled by addition of trace amounts of the fluorescent DNA probe POPO-1 (Ext 434 nm, Ems 456 nm; Molecular Probes, Eugene, OR) sufficient to label 1 in 1600 nucleotides. Dual-labeled complexes were prepared and loaded into the nebulizer reservoir. Aerosol was generated under simulated breathing conditions as described above and generated aerosol was captured via inertial impaction upon the walls of a Dreschel bottle connected in-line between the nebulizer and the ventilator. At intervals during the nebulization procedure, samples of material remaining in the nebulizer reservoir or collected from the aerosol were removed and the concentration of pDNA and lipid was assayed using a Gemini XPS fluorescent plate reader (Molecular Devices). Collected samples were quantified relative to standard curves prepared from the initial fluorescently labeled aerosol formulation.

Aerosol size characteristics were determined using a chilled (4–7C) Next-Generation Pharmaceutical Impactor (NGI; Copley Scientific Ltd., Nottingham, United Kingdom) operating at 15 liters/min ( – 5%) (Berg et al., 2007). Briefly, aerosol was collected over a period of 4 minutes with deposited material subsequently eluted from each impactor stage using 10 ml water. For pDNA/GL67A studies the mass of pDNA deposited upon each stage was quantified using Quant-it Picogreen dsDNA assay kit (Invitrogen, Eugene, OR). Salbutamol was quantified by UV spectrophotometry at 224 nm (Lange and Finlay, 2000). Aerosol size characteristics, including the mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF), were determined from log-probability graphs of cumulative mass fraction versus stage cutoff diameter using CITDAS particle analysis software (Copley Scientific Ltd.). Cutoff diameters of 14.1, 8.61, 5.39, 3.29, 2.07, 1.35, and 0.97 lm were utilized for NGI stages 1–7, respectively (Marple et al., 2004). Aerosol delivery rates

Aerosol delivery rates were determined under simulated breathing conditions. Briefly, nebulizers were attached directly to a large-animal volume-controlled ventilator (Harvard Apparatus, Holliston, MA) with inhaled air entrained directly through the nebulizer device. Aerosol was generated under simulated breathing conditions with a tidal volume of 500 ml, a breathing frequency of 15 breaths/min, and an inhalation/exhalation ratio of 1:1. Rate of aerosol generation was calculated by weighing the nebulizer reservoir at intervals during aerosol delivery. Nebulizers were run continuously until no further aerosol production was observed and the dead volume of the device was determined based on the mass of formulation remaining in the reservoir. A minimum of three separate assessments was performed for each nebulizer operating condition with a new nebulizer used for each measurement. Aerosol delivery efficiency

Oral delivery efficiency was assessed by inclusion of aerosol collection filters (Pari) on the inspiratory and expiratory arms of the breathing circuit (Fig. 2C). Nebulizers were run for 1–3 min with collection filters in situ before the

Quantification of pDNA/GL67A components during nebulization

Gel analysis and densitometry

The pDNA component of collected pDNA/GL67A samples was isolated using phenol/chloroform extraction to remove lipids. Briefly, 75 ll of collected sample was vortexed for 10 sec with 500 ll of phenol/chloroform (Ambion, Austin, TX) before centrifugation at 16,000 g for 5 min. The aqueous phase incorporating isolated pDNA was removed and equivalent pDNA mass of each sample was size-fractionated by electrophoresis at 2–10 V/cm through a 0.7% agarose gel containing 0.5 lg/ml ethidium bromide. For quantification of plasmid degradation in the nebulizer reservoir, the intensity of bands corresponding to covalently closed circular pDNA was measured using NIH Image 1.62 software (http://rsb:info .nih.gov/nih-image/) as described (Davies et al., 2005). The amount of supercoiled plasmid present in samples taken before nebulization was arbitrarily designated as 100% and the amount remaining at time points during nebulization was expressed as a percentage of this value. Estimation of total plasmid dose delivered without degradation was performed using area under the curve (AUC) analysis of individual plasmid degradation curves using GraphPad Prism software v6 (GraphPad, San Diego, CA).

