Gene Transfer To Lung Research Articles

144. Duration of Reporter Gene Expression from Naled pDNA in the Mouse Lung Following Direct Electroporation and Development or Wire Electrodes for Sheep Lung Electroporation Studies

Current non-viral vectors for lung gene transfer are limited by inefficient uptake, and low levels of expression in cells throughout the lung. The use of electroporation to enhance reporter gene expression from naked plasmid DNA (pDNA) in the mouse lung has been assessed. A direct electroporation model has been established, in which following delivery of pDNA and electroporation of the left lung, luciferase (Lux) reporter gene expression has been enhanced by up to 500 fold compared to the non-electroporated right lung depending on the combination of plasmid, field strength (V/cm) and pulse length employed. Furthermore, following delivery of a GFP expressing plasmid, up to 5% of the left lung cells were positive for GFP after electroporation. Now, the duration of Lux activity following electroporation of the mouse lung has been assessed. Female BALB/c mice were anaesthetised with isoflurane and received 100μg pUbLux (human ubiquitin C promoter, Lux transgene) in 150μl water. The left lungs of mice were exposed by performing a lateral thoracotomy. Electroporation of the left lung (8 pulses of 800V/cm, 2ms with a 1sec interval) was carried out using the BTX ECM®830 Generator with 2-Needle electrodes with 5mm gap with the right lungs remaining non-electroporated. Lux activity was measured at 2, 14 and 28 days post dosing. At day 2 post dosing, mean Lux activity in the left lungs of mice was 40 fold higher than the right lung (Mann-Whitney U: P = 0.016). The Lux activity in the left lungs at day 28 post dosing was no different to the level at day 2 (P=0.512) and still significantly higher than the activity observed in the right lung (P = 0.050). Therefore this study has established that direct electroporation of the mouse lung can result in high levels of Lux activity from naked pDNA and that this level persists for at least 28 days post dosing. In similar studies with the plasmid pCIKLux (CMV promoter, Lux transgene), electroporation resulted in far greater levels of expression at day 2 post dosing, but by day 14, the Lux activity had fallen to background. The safety and efficacy of lung electroporation has therefore been well established in the mouse lung. Further development of lung electroporation is now planned in the sheep lung, as a larger animal model will be more relevant for clinical development of this approach. A series of wire electrodes have been constructed from Teflon insulated stainless steel up to 80cm in length with a diameter of just 0.3mm. In an ex vivo mouse lung model, electroporation with these wires following pCIKLux intranasal delivery (100μg pDNA/150μl) resulted in equivalent 200 fold increases in Lux activity to commercially available needle electrodes. These wire electrodes will be placed within the sheep airways with a bronchoscope allowing for a range of electroporation studies to be carried out. These studies will determine if the electric field can be efficiently and safely applied over a large area to allow electroporation to be used for improving gene transfer throughout the whole lung.

505. Effective Transgene Expression in the Murine Lung Using Compacted DNA Nanoparticles Formulated with Plasmid DNAs of 5, 10, and 20 kbp

One concern of non-viral gene therapies is the potential limitation on the size of the DNA construct used in formulating an effective therapeutic. Some open reading frames, such as the coding sequence of the cystic fibrosis transmembrane regulator (4.4 kbp), are quite large and, when combined with other required regulatory elements, result in a therapeutic construct of significant size (> 8 kbp). Gene transfer optimization studies are usually performed using reporter gene constructs. Typical reporter genes have much smaller open reading frames (luciferase 1.7 kbp, green fluorescent protein 0.7 kbp) than many therapeutic genes. Therefore, we wanted to determine if our strategies for gene therapy of multiple inherited and acquired diseases will be limited in vivo by the required size of the plasmid DNA payload. To address this aim, we constructed three luciferase reporter plasmids of increasing size—~5, 10, and 20 kbp—using lambda DNA stuffer fragments inserted into the 5 kbp plasmid. These plasmids were compacted to DNA nanoparticles with a 10 kDa polyethylene glycol (PEG) substituted 30-mer lysine polymer containing either trifluoroacetate or acetate counterions at the time of DNA mixing. As evaluated by transmission electron microscopy, these formulations generate compacted DNA that appears as either ellipsoids or rods, respectively. These formulations were delivered to the lungs of Balb/C mice via the intranasal route, and the lungs were harvested and luciferase activity evaluated 2 days after gene transfer. All DNA nanoparticles tested—~5 kbp to 20 kbp plasmids formulated using PEG-lysine polymers with either counterion—had luciferase reporter activities of ~104 RLU/mg protein/pmol DNA above background. Within each formulation group, the reporter gene activity was equivalent (no statistically significant differences) among all three compacted plasmid DNAs. These results indicate that the size of the plasmid used, up to at least 20 kbp, should not be a limitation for lung gene transfer using this non-viral gene therapy platform.

Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung

A human immunodeficiency virus (HIV)-based vector pseudotyped with the Ebola Zaire (EboZ) viral envelope glycoprotein (GP) was recently shown to transduce murine airway epithelia cells in vivo. In this study, the vector was further redesigned to improve gene transfer and also to increase safety. We used mutant EboZ envelopes for pseudotyping, which resulted in higher titers and increased transduction of airway cells in vivo compared to vectors pseudotyped with wild-type EboZ GP. As these envelopes lack regions associated with toxicity of the wild-type EboZ GP, they should also be safer to use for pseudotyping of lentiviral vectors. In addition, lentiviral vectors were created based on feline immunodeficiency virus and shown to have similar efficiency of transduction compared to HIV-based vectors. The creation of lentiviral vectors with highly engineered EboZ envelopes improved the performance of the system and should also increase its safety since only minimal regions of the EboZ envelope, which lack the toxic domain, are used.

Large Animal Models: Bridging the Gap

als is a perplexing finding. While studies of murine models have led to tremendous advances in our understanding of CF, they have been poor models of respiratory gene transfer in man, because they do not develop the severe inflammatory response and thickened mucus associated with CF lung disease [14]. Anatomical and physiological features underpin these species differences. Mice lack bronchial submucosal glands, leading to the production of fewer secretions in their lungs compared to humans [15]. In addition, Clsecretion in the murine lung is performed predominantly by an alternative Clchannel, not the CFTR channel, and CFTR mRNA levels are lower throughout the murine pulmonary epithelium than in the lungs of man [16]. Therefore, model systems that are more representative of the barriers to gene transfer in the human airway are needed to develop protocols and vectors for gene therapy of CF. The report in this issue by Emerson et al., from the UK CF Gene Therapy Consortium (www.cfgenetherapy.org.uk), investigates gene transfer in the sheep lung as an alternative large animal model for respiratory gene transfer. The size, anatomy, and physiology of sheep and human lungs are similar, and human and sheep CFTR share similarities of sequence and developmental expression. Naked DNA, or a formulation of DNA and the cationic liposome GL-67/DOPE, was deposited in specific lung segments by bronchoscopy. Levels of the reporter gene, chloramphenicol transacetylase (CAT), were assessed quantitatively at the levels of both protein and mRNA, and the inflammatory response was assessed at increasing DNA dosage levels. The authors compared their findings in the sheep with previous murine studies using similar vectors and with a similar study in the pig lung performed with a different, nonviral vector system [17]. One of the more striking differences between the studies in sheep and mice was that transfections with GL67 along with 1mM or 2 mM DNA, produced 500 or 4,400-fold less CAT protein, respectively, than was achieved in mice. Further, CAT protein levels in mouse lung after GL67 transfection were ten-fold higher than naked DNA transfection, while in sheep the difference was only three fold. By contrast, naked DNA performed equally as well as GL67 in clinical studies [18]. While further data is required for a comparison of sheep and pig lung transfections, both studies highlight how sheep and/or pig models may help bridge the gap between results in murine models of CF and results in the clinic. The use of large animal models potentially heralds a new direction and new opportunities in preclinical studies of CF gene therapy, particularly in the key area of vector developPERSPECTIVE

Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung

Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung

1186. Lentiviral Vectors Pseudotyped with Minimal Filovirus Envelopes Increased Gene Transfer in Murine Lung

1186. Lentiviral Vectors Pseudotyped with Minimal Filovirus Envelopes Increased Gene Transfer in Murine Lung

Folate-targeted gene transfer in vivo.

Nonviral systemic delivery is one of the most attractive approaches for cancer gene therapy. To achieve this goal, various laboratories have developed cationic liposomes. However, when injected intravenously, cationic lipid-DNA complexes accumulate mostly into and transfect lung tissue. Here, we describe a method by which these complexes can be targeted to tumors using folic acid. Adding polyethylene glycol (PEG)-lipids to the complexes dramatically reduced both lung accumulation and gene transfer to lungs and tumors after intravenous administration. The presence of folic acid at the distal end of the PEG-lipid did not modify tumor accumulation of the complexes. However, with folate-targeted complexes, gene transfer activity was restored in tumors while the activity in lungs was reduced by 50- to 100-fold compared with nontargeted lipid-DNA complexes. This approach provides a first in vivo proof of concept to achieve targeted tumor gene delivery.

