Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier
Graphical abstract
Introduction
Pulmonary complications are the primary cause of morbidity and mortality associated with cystic fibrosis (CF), asthma, and other life-threatening disorders [1], [2]. A great deal of effort has aimed at development of gene therapy strategies for the airways, including clinical evaluation of a large number of viral and non-viral gene delivery systems in patients with lung diseases [3], [4]. The majority of these trials have utilized recombinant viruses, but immunogenicity, safety concerns, and inefficient gene transfer have precluded their success [3], [5]. Alternatively, non-viral gene carriers offer potential for improved safety, ease of manufacturing and scale up, possibility of repeated dosing without generating a therapy-disabling immune response, and ability to accommodate larger plasmid DNA compared to commonly tested viruses, such as adeno-associated virus. A polymeric gene delivery platform, based on a copolymer of 10 kDa polyethylene glycol (PEG) conjugated to a 30-mer poly-l-lysine (PLL) via a cysteine residue (PEG10k–CK30/DNA), was safely administered to the nares of CF patients with negligible serum or nasal inflammatory responses in a clinical trial [6], thus encouraging further development of similar strategies.
A largely overlooked barrier to efficient gene delivery to the lung airways is the highly adhesive and hyperviscoelastic mucus gel in the airways of patients, including those with CF, asthma and chronic obstructive pulmonary disease (COPD), that traps and prevents the access of gene carriers to the underlying epithelium. Mucus in patients with advanced obstructive lung diseases is composed of a dense mesh of mucin fibers, large macromolecules containing a high density of negatively charged glycans interspersed with periodic hydrophobic regions [7]. In the airways of CF [8], [9] and COPD [10] patients, elevated levels of bacterial and endogenous DNA, as well as actin filaments from degraded neutrophils, further contribute to the dense mucus mesh structure and increased adhesivity of the mucus gel layer. We previously estimated the average pore size in CF mucus to be 140 ± 50 nm (range: 60–300 nm) [11], markedly smaller than the average pore size of 340 ± 70 nm for human cervicovaginal mucus secretions from healthy, non-CF volunteers [12]. As a consequence of the elevated adhesivity and tighter mesh size, we recently found that several clinically tested viral [13] and non-viral [14], [15] gene carriers are incapable of efficiently penetrating human CF mucus.
We sought to engineer polymeric gene carriers that are sufficiently small to diffuse through the mucus mesh and enter cells via non-specific endocytic pathways, and that possess a muco-inert surface to avoid adhesion to mucus constituents. Cationic polymers possess a high density of protonable amines that facilitate efficient DNA condensation [16], [17], protect cargo DNA from enzymatic degradation [18], [19], and provide proton-buffering that may facilitate endosomal escape of cargo DNA from degradative vesicles in cells [20]. Nevertheless, gene transfer efficiency by cationic polymer-based gene carriers is strongly reduced in mucus-coated cells compared to mucus-depleted tissues [21], likely a consequence of entrapment of cationic gene carriers in the mucus gel via electrostatic association with the negatively charged mucus constituents. We have previously demonstrated that a dense coating of low MW (2–5 kDa) PEG markedly improved the diffusion of polymeric nanoparticles (NP) through human cervicovaginal mucus [22], [23], chronic rhinosinusitis mucus [24] and CF airway mucus [11]. PEG coatings also have shown to reduce toxicity of cationic polymers in vivo [25] and in humans [6] to clinically safe levels. Dense PEG coatings, however, interfere with DNA compaction by cationic polymers, resulting in loosely compacted larger particles [26], [27]. Larger particles with PEG surface coatings have shown inferior cellular uptake compared to smaller but similarly coated particles [28], [29]. We describe an approach to produce small, stable NP with sufficiently dense PEG coatings to allow rapid mucus penetration, and we show it is applicable to two widely used polymer systems, polyethylenimine (PEI) and poly-l-lysine (PLL). We show that mucus penetrating DNA NP mediate efficient gene transfer to airway cells in vitro and in vivo without eliciting acute toxic or inflammatory responses.
Section snippets
CF mucus collection
Mucus spontaneously expectorated from CF patients ages 24–37, was collected at the Johns Hopkins Adult Cystic Fibrosis Program. Mucus collection was performed under informed consent on a protocol approved by the Johns Hopkins Medicine Institutional Review Board. Samples were acquired from the weekly CF outpatient clinic, placed immediately on ice, and studied the same day. The total number of individual samples used for the present study was 7.