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Table 1. Physical Characteristics of the pDNA/GL67A Formulation

Water 1% salbutamol pDNA/PEI pDNA/GL67A

Viscosity (cP)

Surface tension (mN/m)

Particle size (nm)

Zeta potential (mV)

1.00 – 0.05 1.00 – 0.06 0.99 – 0.06 6.32 – 0.09

72.8 – 0.1 69.8 – 0.4 72.5 – 0.4 42.2 – 0.3

N/A N/A 75 – 4 930 – 130

N/A N/A + 47.3 – 0.7 - 19.6 – 1.5

N/A, not applicable; pDNA, plasmid DNA; PEI, polyethylenimine. The viscosity (measured in centipoise or cP), surface tension (measured in millinewtons/meter or mN/m), particle size (measured in nanometers or nm), and surface charge (zeta potential measured in millivolts or mV) of pDNA/GL67A formulations were determined. Alternative formulations were also characterized, including a standard pharmaceutical aerosol formulation of 1% salbutamol in 0.9% NaCl; an aerosol gene therapy formulation consisting of pDNA complexed with 25 kDa branched cationic polymer polyethylenimine (PEI; 0.2 mg/ml pDNA concentration and PEI nitrogen [N] to DNA phosphate [P] molar ratio of 10:1 in sterile water), and water alone. Data represent mean – SEM for n = 5 replicates except surface tension, where n = 3.

Statistical analysis

Graphed data represent mean – standard error of the mean (SEM) for all data sets. Comparisons between two groups were performed using a Student’s t-test. Multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis when comparing all groups with each other, or Dunnett’s multiple comparison test when comparing groups to a single control group. Analysis of covariance (ANCOVA) followed where appropriate with Bonferroni corrected post hoc tests was performed using SPSS Statistics software v22 (IBM.com). All other tests were carried out using GraphPad prism software v6 (GraphPad, San Diego, CA). Results Physical characteristics of pDNA/GL67A complexes

The physical characteristics of pDNA/GL67A complexes, prepared at a previously optimized molar ratio of 8 mM:6 mM

(Eastman et al., 1998) were investigated (Table 1). An alternative aerosol gene therapy formulation, whereby pDNA is complexed with the cationic polymer PEI, was included for comparative purposes, along with a standard pharmaceutical aerosol formulation (1% [w/v] salbutamol in 0.9% NaCl solution) (Berg et al., 2007). While pDNA/PEI and salbutamol formulations demonstrated physical properties similar to water, pDNA/GL67A formulations were significantly ( p < 0.0001 ANOVA with Dunnett’s multiple comparison test) more viscous (6.32 – 0.09 cP) and had significantly lower ( p < 0.0001 ANOVA with Dunnett’s multiple comparison test) surface tension (42.2 – 0.3 mN/m) than the other formulations (Table 1). In addition, particle size analysis revealed that pDNA/GL67A formulations contained relatively large particles (930 – 130 nm) compared with pDNA/PEI complexes (75 – 4 nm). Further analysis by electron microscopy suggested that pDNA/GL67A particulates resulted from the aggregation of smaller (*200 nm) multilamellar lipoplexes formed during the complexation process (Fig. 1A).

FIG. 1. Aerosol characteristics of pDNA/GL67A complexes. (A) Electron micrograph of pDNA/GL67A aerosol formulation (scale bar represents 400 nm). (B) Aerosol droplet sizes of pDNA/ GL67A formulation and a 1% salbutamol solution nebulized over a range of operating pressures using a Pari LC+ nebulizer. (C) MMAD and (D) FPF of pDNA/GL67A formulation nebulized using a variety of clinical jet nebulizers operated using compressed air or clinical compressor devices. The data represent mean – SEM of a minimum of three replicates for each measurement. FPF, fine particle fraction; MMAD, mass median aerosol diameter; pDNA, plasmid DNA.

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Performance of pDNA/GL67A complexes in nebulizers

To determine if stable pDNA/GL67A aerosols could be generated using commercially available nebulizer devices, complexes were loaded into the reservoirs of one ultrasonic, two vibrating mesh, and seven pneumatic nebulizers using half the recommended reservoir volume (Table 2). Nebulizers were operated according to manufacturers’ instructions, and the ability to generate stable aerosols was recorded. All nebulizers generated stable aerosols of pDNA/PEI or a 1% salbutamol solution. With the pDNA/GL67A formulation, no aerosol was generated using the ultrasonic nebulizer, and with the vibrating mesh nebulizers a small amount of aerosol was observed initially but output rapidly decreased and stopped after a few seconds of operation. In contrast, considerable pDNA/GL67A aerosol output was observed with all pneumatic nebulizers tested, although with both the Aerotech II and Up-Mist nebulizers, aerosol production was repeatedly interrupted by the tendency of pDNA/GL67A formulations to foam and ‘‘splutter’’ out of the nebulizer resulting in substantial loss of material. Although moderate foaming was also observed in the other pneumatic nebulizers, the associated reservoir geometries prevented gross loss of material and stable aerosol production was achieved. Aerosol size characterization