807. Induction of Apoptosis and Cell Cycle Stopped with Adenovirus Mediated SART1 Gene Transfer in Human Lung and Breast Tumor Cell Line

807. Induction of Apoptosis and Cell Cycle Stopped with Adenovirus Mediated SART1 Gene Transfer in Human Lung and Breast Tumor Cell Line

Enhanced protection against fatal mycobacterial infection in SCID beige mice by reshaping innate immunity with IFN-gamma transgene.

Humans with immune-compromised conditions such as SCID are unable to control infection caused by normally nonpathogenic intracellular pathogens such as Mycobacterium bovis bacillus Calmette-Guérin. We found that SCID beige mice lacking both lymphocytes and NK cells had functionally normal lung macrophages and yet a selectively impaired response of type 1 cytokines IFN-gamma and IL-12, but not TNF-alpha, during M. bovis bacillus Calmette-Guérin infection. These mice succumbed to such infection. A repeated lung gene transfer strategy was designed to reconstitute IFN-gamma in the lung, which allowed investigation of whether adequate activation of innate macrophages could enhance host defense in the complete absence of lymphocytes. IFN-gamma transgene-based treatment was initiated 10 days after the establishment of mycobacterial infection and led to increased levels of both IFN-gamma and IL-12, but not TNF-alpha, in the lung. Lung macrophages were activated to express increased MHC molecules, type 1 cytokines and NO, and increased phagocytic and mycobactericidal activities. Activation of innate immunity markedly inhibited otherwise uncontrollable growth of mycobacteria and prolonged the survival of infected SCID hosts. Thus, our study proposes a cytokine transgene-based therapeutic modality to enhance host defense in immune-compromised hosts against intracellular bacterial infection, and suggests a central effector activity played by IFN-gamma-activated macrophages in antimycobacterial cell-mediated immunity.

Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components.

We introduce a lung inflation-fixation protocol to examine the distribution and gene transfer efficiency of fluorescently tagged lipoplexes using fluorescence confocal microscopy within thick lung tissue sections. Using this technique, we tested the hypothesis that factors related to lipoplex distribution were the predominant reason that intravenous (i.v.) administration of lipoplex was superior to intratracheal (i.t.) administration for gene transfer in the murine lung. Lipoplex distribution was analyzed using digitized images of overlapping fields, reconstructed to view an entire lung lobe. Intravenously administered lipoplexes were confined to the capillary network and homogenously distributed throughout the lung lobe. In contrast, i.t. administration resulted in regional distribution of lipoplex, concentrated around bronchioles and distal airways. Not all the bronchioles were stained with lipoplex, suggesting that the airway-administered solution became channeled through certain bronchiolar pathways. A fluorescent oligonucleotide was used as a marker for cytoplasmic release of nucleic acids. Quantification of the resulting fluorescent nuclei was used to define the relationship between cytoplasmic release of nucleic acids and gene expression. Endothelial cells were stained after i.v. administration, and epithelial cells were stained after i.t. administration. The delivery of nucleic acids was also more homogeneous with i.v. administration of lipoplex than with i.t. administration. After i.t. administration, it was notable that high concentrations of fluorescent nuclei correlated with low GFP expression. This suggested that toxicity was associated with high local concentrations of cationic lipoplexes. The ratio of GFP-expressing cells to fluorescent nuclei indicated that capillary endothelial cells were more efficient in gene expression per delivery event than were pulmonary epithelial cells. Thus, the greater gene expression efficiency of i.v. administered lipoplexes was due not only to the initial distribution but also to the greater efficiency of the vascular endothelial cells to appropriately traffic and express the foreign gene.

Use of perfluorochemical liquid allows earlier detection of gene expression and use of less vector in normal lung and enhances gene expression in acutely injured lung.

One of the obstacles to successful lung gene transfer is effective delivery of vector to lung, particularly injured or diseased lung. We have previously demonstrated that intratracheal instillation of perfluorochemical (PFC) liquids along with instillation of recombinant adenovirus and adeno-associated virus vectors, or with cationic liposome vectors, increased total lung gene expression and enhanced distribution of gene expression throughout the lung. To further explore the potential benefits of PFC liquid use, we evaluated the effect of PFC liquid instillation on several other aspects of adenovirus-mediated gene expression in lung. Use of PFC liquid resulted in earlier detection of gene expression and allowed the use of less vector to achieve expression comparable to that observed with the use of higher amounts of vector alone. Using PFC liquid also enhanced gene expression in a rodent model of acute lung injury. PFC liquid did cause a transient inflammation when instilled into normal lungs but did not cause any additional inflammation when instilled alone or with adenovirus vector into acutely injured lungs. Thus, PFC liquid may be a useful adjunct for clinical lung gene transfer, particularly for injured or diseased lungs.

