Optimised protocols to generate high titre lentiviral vectors using a novel transfection agent enabling extended HEK293T culture following transient transfection and suspension culture.

HIV-1 based lentiviral viruses are considered powerful and versatile gene therapy vectors to deliver therapeutic genes to patients with hereditary or acquired diseases. These vectors can efficiently transduce a variety of cell types when dividing or non-dividing to provide permanent delivery and long-term gene expression. Demand for scalable manufacturing protocols able to generate enough high titre vector for widespread use of this technology is increasing and considerable efforts to improve vector production cost-effectively, is ongoing. Current methods for LV production mainly use transient transfection of producer cell lines. Cells can be grown at scale, either in 2D relying on culturing producer cells in multi-tray flask cell culture factories or in roller bottles or can be adapted to grow in 3D suspensions in large batch fermenters. This suits rapid production and testing of new vector constructs pre-clinically for their efficacy, particle titre and safety. In this study, we sought to improve lentiviral titre over time by testing two alternative commercially available transfection reagents Fugene® 6 and Genejuice® with the commonly used polycation, polyethyleneimine. Our aim was to identify less cytotoxic transfection reagents that could be used to generate LV particles at high titre past the often used 72 h period when vector is usually collected before producer cell death is caused due to post transfection cytotoxicity. We show that LV could be produced in extended culture using Genejuice® and even by transfected cells in glass flasks in suspension. Because this delivery agent is less toxic to 293 T producer cells, following optimisation of transfection we found that LV can be harvested for more than 10 days at high titre. Using our protocol, titres of 109 TU/ml and 108 TU/ml were routinely reached via traditional monolayer conditions or suspension cultures, respectively. We propose, this simple change in vector production enables large volumes of high titre vector to be produced, cost effectively for non-clinical in vivo and in vitro applications or for more stringent downstream clinical grade vector purification.

HIV- 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival.

Lentiviral vectors (LV) are attractive for permanent and effective gene therapy. However, integration into the host genome can cause insertional mutagenesis highlighting the importance of understanding of LV integration. Insertion site (IS) tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre-integration complexes (PICs) helping to target the virus genome. Transcription factors (TF) that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites (pTFBS) near to IS chosen by HIV-1 LV using a narrow 20 bp window in infected human induced pluripotent stem cells (iPSCs) and their hepatocyte-like cell (HLC) derivatives. We then aligned the pTFBS with these sequences found in the LTRs of native and self-inactivated LTRs. We found significant enrichment of these sequences for pTFBS essential to HIV-1 life cycle and virus survival. These same sites also appear in HIV-1 patient IS and in mice infected with HIV-1 based LV. This in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV-1 LV are important to virus survival and propagation.

Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology.

Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH4)2SO4 (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at -20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines.

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.  Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia.

Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5' region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3' gene can be significantly increased to similar levels as the cap-dependent expression of the 5' gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18-141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5' and 3' ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation.

Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes.

Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136-370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.

Three-dimensional cell culture: from evolution to revolution.

Recent advances in the isolation of tissue-resident adult stem cells and the identification of inductive factors that efficiently direct differentiation of human pluripotent stem cells along specific lineages have facilitated the development of high-fidelity modelling of several tissues in vitro Many of the novel approaches have employed self-organizing three-dimensional (3D) culturing of organoids, which offer several advantages over conventional two-dimensional platforms. Organoid technologies hold great promise for modelling diseases and predicting the outcome of drug responses in vitro Here, we outline the historical background and some of the recent advances in the field of three-dimensional organoids. We also highlight some of the current limitations of these systems and discuss potential avenues to further benefit biological research using three-dimensional modelling technologies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Genome wide classification and characterisation of CpG sites in cancer and normal cells.

