Assessment of the nuclear pore dilating agent trans-cyclohexane-1,2-diol in differentiated airway epithelium.

BACKGROUND: The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. METHODS: We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). RESULTS: TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. CONCLUSIONS: Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications.

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.

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.

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.

Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.

Gene therapy is being investigated in the treatment of lung-related aspects of the genetic disease, Cystic fibrosis (CF). Clinical studies have demonstrated CF transmembrane conductance regulator (CFTR) expression in the airways of adults with CF using a variety of gene transfer agents. In utero gene therapy is an alternative approach that facilitates vector transduction of rapidly expanding populations of target cells while avoiding immune recognition of the vector. In CF, in utero gene transfer could potentially delay the onset of disease symptoms in childhood and compensate for the role, if any, that CFTR plays in the developing organs. Previously published studies have suggested that transient expression of CFTR in utero was sufficient to rescue the fatal intestinal defect in S489X Cftr(tm1Unc)/Cftr(tm1Unc) knockout mice. We replicated these studies using an identical CFTR-expressing adenoviral vector and CF mouse strain in sufficiently large numbers to provide robust Kaplan-Meier survival data. Although each step of the procedure was carefully controlled and vector-specific CFTR expression was confirmed in the fetal organs after treatment, there was statistically no significant improvement in the survival of mice treated in utero with AdCFTR, compared with contemporaneous control animals.

A novel tankyrase inhibitor, MSC2504877, enhances the effects of clinical CDK4/6 inhibitors.

Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/ß-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G1 cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo. However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance.

Assessment of CFTR function after gene transfer in vitro and in vivo.

Cystic fibrosis (CF) a monogenic lethal disease and, therefore, ideally suited for the development of gene therapy. The first clinical trials were carried out shortly after cloning the CF gene in 1989. Since then, 25 trials have been carried out. Proof of principle for low-level airway gene transfer was established in most, but not all, trials. It is currently unclear whether current gene transfer efficiency will lead to improvements in clinically relevant endpoints such as inflammation or infection. In addition to addressing this important question, we and others are further improving airway gene transfer, by modifying existing and developing new gene transfer agents. Here, we describe pre-clinical methods related to assessing correction of the CF chloride transport defect.

Vascular oligonucleotide transfer facilitated by a polymer-coated stent.

To evaluate the potential of clinically used phosphorylcholine (PC)-coated stents for their ability to load and release small decoy oligonucleotides (ODNs). Stents were loaded with 41 +/- 6 microg ODNs. Ex vivo deployment of ODN-loaded stents in explanted rabbit aortas showed significant vascular ODN transfer, with 18 +/- 12% of intimal or medial cell nuclei containing ODNs. In proof-of-principle in vivo experiments (using the double-injury rabbit model) there was no difference in fluorescent signal intensity between animals receiving ODNloaded stents or controls. However, a significant increase in signal intensity was detected in the kidneys of animals receiving ODN-loaded stents. PC-coated stents can be loaded with ODNs. Despite successful ex vivo ODN deposition and nuclear uptake in the vessel wall, in vivo vascular ODN transfer was not achieved. Rapid intravascular release of ODN before implantation and potential vascular barriers for gene transfer are most likely responsible for the currently unsatisfactory in vivo release kinetics.

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.

Measurement of halide efflux from cultured and primary airway epithelial cells using fluorescence indicators.

The use of the halide-sensitive fluorescent probes (6-methoxy-N-(-sulphopropyl)quinolinium (SPQ) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)) to measure chloride transport in cells has now been established as an alternative to the halide-selective electrode technique, radioisotope efflux assays and patch-clamp electrophysiology. We report here procedures for the assessment of halide efflux, using SPQ/MQAE halide-sensitive fluorescent indicators, from both adherent cultured epithelial cells and freshly obtained primary human airway epithelial cells. The procedure describes the calculation of efflux rate constants using experimentally derived SPQ/MQAE fluorescence intensities and empirically derived Stern-Volmer calibration constants. These fluorescence methods permit the quantitative analysis of CFTR function.

The use of carboxymethylcellulose gel to increase non-viral gene transfer in mouse airways.

