Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia.     

Sendai virus vectors as an emerging negative-strand RNA viral vector system

The power to manipulate the genome of negative-strand RNA viruses, including the insertion of additional non-viral genes, has led to the development of a new class of viral vectors for gene transfer approaches. The murine parainfluenza virus type I, or Sendai virus (SeV), has emerged as a prototype virus of this vector group, being employed in numerous in vitro as well as animal studies over the last few years. Extraordinary features of SeV are the remarkably brief contact time that is necessary for cellular uptake, a strong but adjustable expression of foreign genes, efficient infection in the respiratory tract despite a mucus layer, transduction of target cells being independent of the cell cycle, and an exclusively cytoplasmic replication cycle without any risk of chromosomal integration. In this review we describe the current knowledge of Sendai virus vector (SeVV) development as well as the results of first-generation vector applications under both in vitro and in vivo conditions. So far, Sendai virus vectors have been identified to be a highly efficient transduction tool for a broad range of different tissues and applications. Future directions in vector design and development are discussed.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

Gene Therapy of c-myc Suppressor FUSE-Binding Protein-Interacting Repressor by Sendai Virus Delivery Prevents Tracheal Stenosis

Acquired tracheal stenosis remains a challenging problem for otolaryngologists. The objective of this study was to determine whether the Sendai virus (SeV)-mediated c-myc suppressor, a far upstream element (FUSE)-binding protein (FBP)-interacting repressor (FIR), modulates wound healing of the airway mucosa, and whether it prevents tracheal stenosis in an animal model of induced mucosal injury. A fusion gene-deleted, non-transmissible SeV vector encoding FIR (FIR-SeV/ΔF) was prepared. Rats with scraped airway mucosae were administered FIR-SeV/ΔF through the tracheostoma. The pathological changes in the airway mucosa and in the tracheal lumen were assessed five days after scraping. Untreated animals showed hyperplasia of the airway epithelium and a thickened submucosal layer with extensive fibrosis, angiogenesis, and collagen deposition causing lumen stenosis. By contrast, the administration of FIR-SeV/ΔF decreased the degree of tracheal stenosis (P < 0.05) and improved the survival rate (P < 0.05). Immunohistochemical staining showed that c-Myc expression was downregulated in the tracheal basal cells of the FIR-SeV/ΔF-treated animals, suggesting that c-myc was suppressed by FIR-SeV/ΔF in the regenerating airway epithelium of the injured tracheal mucosa. The airway-targeted gene therapy of the c-myc suppressor FIR, using a recombinant SeV vector, prevented tracheal stenosis in a rat model of airway mucosal injury.     

Lysophosphatidylcholine as an adjuvant for lentiviral vector mediated gene transfer to airway epithelium: effect of acyl chain length

Background

Poor gene transfer efficiency has been a major problem in developing an effective gene therapy for cystic fibrosis (CF) airway disease. Lysophosphatidylcholine (LPC), a natural airway surfactant, can enhance viral gene transfer in animal models. We examined the electrophysiological and physical effect of airway pre-treatment with variants of LPC on lentiviral (LV) vector gene transfer efficiency in murine nasal airways in vivo.

Methods

Gene transfer was assessed after 1 week following nasal instillations of a VSV-G pseudotype LV vector pre-treated with a low and high dose of LPC variants. The electrophysiological effects of a range of LPC variants were assessed by nasal transepithelial potential difference measurements (TPD) to determine tight junction permeability. Any physical changes to the epithelium from administration of the LPC variants were noted by histological methods in airway tissue harvested after 1 hour.

Results

Gene transduction was significantly greater compared to control (PBS) for our standard LPC (palmitoyl/stearoyl mixture) treatment and for the majority of the other LPC variants with longer acyl chain lengths. The LPC variant heptadecanoyl also produced significantly greater LV gene transfer compared to our standard LPC mixture. LV gene transfer and the transepithelial depolarization produced by the 0.1% LPC variants at 1 hour were strongly correlated (r² = 0.94), but at the 1% concentration the correlation was less strong (r² = 0.59). LPC variants that displayed minor to moderate levels of disruption to the airway epithelium were clearly associated with higher LV gene transfer.

Conclusions

These findings show the LPC variants effect on airway barrier function and their correlation to the effectiveness of gene expression. The enhanced expression produced by a number of LPC variants should provide new options for preclinical development of efficient airway gene transfer techniques.     

Glycocalyx restricts adenoviral vector access to apical receptors expressed on respiratory epithelium in vitro and in vivo: role for tethered mucins as barriers to lumenal infection

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.     

Recombinant Sendai virus vectors deleted in both the matrix and the fusion genes: efficient gene transfer with preferable properties

BACKGROUND: Sendai virus (SeV) is a new type of cytoplasmic RNA vector, which infects and replicates in most mammalian cells, directs high-level expression of the genes on its genome and is free from genotoxicity. In order to improve this vector, both the matrix (M) and fusion (F) genes were deleted from its genome. METHODS: For the recovery of the M and F genes-deleted SeV (SeV/DeltaMDeltaF), the packaging cell line was established by using a Cre/loxP induction system. SeV/DeltaMDeltaF was characterized and compared with wild-type and F or M gene-deleted SeV vectors in terms of transduction ability, particle formation, transmissible property and cytotoxicity. RESULTS: SeV/DeltaMDeltaF was propagated in high titers from the packaging cell line. When this vector was administered into the lateral ventricle and the respiratory tissue, many of the ependymal and epithelial cells were transduced, respectively, as in the case of wild-type SeV. F gene-deletion made the SeV vector non-transmissible, and M gene-deletion worked well to inhibit formation of the particles from infected cells. Simultaneous deletions of these two genes in the same genome resulted in combining both advantages. That is, both virus maturation into particles and transmissible property were almost completely abolished in cells infected with SeV/DeltaMDeltaF. Further, the cytopathic effect of SeV/DeltaMDeltaF was significantly attenuated rather than that of wild type in vitro and in vivo. CONCLUSIONS: SeV/DeltaMDeltaF is an advanced type of cytoplasmic RNA vector, which retains efficient gene transfer, gains non-transmissible properties and loses particle formation with less cytopathic effect.     

Sendai virus vector-mediated gene transfer of glial cell line-derived neurotrophic factor prevents delayed neuronal death after transient global ischemia in gerbils.

We have developed a cytoplasmic replicating virus vector of Sendai virus (SeV) that infects and replicates in most mammalian cells, including neurons, and directs high-level gene expression. To investigate the protective effect of SeV vector-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) on the delayed neuronal death caused by transient global ischemia in gerbils, SeV vectors carrying either GDNF (SeV/GDNF) or enhanced green fluorescent protein gene (SeV/GFP) were stereotaxically microinjected into the lateral ventricle. Four days after injection, occlusion of the bilateral common carotid arteries for 5 min produced transient global forebrain ischemia. Treatment with SeV/GDNF significantly decreased the delayed neuronal death of the hippocampal CA1 pyramidal neurons observed 6 days after the operation. TUNEL staining demonstrated that SeV/GDNF treatment markedly reduced the number of apoptotic cells in the hippocampal CA1 neurons, indicating that SeV/GDNF treatment prevented apoptosis. Furthermore, delayed neuronal death on the contralateral side of the hippocampal CA1 was also prevented to a similar extent as that on the ipsilateral side. These results suggest that SeV/GDNF prevents the delayed neuronal death induced by ischemia and is potentially useful for gene therapy for stroke.