pDNA/GL67A complexes were nebulized using the Pari LC + nebulizer over a range of operating pressures, and the resultant aerosol was analyzed by cascade impaction using a chilled NGI. The MMAD of resultant aerosols was compared with aerosols generated using a 1% salbutamol solution. The MMAD for both pDNA/GL67A and salbutamol formulations was inversely proportional to the nebulizer operating pressure (linear regression analysis with R2 values of 0.94 and 0.71 for pDNA/GL67A and salbutamol, respectively) with smaller aerosol droplets produced at higher operating pressures (Fig. 1B). Using the manufacturers’ recommended operating pressure of 29 psi, the MMAD of salbutamol formulations (4.0 – 0.2 lm) was consistent with the published literature. However, pDNA/GL67A formula-

tions generated substantially larger droplets (5.5 – 0.2 lm), and after controlling for the effect of nebulizer operating pressure, a significant difference in MMAD was observed between salbutamol and pDNA/GL67A ( p = 0.0004, ANCOVA). Droplets >5 lm are generally considered too large for respiratory delivery under normal breathing conditions; thus, to optimize nebulizer parameters for pDNA/GL67A aerosols suitable for patient delivery, the MMAD and FPF (defined as the proportion of aerosol contained in droplets < 5 lm in diameter) of pDNA/GL67A aerosols generated by a variety of nebulizers were determined. Five jet nebulizers were assessed at operating pressures of 29 and 50 psi using compressed air from a gas cylinder for aerosol generation, and all nebulizers were also assessed using two portable medical compressors (TurboBoy S and Boy SX; Pari). For each nebulizer, an increased operating pressure was associated with a reduction in MMAD and an increase in the proportion of respirable pDNA/GL67A aerosol generated (Fig. 1C and D). The largest droplets were observed for each nebulizer when using the portable TurboBoy S and Box SX compressor systems (nominal operating pressures of 17 and 22 psi, respectively). Conversely, the smallest droplets, and hence the greatest proportion of respirable aerosol, were generated when the nebulizer was operating at 50 psi. Although 50 psi is greater than the maximum recommended operating pressure for all nebulizers except the AeroEclipse II (Table 2), under these conditions the Junior (MMAD 3.4 – 0.1 lm; FPF 74.9 – 1.4%), Star (MMAD 3.4 – 0.1 lm; FPF 78.3 – 1.6%), and AeroEclipse II (MMAD 3.4 – 0.1 lm; FPF 71.4 – 1.5%) nebulizers all generated pDNA/GL67A aerosols significantly smaller ( p = 0.002, p = 0.001, and p = 0.002, respectively; ANOVA with Dunnett’s multiple comparison test) and more appropriate for clinical delivery than the LC+ (MMAD 4.3 – 0.4 lm; FPF 60.9 – 5.8%) used in previous clinical studies (Alton et al., 1999). It was also noted that the Star nebulizer was susceptible to intermittent ‘‘spluttering’’ when formulations were aerosolized over extended periods. A combination of the aerosol size data and stability of pDNA/GL67A aerosols over a range of

Table 2. Initial Comparison of Clinical Nebulizers for Aerosolization of pDNA/GL67A Formulations

Nebulizer EuroNeb eFlow I-neb Aerotech II Sidestream LC+ Sprint Junior Star AeroEclipse II

Manufacturer

Type

Medikare Pari Philips Respironics Biodex Systems Philips Respironics Pari Pari Pari Pari Trudell

Ultrasonic Vibrating mesh Vibrating mesh Jet Jet Jet Jet Jet Jet Jet

Nebulize Nebulize Reservoir Max vol. operating 1% pDNA/ Mode (ml) pressure (psi) salbutamol PEI CO CO COa CO CO BE BE BE BE BA