This study identifies common methylation patterns across different cancer types in an effort to identify common molecular events in diverse types of cancer cells and provides evidence for the sequence surrounding a CpG to influence its susceptibility to aberrant methylation. CpG sites throughout the genome were divided into four classes: sites that either become hypo or hyper-methylated in a variety cancers using all the freely available microarray data (HypoCancer and HyperCancer classes) and those found in a constant hypo (Never methylated class) or hyper-methylated (Always methylated class) state in both normal and cancer cells. Our data shows that most CpG sites included in the HumanMethylation450K microarray remain unmethylated in normal and cancerous cells; however, certain sites in all the cancers investigated become specifically modified. More detailed analysis of the sites revealed that majority of those in the never methylated class were in CpG islands whereas those in the HyperCancer class were mostly associated with miRNA coding regions. The sites in the Hypermethylated class are associated with genes involved in initiating or maintaining the cancerous state, being enriched for processes involved in apoptosis, and with transcription factors predicted to bind to these genes linked to apoptosis and tumourgenesis (notably including E2F). Further we show that more LINE elements are associated with the HypoCancer class and more Alu repeats are associated with the HyperCancer class. Motifs that classify the classes were identified to distinguish them based on the surrounding DNA sequence alone, and for the identification of DNA sequences that could render sites more prone to aberrant methylation in cancer cells. This provides evidence that the sequence surrounding a CpG site has an influence on whether a site is hypo or hyper methylated.

Transduction of fetal mice with a feline lentiviral vector induces liver tumors which exhibit an E2F activation signature.

Lentiviral vectors are widely used in basic research and clinical applications for gene transfer and long-term expression; however, safety issues have not yet been completely resolved. In this study, we characterized hepatocarcinomas that developed in mice 1 year after in utero administration of a feline-derived lentiviral vector. Mapped viral integration sites differed among tumors and did not coincide with the regions of chromosomal aberrations. Furthermore, gene expression profiling revealed that no known cancer-associated genes were deregulated in the vicinity of viral integrations. Nevertheless, five of the six tumors exhibited highly significant upregulation of E2F target genes, of which a majority are associated with oncogenesis, DNA damage response, and chromosomal instability. We further show in vivo and in vitro that E2F activation occurs early on following transduction of both fetal mice and cultured human hepatocytes. On the basis of the similarities in E2F target gene expression patterns among tumors and the lack of evidence implicating insertional mutagenesis, we propose that transduction of fetal mice with a feline lentiviral vector induces E2F-mediated major cellular processes that drive hepatocytes toward uncontrolled proliferation culminating in tumorigenesis.

Monitoring for potential adverse effects of prenatal gene therapy: genotoxicity analysis in vitro and on small animal models ex vivo and in vivo.

Gene delivery by integrating vectors has the potential to cause genotoxicity in the host by insertional mutagenesis (IM). Previously, the risk of IM by replication incompetent retroviral vectors was believed to be small. However, the recent observation of leukaemic events due to gamma retroviral vector insertion and activation of the LMO-2 proto-oncogene in patients enrolled in the French and British gene therapy trials for X-SCID demonstrates the need to understand vector associated genotoxicity in greater detail. These findings have led to the development of in vitro, ex vivo, and in vivo assays designed to predict genotoxic risk and to further our mechanistic understanding of this process at the molecular level. In vitro assays include transformation of murine haematopoietic stem cells by integrating retroviral (RV) or lentiviral (LV) vectors and measurement of cell survival resulting from transformation due to integration mainly into the Evi1 oncogene. Ex vivo assays involve harvesting haematopoietic stem cells from mice followed by gene transfer and re-infusion of RV or LV infected cells to reconstitute the immune system. Insertional mutagenesis is then determined by analysis of clonally dominant populations of cells. The latter model has also been made highly sensitive using cells from mice predisposed to oncogenesis by lack of the P53 and Rb pathways. Our investigations on fetal gene therapy discovered a high incidence of liver tumour development that appears to be associated with vector insertions into cancer-related genes. Many genes involved in growth and differentiation are actively transcribed in early developmental and are therefore in an open chromatin configuration, which favours provirus insertion. Some of these genes are known oncogenes or anti-oncogenes and are not usually active during adulthood. We found that in utero injection of primate HIV-1, HR'SIN-cPPT-S-FIX-W does not result in oncogenesis as opposed to administration of non-primate equine infectious anaemia virus (EIAV), SMART 2 lentivirus vectors and, most recently, the non-primate pLIONhAATGFP (FIV) vector, which both give rise to high frequency hepatocellular carcinoma. The peculiar integration pattern into cancer-related genes observed in this model makes the fetal mouse a sensitive tool, not only to investigate long-term vector-mediated gene expression, but also vector safety in an in vivo system with minimal immunological interference. The identification of distinct differences in genotoxic outcome between the applied vector systems i.e. EIAV or FIV vectors versus HIV may indicate a particular biosafety profile of the HIV-1-based vector, which renders it potentially suitable for safe prenatal gene therapy.