We have assessed whether viscoelastic gels known to inhibit mucociliary clearance can increase lipid-mediated gene transfer. Methylcellulose or carboxymethylcellulose (0.25-1.5%) was mixed with complexes of the cationic lipid GL67A and plasmids encoding luciferase and perfused onto the nasal epithelium of mice. Survival after perfusion with 1% CMC or 1% MC was 90 and 100%, respectively. In contrast 1.5% CMC was uniformly lethal likely due to the viscous solution blocking the airways. Perfusion with 0.5% CMC containing lipid/DNA complexes reproducibly increased gene expression by approximately 3-fold (n=16, p<0.05). Given this benefit, likely related to increased duration of contact, we also assessed the effect of prolonging contact time of the liposome/DNA complexes by delivering our standard 80 microg DNA dose over either approximately 22 or 60 min of perfusion. This independently increased gene transfer by 6-fold (n=8, p<0.05) and could be further enhanced by the addition of 0.5% CMC, leading to an overall 25-fold enhancement (n=8, p<0.001) in gene expression. As a result of these interventions CFTR transgene mRNA transgene levels were increased several logs above background. Interestingly, this did not lead to correction of the ion transport defects in the nasal epithelium of cystic fibrosis mice nor for immunohistochemical quantification of CFTR expression. To assess if 0.5% CMC also increased gene transfer in the mouse lung, we used whole body nebulisation chambers. CMC was nebulised for 1h immediately before, or simultaneously with GL67A/pCIKLux. The former did not increase gene transfer, whereas co-administration significantly increased gene transfer by 4-fold (p<0.0001, n=18). This study suggests that contact time of non-viral gene transfer agents is a key factor for gene delivery, and suggests two methods which may be translatable for use in man.

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 (>or=56 d) in vivo transgene expression in the absence of lung inflammation.

Nondisruptive, sequence-specific coupling of fluorochromes to plasmid DNA.

A method to attach a fluorochrome sequence-specifically to supercoiled plasmid DNA (pDNA) without perturbing transgene expression would provide an invaluable aid in a variety of applications requiring probes for the intracellular tracking of transfected pDNA. Here we report a method to couple commercially available fluorochromes covalently and sequence-specifically to pDNA using a peptide nucleic acid (PNA) as a linker molecule. The terminal cysteine thiol group on the PNA peptide backbone is reacted with a maleimide moiety on the fluorochrome to produce a fluorescent conjugate which is in turn hybridized to a plasmid expression vector containing an 11-bp target sequence. Spectroscopic evaluation and an electrophoretic mobility shift assay showed that the pDNA hybridized to one PNA-fluorochrome conjugate molecule. The fluorescence signal comigrated with pDNA on acrylamide gels, confirming the stable attachment of the fluorescent conjugate to the pDNA. The utility of one of the conjugates, PNA-Oregon green 488/pCMVbeta-DTS, to probe pDNA transport across the nuclear envelope, a significant barrier to gene transfer, was undertaken using a digitonin-permeabilized HeLa cell assay. The PNA-Oregon green 488/pCMVbeta-DTS conjugate is able to efficiently traverse the nuclear membrane of the permeabilized cells, accumulating in the nuclei within 30 min and reaching maximal levels by 1h. When transfected into HeLa cells, the PNA-Oregon green 488/pCMVbeta-DTS conjugate retained 55% of the native plasmid's biological activity, as determined by a beta-galactosidase assay. Thus, this method allows for the sequence-specific coupling of commercially available fluorochromes to DNA expression vectors while retaining biological function.

The efficacy of a 'master switch gene' HIF-1alpha in a porcine model of chronic myocardial ischaemia.

AIMS: Therapeutic angiogenesis is a potential new treatment for patients unsuitable for conventional revascularization strategies. We investigated angiogenesis via a 'master switch gene' hypoxia inducible factor (HIF-1alpha). METHODS AND RESULTS: Ameroid occluders were placed around the left circumflex coronary artery of 74 pigs. Three weeks later, pigs were randomized to receive (i) adenovirus encoding HIF-1alpha (Ad2/HIF-1alpha VP-16 10(10) particles); (ii) plasmid DNA encoding HIF-1alpha (pHIF-1alpha NFkappaB 500 microg); (iii) pHIF-1alpha NFkappaB 2500 microg; and (iv) adenoviral control (Ad2/CMV-empty vector 10(10) particles). Twenty injections (50 microL each) were administered epicardially via re-thoracotomy. Three weeks after gene delivery significant (ANOVA P=0.02) changes in myocardial perfusion during stress were seen in the area adjacent to injections. Post hoc testing (Bonferroni) demonstrated that the AdHIF-1alpha group was significantly (P=0.02) different from the Ad2/control. There were also significant (ANOVA P=0.02) differences in resting left ventricular (LV) function. Post hoc (Bonferroni) showed that the AdHIF-1alpha group was significantly different from the Ad2/control (P=0.03). No significant changes in any parameter were seen with plasmid HIF-1alpha. There were no differences in collateralization or capillary growth. CONCLUSION: Ad2/HIF-1alpha increased myocardial perfusion and improved LV function. Plasmid HIF-1alpha was not associated with improvements in any bioactivity endpoints.