6 6 3 10 10 8 8 8 8 6

N/A N/A N/A 50 33 29 29 29 29 50

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Nebulize pDNA/ GL67A No No No Spluttered Spluttered Yes Yes Yes Yes Yes

BA, breath actuated; BE, breath enhanced; CO, constant output. Nebulizers were operated according to manufacturers’ instructions with a fill volume equal to half the maximum recommended volume. Jet nebulizers were operated at the maximum pressure recommended for that device. For comparison, 1% salbutamol solution (in 0.9% NaCl) and pDNA/PEI complexes (0.2 mg/ml pDNA and N:P of 10:1) were also assessed under identical conditions. a Although the I-neb device is capable of complex breath coordination, to simplify comparison, it was operated in the constant output mode for the purposes of this study.

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FIG. 2. Nebulizer output utilizing pDNA/GL67A formulations. The pDNA/GL67A aerosol output rates for the LC+ (filled circles), AeroEclipse II (filled squares), and Junior (open triangles) nebulizers were measured under simulated breathing conditions using (A) compressed air at 50 psi or (B) the Pari Boy SX medical compressor. The data represent mean – SEM of three replicates for all measurements. (C) Aerosol collection filters incorporated into the inspiratory (INS) or expiratory (EXP) arms of the nebulizer breathing circuit were utilized to determine pDNA/GL67A aerosol delivery efficiency under simulated breathing conditions as shown in (D). Data represent mean – SEM for n = 3 for each nebulizer and statistical analysis was performed using the Student’s t-test. operating conditions led to the selection of the Pari Junior and AeroEclipse II nebulizers for further investigation. The Pari LC + was also included for comparison with previous nebulization studies (Eastman et al., 1998). Nebulizer output rate

Aerosol delivery of a gene therapy formulation needs to be feasible in a clinically acceptable time frame; thus, delivery rate is a key determinant of suitability for clinical studies. However, as aerosol output from many nebulizer designs varies throughout the respiratory cycle, it is essential that aerosol output rate is measured under simulated breathing conditions. To determine aerosol output rates for pDNA/GL67A formulations, the Junior, AeroEclipse II, and LC + nebulizers were connected to a ventilator set to mimic

sinusoidal human breathing at 15 breaths/min, 1:1 inspiration:expiration ratio, and a tidal volume of 500 ml. Nebulizers were loaded with pDNA/GL67A (10 ml in LC + and Junior, 5 ml in AeroEclipse II) and operated continuously until no more aerosol was generated and the total aerosol output was measured throughout by determining the weight of material remaining in the nebulizer reservoir. At 50 psi, aerosol output rates for the LC + (0.38 – 0.01 ml/min) and Junior nebulizers (0.34 – 0.01 ml/min) were similar, but the AeroEclipse II was considerably slower (0.17 – 0.01 ml/min) (Fig. 2A). In the context of an uninterrupted clinical aerosol delivery study, these rates would equate to minimum delivery times of 26 min (LC + ), 29 min ( Junior), and 59 min (AeroEclipse II) for a nominal 10 ml aerosol dose (Table 3). In contrast, aerosol delivery using the Pari Boy SX compressor was significantly slower for each nebulizer at

Table 3. Clinical Delivery Characteristics of Selected Nebulizer Devices Boy SX compressor

MMAD (lm) FPF (%) Delivery rate (ml/min) Delivery efficiency (% total aerosol) Dead volume (ml) 10 ml delivery time (min) Estimated lung delivery volume (ml) Estimated lung pDNA delivery (mg)