Monitoring for potential adverse effects of prenatal gene therapy: mouse models for developmental aberrations and inadvertent germ line transmission.

So far no systematic studies have been conducted to investigate developmental aberrations after prenatal gene transfer in mice. Here, we suggest procedures for such observations to be applied, tested and improved in further in utero gene therapy experiments. They are based on our own experience in husbandry for transgenic human diseases mouse models and breading, rearing, and observing mice after fetal gene transfer as well as on the systematic screens for monitoring of knock-out mutant mouse phenotypes established in international mutagenesis projects (EUMORPHIA and EUMODIC and subsequently the International Mouse Phenotyping Consortium). We also describe here the analysis procedures for detection of germ line mutations based on quantitative PCR (qPCR) by sperm-DNA analysis and breeding studies.

LDLR-Gene therapy for familial hypercholesterolaemia: problems, progress, and perspectives.

Coronary artery diseases (CAD) inflict a heavy economical and social burden on most populations and contribute significantly to their morbidity and mortality rates. Low-density lipoprotein receptor (LDLR) associated familial hypercholesterolemia (FH) is the most frequent Mendelian disorder and is a major risk factor for the development of CAD. To date there is no cure for FH. The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved. The first ex vivo clinical trial of gene therapy in FH was conducted nearly 18 years ago. Patients who had inherited LDLR gene mutations were subjected to an aggressive surgical intervention involving partial hepatectomy to obtain the patient's own hepatocytes for ex vivo gene transfer with a replication deficient LDLR-retroviral vector. After successful re-infusion of transduced cells through a catheter placed in the inferior mesenteric vein at the time of liver resection, only low-level expression of the transferred LDLR gene was observed in the five patients enrolled in the trial. In contrast, full reversal of hypercholesterolaemia was later demonstrated in in vivo preclinical studies using LDLR-adenovirus mediated gene transfer. However, the high efficiency of cell division independent gene transfer by adenovirus vectors is limited by their short-term persistence due to episomal maintenance and the cytotoxicity of these highly immunogenic viruses. Novel long-term persisting vectors derived from adeno-associated viruses and lentiviruses, are now available and investigations are underway to determine their safety and efficiency in preparation for clinical application for a variety of diseases. Several novel non-viral based therapies have also been developed recently to lower LDL-C serum levels in FH patients. This article reviews the progress made in the 18 years since the first clinical trial for gene therapy of FH, with emphasis on the development, design, performance and limitations of viral based gene transfer vectors used in studies to ameliorate the effects of LDLR deficiency.

Ultrasonographic development of the fetal sheep stomach and evaluation of early gestation ultrasound-guided in utero intragastric injection.

OBJECTIVE: Safely targeting the fetal gastrointestinal tract during early gestation is essential to develop effective prenatal gene therapy for gastrointestinal diseases. In this study, we aimed to characterize the development of the fetal sheep stomach sonographically and to determine the optimum gestational age, as well as the shortterm morbidity and mortality of early-gestation ultrasound-guided intragastric injection. MATERIALS AND METHODS: In experiments investigating ultrasound-guided prenatal gene therapy, we studied the size and development of the stomach of 185 sheep fetuses (33-144 days' gestational age [GA]; term is 145 days). Ultrasound-guided intragastric injection was performed in 12 fetuses at 55-62 days' GA and postmortem examinations were performed 48 hours later. RESULTS: The stomach was not visible at or before 40 days' GA, but it was seen in all fetuses at 55 days' GA or more. The anteroposterior, transverse and longitudinal diameters of the stomach increased in a quasi-linear fashion throughout gestation. Intragastric injection was successful in 10 out of the 11 fetuses (91%) injected at 60-62 days' GA, with nine fetuses (91%) surviving this procedure. CONCLUSION: In the early-gestation sheep fetus, ultrasound-guided intragastric injection has a good success rate with a low short-term mortality and morbidity.

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype.

The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by preexposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy.