FACTORS INFLUENCING THE EFFICIENCY OF LIPOPLEXES MEDIATED GENE TRANSFER IN LUNG AFTER INTRAVENOUS ADMINISTRATION*

The objectives of this study were to test the influence of different parameters on the in vivo cationic lipid mediated gene transfer in lung after intravenous administration. Luciferase activity was evaluated in lung tissue 24 hours after intravenous administration of different types of lipoplexes. These included lipoplexes prepared using cationic phosphonolipids or DOTAP and various amounts of plasmid DNA. Using two different plasmids we tested the influence of plasmid size on transfection efficiency in vivo. In a last series of experiments, lipoplexes were prepared using different excipients (water, NaCl or 5% glucose solution) and three injection volumes were tested. We demonstrate that chemical structure modifications such as cation substitution and increment of the aliphatic chain length significantly improve transfection efficiency. High luciferase levels are obtained by increasing lipid to DNA charge ratio and plasmid DNA dose and decreasing plasmid size. Lipoplexes prepared in physiological NaCl solution and injected using a volume of 800μl are significantly the most effective.Cationic lipid mediated gene transfer in lung tissue after intravenous administration is influenced by factors including cationic lipid chemical structure, lipid to DNA ratio and plasmid dose. Nevertheless, plasmid size, injection volume and the excipient, used for the lipoplexes preparation, are also important factors and must be considered for an optimization of in vivo gene delivery using intravenous administration.

Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody.

To achieve efficient systemic gene delivery to the lung with minimal toxicity, a vector was developed by chemically conjugating a cationic polymer, polyethylenimine (PEI), with anti-platelet endothelial cell adhesion molecule (PECAM) antibody (Ab). Transfection of mouse lung endothelial cells with a plasmid expression vector with cDNA to luciferase (pCMVL) complexed with anti-PECAM Ab-PEI conjugate was more efficient than that with PEI-pCMVL complexes. Furthermore, the anti-PECAM Ab-PEI conjugate mediated efficient transfection at lower charge plus-to-minus ratios. Conjugation of PEI with a control IgG (hamster IgG) did not enhance transfection of mouse lung endothelial cells, suggesting that the cellular uptake of anti-PECAM Ab-PEI-DNA complexes and subsequent gene expression were governed by a receptor-mediated process rather than by a nonspecific charge interaction. Conjugation of PEI with anti-PECAM Ab also led to significant improvement in lung gene transfer to intact mice after intravenous administration. The increase in lung transfection was associated with a decrease compared with PEI-pCMVL with respect to circulating proinflammatory cytokine (tumor necrosis factor-alpha) levels. These results indicate that targeted gene delivery to the lung endothelium is an effective strategy to enhance gene delivery to the pulmonary circulation while simultaneously reducing toxicity.

In Vivo Gene Transfer of Prepro-Calcitonin Gene–Related Peptide to the Lung Attenuates Chronic Hypoxia-Induced Pulmonary Hypertension in the Mouse

Calcitonin gene-related peptide (CGRP) is believed to play an important role in maintaining low pulmonary vascular resistance (PVR) and in modulating pulmonary vascular responses to chronic hypoxia; however, the effects of adenovirally mediated gene transfer of CGRP on the response to hypoxia are unknown. In the present study, an adenoviral vector encoding prepro-CGRP (AdRSVCGRP) was used to examine the effects of in vivo gene transfer of CGRP on increases in PVR, right ventricular mass (RVM), and pulmonary vascular remodeling that occur in chronic hypoxia in the mouse. Intratracheal administration of AdRSVCGRP, followed by 16 days of chronic hypoxia (FIO(2) 0.10), increased lung CGRP and cAMP levels. The increase in pulmonary arterial pressure (PAP), PVR, RVM, and pulmonary vascular remodeling in response to chronic hypoxia was attenuated in animals overexpressing prepro-CGRP, whereas systemic pressure was not altered while in chronically hypoxic mice, angiotensin II and endothelin-1-induced increases in PAP were reduced, whereas decreases in PAP in response to CGRP and adrenomedullin were not changed and decreases in PAP in response to a cAMP phosphodiesterase inhibitor were enhanced by AdRSVCGRP. In vivo CGRP lung gene transfer attenuates the increase in PVR and RVM, pulmonary vascular remodeling, and pressor responses in chronically hypoxic mice, suggesting that CGRP gene transfer alone and with a cAMP phosphodiesterase inhibitor may be useful for the treatment of pulmonary hypertensive disorders.