50 psi compressed air

LC+

Junior

AeroEclipse II

LC+

Junior

AeroEclipse II

5.5 – 0.1 44.8 – 1.5 0.25 – 0.01 63.8 – 0.4 0.64 – 0.05 40 2.6 7.07

4.4 – 0.1 58.3 – 1.4 0.15 – 0.01 66.8 – 1.1 0.54 – 0.11 67 3.68 9.72

3.8 – 0.1 68.7 – 1.5 0.11 – 0.01 83.7 – 2.7 0.48 – 0.12 90 5.16 13.3

4.3 – 0.4 60.9 – 5.8 0.38 – 0.01 57.4 – 0.4 0.61 – 0.05 26 3.28 8.66

3.4 – 0.1 74.9 – 1.4 0.34 – 0.01 55.3 – 2.2 0.74 – 0.01 29 3.83 10.11

3.4 – 0.1 71.4 – 1.5 0.17 – 0.01 83.0 – 2.3 0.53 – 0.10 59 5.3 14.00

FPF, fine particle fraction; MMAD, mass median aerodynamic diameter. Aerosols of pDNA/GL67A generated using the LC+ , Junior, and AeroEclipse II nebulizers in conjunction with the Boy SX compressor or a 50 psi compressed air source were used to estimate clinical delivery parameters based upon a standard 10 ml starting volume of formulation. Calculations are based on a single 10 ml reservoir dose for the LC+ and Junior nebulizers and two consecutive 5 ml doses delivered in separate nebulizers for the AeroEclipse II. Estimated lung delivery volume was calculated using (reservoir volume - dead volume) · FPF · delivery efficiency, and estimated pDNA delivery was based on pDNA/GL67A formulation containing 2.64 mg/ml pDNA.

DNA/LIPOSOME AEROSOL DELIVERY

0.25 – 0.01 ml/min (LC + ), 0.15 – 0.01 ml/min ( Junior), and 0.11 – 0.01 ml/min (AeroEclipse II) (Fig. 2B) requiring 40, 67, and 90 min for delivery of 10 ml. Although nebulizers were initially filled to capacity with pDNA/GL67A, a quantity of pDNA/GL67A remained in the reservoir of each device after aerosol generation had ceased. This ‘‘dead’’ volume varied between nebulizers (Table 3) but amounted to approximately 5–10% of the initial reservoir load. Delivery efficiency

To assess the aerosol delivery efficiency of the LC + , Junior, and AeroEclipse II nebulizers, the oral bioavailability (percentage of generated aerosol delivered to the mouthpiece) of pDNA/GL67A aerosols was measured. Aerosol collection filters were introduced on the inspiratory and expiratory arms of the breathing circuit (Fig. 2C) to capture inhaled aerosol and aerosol that would normally bypass the patient and be lost to the environment. Comparative analysis of aerosol deposition on the two filters allowed the delivery efficiency of a nebulizer to be measured under simulated breathing conditions. As would be expected for breath-enhanced nebulizers that increase the proportion of aerosol generated during patient inhalation and minimize losses during patient exhalation (Dennis, 1995), the LC + and Junior nebulizers demonstrated relatively high oral bioavailability (65–70%) when used in conjunction with the Boy SX compressor (Fig. 2D). However, when operated at 50 psi there was a significant ( p = 0.006 [LC + ] and p = 0.009 [Junior] Student’s t-test) fall in delivery efficiency to around 55% in both devices, which may be partly explained by the inefficiency of the breath enhancement mechanism when operated at pressures in excess of the manufacturers’ guidelines (Table 2). The breath-actuated nebulizer AeroEclipse II, which produces aerosol only during the inspiratory phase (Leung et al., 2004), demonstrated the highest delivery efficiencies with around 83% of aerosolized material collected on the inspiratory filter using the Boy SX compressor. In contrast to the other nebulizers tested, no loss of delivery efficiency was observed at the higher 50 psi delivery pressure ( p = 0.86 Student’s t-test). Stability of pDNA/GL67A formulations during nebulization

Jet nebulizers have been shown to concentrate drugs in the nebulizer reservoir because of the preferential aerosolization of the formulation solvent (Dennis, 1995), which in turn affects the rate of drug delivery to patients. Excessive concentration can also lead to precipitation of colloidal suspensions such as pDNA/GL67A. In addition, the ratio of pDNA and lipid components in pDNA/GL67A formulations is a key determinant of formulation efficacy (Eastman et al., 1997a), and any propensity for differential concentration of components during nebulization could impact formulation viability. To investigate the effects of nebulization upon the pDNA/GL67A formulations, the concentrations of the individual components were measured in the reservoir and in the generated aerosol of the LC + , Junior, and AeroEclipse II nebulizers. Aerosolization of pDNA/GL67A complexes with all three nebulizers was associated with a steady increase in the concentration of both pDNA (Fig. 3A) and