Widespread distribution and muscle differentiation of human fetal mesenchymal stem cells after intrauterine transplantation in dystrophic mdx mouse.

Duchenne muscular dystrophy (DMD) is a common X-linked disease resulting from the absence of dystrophin in muscle. Affected boys suffer from incurable progressive muscle weakness, leading to premature death. Stem cell transplantation may be curative, but is hampered by the need for systemic delivery and immune rejection. To address these barriers to stem cell therapy in DMD, we investigated a fetal-to-fetal transplantation strategy. We investigated intramuscular, intravascular, and intraperitoneal delivery of human fetal mesenchymal stem cells (hfMSCs) into embryonic day (E) 14-16 MF1 mice to determine the most appropriate route for systemic delivery. Intramuscular injections resulted in local engraftment, whereas both intraperitoneal and intravascular delivery led to systemic spread. However, intravascular delivery led to unexpected demise of transplanted mice. Transplantation of hfMSCs into E14-16 mdx mice resulted in widespread long-term engraftment (19 weeks) in multiple organs, with a predilection for muscle compared with nonmuscle tissues (0.71% vs. 0.15%, p < .01), and evidence of myogenic differentiation of hfMSCs in skeletal and myocardial muscle. This is the first report of intrauterine transplantation of ontologically relevant hfMSCs into fully immunocompetent dystrophic fetal mice, with systemic spread across endothelial barriers leading to widespread long-term engraftment in multiple organ compartments. Although the low-level of chimerism achieved is not curative for DMD, this approach may be useful in other severe mesenchymal or enzyme deficiency syndromes, where low-level protein expression may ameliorate disease pathology.

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.

Coupled analysis of bacterial transformants and ligation mixture by duplex PCR enables detection of fatal instability of a nascent recombinant plasmid.

When a DNA cloning experiment fails, it is often difficult to distinguish between an inadequate cloning protocol and instability of the new recombinant plasmid. The identification of plasmid instability is particularly challenging when the instability is fatal and no DNA of the expected construct can be isolated. We have effectively addressed this problem by employment of duplex PCR (insert-insert, vector-insert) to analyse both the ligation mixture and the resultant bacterial transformants. Using this approach we found a fatal maintenance instability of one of the plasmids generated during subcloning of the cDNA for human LDLR in Escherichia coli STBL2. The described duplex PCR screening method allows monitoring of the fate of nascent recombinant plasmid from ligation, through the initial bacterial colony and the subsequent overnight culture.

In utero gene therapy: current challenges and perspectives.

Over the past few years, considerable progress in prenatal diagnosis and surgery combined with improvements in vector design vindicate a reappraisal of the feasibility of in utero gene therapy for serious monogenetic diseases. As adult gene therapy gathers pace, several apparent obstacles to its application as a treatment may be overcome by pre- or early postnatal treatment. This review will examine the concepts and practice of prenatal vector administration. We aim to highlight the advantages of early therapeutic intervention focusing on diseases that could benefit greatly from a prenatal gene therapy approach. We will pay special attention to the strategies and vectors that are most likely to be used for this application and will speculate on their expected developments for the near future.

Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo.

Gene transfer to the trachea and airways by adenoviral vectors is limited by the basolateral localization of viral receptors, resulting in relatively low levels of transduction. Modification of paracellular permeability by sodium caprate, which opens tight junctions, enhances gene transfer from the apical side of cultured human airway epithelial cells. Based on this observation we investigated whether Na-caprate could also increase gene transfer when applied to the luminal surface of the airway epithelia in vivo and compared these results with EGTA, which has previously been shown to enhance adenovirus transduction. Transgene expression in the trachea and upper airways was increased 25-fold by a 10-min pretreatment with 50 mM Na-caprate, corresponding to a 3-fold improvement over EGTA. In the more peripheral airways EGTA had no effect, whereas expression of beta-gal was increased 3-fold by Na-caprate. When the adenovirus was complexed with DEAE dextran, transduction of the airway epithelia after Na-caprate pretreatment was increased 45-fold over virus alone. In conclusion, Na-caprate facilitates gene transfer to airway epithelia, particularly when adenovirus is complexed with DEAE dextran, and may in future be used in a clinical setting to enhance the efficiency of vectors for gene therapy of cystic fibrosis via airway delivery.