Felix M. Munkonge

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (≥56 d) in vivo transgene expression in the absence of lung inflammation.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.

Emerging significance of plasmid DNA nuclear import in gene therapy

The signal-mediated import of plasmid DNA (pDNA) into nondividing mammalian cell nuclei is one of the key biological obstacles to nonviral therapeutic pDNA delivery. Overcoming this barrier to pDNA transfer is thus an important fundamental objective in gene therapy. Here, we outline the rationale behind current and future strategies for signal-mediated pDNA nuclear import. Results obtained from studies of the nuclear delivery of pDNA coupled to experimentally defined nuclear localisation signal (NLS) peptides, in conjunction with detergent-permeabilised reconstitution cell assays, direct intracellular microinjection, cell-based transfection, and a limited number of in vivo experiments are discussed.

Sendai virus-mediated CFTR gene transfer to the airway epithelium

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (CftrG551D and Cftrtm1UncTgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Identification and Functional Characterization of Cytoplasmic Determinants of Plasmid DNA Nuclear Import

Import of exogenous plasmid DNA (pDNA) into mammalian cell nuclei represents a key intracellular obstacle to efficient non-viral gene delivery. This includes access of the pDNA to the nuclei of non-dividing cells where the presence of an intact nuclear membrane is limiting for gene transfer. Here we identify, isolate, and characterize, cytoplasmic determinants of pDNA nuclear import into digitonin-permeabilized HeLa cells. Depletion of putative DNA-binding proteins, on the basis of their ability to bind immobilized pDNA, abolished pDNA nuclear import supporting the critical role of cytoplasmic factors in this process. Elution of pDNA-bound proteins, followed by two-dimensional sodium dodecyl polyacrylamide gel electrophoresis identified several candidate DNA shuttle proteins. We show that two of these, NM23-H2, a ubiquitous c-Myc transcription-activating nucleoside diphosphate kinase, and the core histone H2B can both reconstitute pDNA nuclear import. Further, we demonstrate a significant increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from two of the DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10. These data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus using the minimal import machinery. Importantly, increasing the binding of pDNA to shuttle proteins by re-engineering reporter plasmids with shuttle binding sequences enhances gene transfer. Increasing the potential for exogenously added pDNA to bind intracellular transport cofactors may enhance the potency of non-viral gene transfer.

Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import

Although transfections are routinely used in the laboratory, the mechanism(s) by which exogenous DNA is transported into the nucleus is poorly understood. By improving our understanding of how vectors circumvent the numerous cellular barriers to gene transfer, more efficient gene delivery methods can be devised. We have begun to design plasmid constructs that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing levels of expression. We have shown that inclusion of specific DNA sequences in plasmid constructs mediates nuclear import both in vitro and in vivo. Here, we use plasmid affinity chromatography, mass spectrometry (MS), and live-cell pulldowns of transfected plasmid constructs to identify protein cofactors that interact in a sequence-specific manner with these DNA nuclear targeting sequences (DTSs). Importin beta(1), importin 7, and the small guanosine triphosphatase Ran all demonstrate DTS-specific interaction in both MS and pull-down assays, consistent with our model of plasmid nuclear import. In addition, knockdown of importin beta(1) with small interfering RNA (siRNA) abrogates plasmid nuclear import, indicating that it is a necessary cofactor. Our discovery that specific karyopherins mediate plasmid nuclear import can be used to design more effective vectors for gene delivery.

Expression and maturation of Sendai virus vector-derived CFTR protein : functional and biochemical evidence using a GFP-CFTR fusion protein

Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.

Limitations of the murine nose in the development of nonviral airway gene transfer.

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.

Lack of inhibition by dideoxy-forskolin and verapamil of DIDS-sensitive volume-activated Cl- secretion in human squamous lung carcinoma epithelial cells.

The effect of osmotic stress on Cl- permeability in human squamous lung carcinoma epithelial (S1) cells was investigated using a macroscopic 125I efflux assay. Hypotonic challenge of monolayers led to a significant (P < 0.01) dose-related increase in efflux from pre-loaded cells, returning to pre-activation rates within 10 min. A similar magnitude of response could be produced by challenge with an isotonic low chloride-containing solution. Neither 100 mM dideoxy-forskolin nor 100 mM verapamil inhibited the increase in Cl- secretion after hypotonic challenge, whereas 100 mM DIDS inhibited volume-activated Cl- secretion by 55%. Both Northern and Western blot analysis confirmed the absence of MDR1 mRNA and P-glycoprotein in the S1 cells. We conclude that these cells have a volume-regulated Cl- secretory pathway that is independent of the ABC transporter, P-glycoprotein.

Transient in utero expression of CFTR does not improve survival of CF knockout mice

Lentiviral (LV) vectors can transduce slowly dividing cells such as those of the airway epithelium, the recognised target of CF lung disease. However, efficient LV-mediated transduction of airway epithelial cells is hampered by extra-cellular barriers. Thus, the local confinement on the cell surface of LV vectors is an important requirement for gene delivery to the airway epithelium. To address this issue we have exploited the magnetofection technique in in vitro models of airway epithelium applying a LV vector carrying the Green Fluorescent Protein (GFP) gene. Magnetofectins were formed by non-covalent interaction between LV particles and polycation-coated iron oxide nanoparticles. Bronchial 16HBE14oand bronchiolar H441 cells were tranduced with magnetofectins in the presence of a magnetic field. Magnetofectins were more efficient than LV alone up to 3.9 fold and magnetofection was able to significantly increase the percentage of transduced cells already after 15 minutes of incubation. We next asked whether magnetofection could also be efficient in polarised 16HBE14ocells. Magnetofection determined a 3-fold increase of GFP+ cells (up to 10%) as compared with LV alone. Upon treatment with 50 nM dexamethasone, H441 cells can form hemicysts containing fluid, called “domes”, which are relatively resistant to LV-mediated transduction (GFP+ cells = 6%). Magnetofection increased up to 4 fold the transduction efficiency. Conversely, in the absence of magnetic field no significant effect could be observed. We conclude that magnetofection can facilitate LV-mediated transduction of airway epithelial cells by lowering vector dose and reducing incubation time to achieve efficient transduction. Supported by EU: project no. 005213.