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lipid (Fig. 3B) in the reservoir. The rate of concentration increased significantly after 80% of the reservoir volume had been aerosolized, most likely because of the increased rate of recycling of material as the devices emptied. The concentration of pDNA in nebulizer reservoirs increased from a starting concentration of 8 mM to a maximum of 18– 24 mM observed in the final 10% of formulation remaining in the nebulizer reservoirs. Lipid concentration over the same time period increased from 6 mM to a maximum of 15–17 mM. Crucially, the ratio of pDNA and lipid did not change over time either in the nebulizer reservoir (Fig. 3C) or in the generated aerosol (Fig. 3D), indicating that neither component was aerosolized preferentially. No visible evidence of precipitation or aggregation of the formulation was apparent even in highly concentrated samples retained in the nebulizer dead volume. Shear-related pDNA degradation

To investigate the impact of jet nebulization on the pDNA component of the complexes, formulations were aerosolized using the LC + , Junior, and AeroEclipse II operating at 50 psi. At intervals during nebulization, samples of formulation were removed from the nebulizer reservoir (Fig. 3E), or collected from the nebulizer aerosol (Fig. 3F), and the structural integrity of the pDNA component was analyzed by gel electrophoresis. Results were similar for both reservoir and aerosol samples. Nebulization in all three devices was associated with a progressive degradation of pDNA indicated by loss of the biologically active covalently closedcircular and open-circular plasmid forms along with appearance of a low-molecular-weight smear indicative of sheared pDNA fragments. As has been observed previously (Eastman et al., 1998), complexation of pDNA with GL67A offers incomplete protection from shear-related degradation during jet nebulization. However, densitometry showed that the rate of pDNA degradation (Fig. 3G) was similar between the three nebulizers ( p = 0.78 ANCOVA) and the proportion of total pDNA dose delivered intact over the course of the aerosol revealed no significant difference ( p = 0.9 ANOVA) between the LC + (67.7 – 5.7%), Junior (66.2 – 1%), and AeroEclipse II (68.5 – 0.9%) nebulizers. In vivo luciferase expression

Assessment of physical stability of formulations during nebulization indicates nebulizer compatibility, but an assessment of performance can only be achieved after aerosol delivery and gene expression in vivo. Mice were exposed to 10 ml aerosols of pCIKLux/GL67A generated using each of the LC + , Junior, and AeroEclipse II nebulizers, and luciferase expression in whole lung homogenates was analyzed 24 hr later. All treated mice demonstrated robust luciferase expression above the background levels observed in naı¨ve mice (Fig. 3H), but expression after aerosol delivery using the Junior (73.1 – 7.9 RLU/mg) and AeroEclipse II (87.7 – 12.1 RLU/mg) nebulizers was significantly higher ( p = 0.0004 and p < 0.0001, respectively; ANOVA with Dunnett’s multiple comparison test) than that observed with the LC + (13.7 – 3.6 RLU/mg). The higher gene expression observed in mice using the Junior and AeroEclipse II can be explained partly by the lower MMAD of the generated aerosols (Fig. 1C), which are more suitable for inhalation and

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FIG. 3. Physical characteristics of pDNA/GL67A formulations during nebulization. The concentrations of pDNA (A) and GL67A (B) were independently determined in the nebulizer reservoir at intervals during nebulization using the LC+ (filled circles), AeroEclipse II (filled squares), and Junior (open triangles) nebulizers. The molar ratio of pDNA and GL67A was determined for material collected in the nebulizer reservoir (C) or from the generated aerosol (D) at intervals during nebulization. The data represent mean – SEM of three replicates for all measurements. Electrophoretic profiles of pDNA samples removed at intervals from the AeroEclipse II reservoir (E) or collected from the pDNA/GL67A aerosol (F) during nebulization. Open circular (oc) and covalently closed circular (ccc) plasmid forms are indicated. Samples were collected after 0–90% of the initial reservoir volume had been nebulized. (G) Densitometric analysis of pDNA nebulized using LC+ , AeroEclipse II and Junior nebulizers indicating the proportion of ccc pDNA form remaining during the course of nebulization. The data represent mean – SEM for three separate aerosol studies utilizing each nebulizer. (H) Lung gene expression in BALB/c mice 24 hr after aerosol delivery of 10 ml pCIKLux/GL67A formulation using the LC+ , AeroEclipse II, or Junior nebulizers operating at 50 psi. Data represent mean – SEM for n = 6 animals in each group with statistical analysis performed using ANOVA with Dunnett’s multiple comparison test. (I) Lung gene expression in mice exposed to increasing volumes of pCIKLux/GL67A delivered using the AeroEclipse II nebulizer operating at 50 psi. The data represent mean – SEM for n = 6 animals in each group.