Polymer preparation
Methoxy PEG N-hydroxysuccinimide (mPEG-NHS, 5 kDa,
Results
To effectively shield the highly positive surface charge of gene carriers based on PEI (uncoated PEI/DNA NP; PEI-UCP hereafter), we first attempted to conjugate multiple 5 kDa PEG molecules to 25 kDa branched PEI molecules at a high PEG to PEI ratio of 50. The reaction yielded PEG5k–PEI copolymers with an average PEG to PEI ratio of 37, which is substantially higher than previously reported (< 20) [26], [38], [39]. We found that surface charge of PEG-coated DNA NP (conventionally coated PEI/DNA
Discussion
Uniform expression of functional genes throughout the airway epithelium is thought to be essential to improved lung gene therapy for several diseases, including correcting phenotypical defects in the airways of CF patients [44]. However, this remains a formidable challenge due to numerous biological barriers, including the tenacious mucus layer that lines the airways and excludes conventional NP and gene carriers [8], [9], [13], [45]. By developing a formulation process that allows highly dense
Acknowledgments
Funding was provided by the National Institutes of Health (R01 EB003558, P01 HL51811 and F32 HL103137) and the Cystic Fibrosis foundation (HANES07XX0). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We are grateful to Meghan Ramsay and Sharon Watts at the Johns Hopkins Adult Cystic Fibrosis Center for CF mucus collection. We also thank Tao Yu and Benjamin Schuster for their help with NMR
References (56)
- et al.
Gene transfer to the lung: lessons learned from more than 2 decades of CF gene therapy
Adv. Drug Deliv. Rev.
(2009) Barrier properties of mucus
Adv. Drug Deliv. Rev.
(2009)- et al.
Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues
Adv. Drug Deliv. Rev.
(2009) - et al.
The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles
Biomaterials
(2009) - et al.
N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles
Mol. Ther.
(2011) - et al.
Polyethylenimine shows properties of interest for cystic fibrosis gene therapy
Biochim. Biophys. Acta
(1999) - et al.
Drug carrier nanoparticles that penetrate human chronic rhinosinusitis mucus
Biomaterials
(2011) - et al.
Inflammatory responses to pulmonary application of PEI-based siRNA nanocarriers in mice
Biomaterials
(2011) - et al.
Intranasal gene delivery with a polyethylenimine–PEG conjugate
J. Control. Release
(2002) - et al.
Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells
J. Control. Release
(2007)
Transfection of airway epithelium by stable PEGylated poly-L-lysine DNA nanoparticles in vivo
Mol. Ther.
Real-time multiple-particle tracking: applications to drug and gene delivery
Adv. Drug Deliv. Rev.
Static and dynamic errors in particle tracking microrheology
Biophys. J.
Single-particle tracking as a quantitative microscopy-based approach to unravel cell entry mechanisms of viruses and pharmaceutical nanoparticles
Mol. Ther.
Multiple-particle tracking and two-point microrheology in cells
Methods Cell Biol.
In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers
Biomaterials
Cystic fibrosis transmembrane regulator missing the first four transmembrane segments increases wild type and DeltaF508 processing
J. Biol. Chem.
Cationic carriers of genetic material and cell death: a mitochondrial tale
Biochim. Biophys. Acta
Extracellular barriers in respiratory gene therapy
Adv. Drug Deliv. Rev.
PEGylation affects cytotoxicity and cell-compatibility of poly(ethylene imine) for lung application: structure–function relationships
Toxicol. Appl. Pharmacol.
Minimal toxicity of stabilized compacted DNA nanoparticles in the murine lung
Mol. Ther.
Enhanced lung gene expression after aerosol delivery of concentrated pDNA/PEI complexes
Mol. Ther.
The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes
Biomaterials
Clinical review: severe asthma
Crit. Care
Pulmonary complications of cystic fibrosis
Respir. Care
Gene therapy prospects—intranasal delivery of therapeutic genes
Adv. Clin. Exp. Med.
Progress and prospects: prospects of repeated pulmonary administration of viral vectors
Gene Ther.
Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution
Hum. Gene Ther.
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- 1
These authors equally contributed to this work.
- 2
Current Address: Department of Neurosurgery, University of Maryland School of Medicine, 22 South Green Street, Baltimore, MD 21201.
- 3
Current Address: Eshelman School of Pharmaceutics, Division of Molecular Pharmaceutics, University of North Carolina at Chapel Hill, 120 Mason Farm Road, Chapel Hill NC, 27599.