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deposition in the murine lung (Raabe et al., 1988). Subsequent studies looking at delivery of increasing volumes of pCIKLux/GL67A using the AeroEclipse II nebulizer (Fig. 3I) demonstrated that lung gene expression was dose dependent (linear regression analysis with R2 value of 0.87), and higher gene expression was achievable by administering multiple reservoir volumes sequentially. Discussion

GL67A is one of the most promising GTAs available for aerosol-mediated nonviral gene transfer; considerable development has resulted in a potent aerosol leading to robust lung gene expression in several animal models, including mice (Eastman et al., 1997a) and sheep (McLachlan et al., 2011). GL67A formulations have previously been aerosolized to the lungs of CF patients (Alton et al., 1999; Ruiz et al., 2001), but developments in aerosol science now permit a more detailed assessment of formulations before entry into the clinic, and advances in nebulizer design can be advantageous in terms of both patient interface and clinical outcome. In this study, we have investigated the compatibility of GL67A formulations with a range of nebulizer devices, including those based on vibrating mesh technology. Being simple to use and capable of delivering drugs more rapidly than other devices, they have proven popular with both clinicians and patients. Unfortunately, both the eFlow and I-neb devices, although capable of generating excellent aerosols using a 1% salbutamol solution or a pDNA/PEIbased gene therapy formulation, were unable to aerosolize pDNA/GL67A formulations. The most likely explanation for this failure is the rather atypical physicochemical properties of the pDNA/GL67A formulation itself (Table 1). Optimization of the formulation to enhance aerosol stability (Eastman et al., 1997b) and increase pDNA concentration in the aerosol (Eastman et al., 1997a) has resulted in a viscous formulation containing relatively large aggregates of pDNA/ lipid complexes (Fig. 1A). While the aerosol generation process in jet nebulizers is relatively unaffected by the physical properties of the formulation, operation of both ultrasonic (McCallion et al., 1995) and vibrating mesh nebulizers is severely impaired by excessively viscous formulations (Ghazanfari et al., 2007). It is also likely that aerosol generation using vibrating mesh nebulizers was further impaired by the presence of large (*900 nm) pDNA/ GL67A aggregates that may have impeded liquid transfer through the micron-sized pores utilized for aerosol production in such devices (Kesser and Geller, 2009). Based upon these results, it would appear that vibrating mesh nebulizers are unsuitable for the aerosol delivery of concentrated pDNA/GL67A formulations. However, the devices remain an attractive alternative to jet nebulization for the aerosol delivery of polymer-based gene therapy formulations, such as pDNA/PEI, which have more compatible physical characteristics (Arulmuthu et al., 2007). In contrast to the vibrating mesh nebulizers, aerosolization of pDNA/GL67A using jet nebulizers was relatively straightforward, although stable aerosol generation was only possible with a subset of devices (Table 2). Because of the high concentration of surface-active agents within GL67A, the formulation has a tendency to foam when exposed to the constant recirculation of material within a nebulizer reser-

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voir. This behavior can lead to substantial loss of material into the aerosol stream, depending on the geometry of the reservoir. Consequently, many of the nebulizers identified as suitable for stable aerosolization of pDNA/GL67A were very similar in terms of design and geometry. Despite similarities in nebulizer design, the physical characteristics of pDNA/GL67A aerosols generated were surprisingly varied (Fig. 1C and D), confirming the need for a thorough investigation of any new combination of aerosol gene therapy formulation and delivery device. In general, aerosols of pDNA/GL67A contained larger droplets than would be achieved using standard aerosol formulations (Fig. 1B), and under many of the conditions tested, the pDNA/GL67A aerosols would be considered too large (MMAD > 5 lm) for practical delivery to the lungs of patients. Interestingly, the pDNA/GL67A MMAD measured in this study, using the Pari LC+ nebulizer operating at 50 psi, was 4.3 – 0.4 lm (Fig. 1B), which was higher than an MMAD of 2.2 lm reported previously (Eastman et al., 1998) using identical conditions. Although different plasmids were used in the two studies, this would not be expected to impact droplet size and it is likely that the observed difference is a result of improvements in aerosol sizing methodologies. The Andersen Cascade Impactor used for aerosol sizing in previous studies (Eastman et al., 1998) has been shown to underestimate droplet size because of heating and evaporation during transit through the device (Finlay and Stapleton, 1999). In contrast, aerosol characteristics in this study were assessed using an NGI operated under conditions designed to prevent aerosol evaporation and accepted as current best practice in the field leading to a more realistic interpretation of aerosol droplet size (Berg et al., 2007). While droplets < 5 lm in diameter are generally regarded as respirable, effective delivery of therapeutic formulations to specific regions of the lung requires aerosols with an appropriate MMAD. Excessively large droplets are filtered from inhaled air by deposition in the upper respiratory tract. Conversely, small droplets can be exhaled without deposition (Geller, 2008). Modeling of aerosol deposition in CF patients is challenging because of the presence of mucus plugs and inflammation in the lungs (Dolovich et al., 2005). However, it has been suggested that aerosol with an MMAD of approximately 3 lm would be optimal for targeting the bronchial epithelium associated with CF lung disease (Geller, 2008). Because of the nature of pDNA/GL67A formulations, such aerosols are difficult to achieve unless nebulizers are operated at relatively high pressures, probably precluding the use of portable compressor devices for clinical studies. While easy to use and convenient for patients, portable compressors cannot generally achieve the required operating pressures and an alternative air source such as a compressed air cylinder would be required. Importantly, the required gas pressures also exceed the manufacturer’s recommended operating conditions for many of the tested nebulizers (Table 2). As well as introducing safety concerns, it would appear that the breath enhancement mechanism that increases the proportion of aerosol generated during patient inhalation in many of the tested nebulizers is impaired at higher pressures (Fig. 2D). Consequently, the efficiency of aerosol delivery to the patient lung is compromised, thereby greatly reducing the appeal of these devices for pDNA/ GL67A delivery.

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In contrast to the other nebulizers examined, the breath actuated AeroEclipse II is certified to operate safely at 50 psi (Table 2) and is capable of efficiently delivering over 80% of generated aerosol to the patient (Fig. 2D). Breath actuation greatly reduces the amount of ‘‘wasted’’ aerosol produced during patient exhalation and simultaneously minimizes environmental contamination and increases the proportion of aerosol delivered to the lung. This is a highly desirable property for delivery of expensive biomaterials such as gene therapy formulations. Conclusions

After assessment of the delivery characteristics of several aerosol devices (Table 3) and their subsequent effects on the aerosol formulation, the AeroEclipse II nebulizer was selected for the delivery of pGM169/GL67A aerosols to the lungs of CF patients. Aerosols of pDNA/GL67A generated by the AeroEclipse II demonstrated appropriate physical characteristics for lung delivery, with the combination of high respirable fraction and high delivery efficiency associated with breath actuation resulting in the highest estimation of lung delivery (Table 3). Similar levels of deposition were indicated using the AeroEclipse II operating at 50 psi from a compressed air cylinder and using the portable Pari Boy SX compressor. However, the 50 psi operating source led to savings in aerosol delivery time to patients and was therefore selected for future clinical trials. The viability of the selected AeroEclipse II nebulizer and operating conditions has recently been demonstrated in preclinical toxicological studies in the mouse lung (Alton et al., 2014), where repeated administration of pGM169/ GL67A aerosols at monthly intervals resulted in reproducible, dose-related, and persistent gene expression in the absence of significant toxicity. After these encouraging data, the AeroEclipse II nebulizer is now being utilized for delivery of the formulation to the lungs of CF patients as part of a phase IIa/b clinical trial (Alton et al., 2013). These studies demonstrate the potential benefit of careful investigation of combinations of drug delivery device and operating conditions to maximize delivery efficiency and therapeutic success. Acknowledgments

This work was funded by a grant from the UK Cystic Fibrosis Trust to the UK Cystic Fibrosis Gene Therapy Consortium (http://cfgenetherapy.org.uk). We would like to thank David Collie and Peter Tennant for helpful discussions, and Alan Coates and Kitty Leung for their considerable assistance with particle sizing technology. Author Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Dr. Deborah R. Gill Gene Medicine Research Group Nuffield Division of Clinical Laboratory Sciences University of Oxford, John Radcliffe Hospital Headley Way Oxford, OX3 9DU United Kingdom E-mail: [email protected] Received for publication February 13, 2014; accepted after revision April 17, 2014. Published online: April 28, 2014.