Adenoviral-Mediated Gene Transfer of Interleukin-6 in Rat Lung Enhances Antiviral Immunoglobulin A and G Responses in Distinct Tissue Compartments

The effect of IL-6 transgene expression following lung gene transfer on anti-adenovirus humoral responses of immunoglobulin (Ig) G and A both in the lung and peripheral blood was investigated. Lung infection by a control adenovirus caused an increased level of circulating anti-adenoviral IgG antibodies. However, the magnitude of this response was many times higher in the peripheral blood of rats receiving an adenovirus engineered to express IL-6 transgene. In comparison, much lower levels of anti-adenoviral IgG were detected in the bronchoalveolar fluids of rats receiving either virus. In contrast, there was no detectable level of anti-adenoviral IgA in the peripheral blood in any cases, yet significantly detectable levels of anti-adenoviral IgA were measured in the lung. The levels of this IgA were much higher in the lung of rats expressing IL-6 than in the lung of control animals (15 times higher by day 14). Our findings thus provide evidence that IL-6 plays a significant role in enhancing specific airways mucosal IgA and systemic IgG responses during local lung viral infection, and provide the rationale for using IL-6 locally at mucosa sites as an immune adjuvant for antiviral vaccination program.

Keratinocyte growth factor induced epithelial proliferation facilitates retroviral-mediated gene transfer to distal lung epithelia in vivo.

Cell proliferation, vector titer and accessibility of target cells represent hurdles for efficient gene transfer to lung epithelia in vivo using recombinant murine leukemia (MuLV)-based retroviruses. We tested the hypothesis that the pulmonary epithelium is susceptible to retroviral-mediated gene transfer when stimulated to proliferate by a mitogen, keratinocyte growth factor (KGF). Rats received keratinocyte growth factor (KGF, 2.5 micrograms/g x 4 doses, two consecutive days) intratracheally followed by high titer amphotropic retrovirus expressing beta-galactosidase. Gene transfer was assessed five days later. KGF stimulated transient proliferation in the bronchiolar and alveolar epithelia (30-40% PCNA positive cells at peak) which decreased to background levels seven days after administration. Gene transfer to epithelia (X-Gal positive cells) occurred more frequently in KGF treated rats, but proliferation exceeded the level of gene transfer. X-gal positive cells were noted in the alveolar epithelium and occasionally in the bronchiolar epithelium. In order to understand the discrepancy between the number of proliferating and transduced cells, primary rat tracheal epithelium cultured at the air-liquid interface was infected from either the apical or basolateral side. Gene transfer was achieved only through basolateral application of vector, suggesting that epithelial polarity represents a barrier to MuLV-based lung gene transfer in vivo. KGF transiently stimulates epithelial proliferation in vivo, facilitating MuLV-based gene transfer. Retroviral vectors may encounter multiple barriers which have evolved to defend the lung from infections.

Surfactant inhibits cationic liposome-mediated gene transfer.

We have demonstrated that tracheal insufflation of recombinant plasmid DNA results in transfection of rat lungs to the same extent as insufflation of plasmid-cationic liposome complex. To understand this observation better, we investigated the in vitro gene transfer of plasmid DNA in the presence and absence of cationic liposome and the effect of surfactant on gene transfer. The chloramphenicol acetyltransferase (CAT) expression plasmids pBL-CAT and pSV-CAT were studied in three cell types: rat fetal lung fibroblast (RFL-6), calf pulmonary artery endothelial cell (CPAE), and rat type II alveolar epithelial cell (type II AE). Three cationic liposomes were tested: DDAB (dimethyl-dioctadecyl ammonium bromide)-liposome, DOTAP (dioleoyltrimethyl ammonium propane)-liposome, and lipofectin. The results revealed that (i) plasmid DNA alone caused a dose-dependent, low-level transfection, most efficiently in RFL-6 followed by CPAE and type II AE, (ii) DDAB-liposome markedly enhanced gene transfer, most efficiently in RFL-6 followed by CPAE and type II AE, (iii) Survanta, a naturally derived surfactant preparation, and Exosurf, a synthetic surfactant, while having no effect on in vitro gene transfer by plasmid DNA alone, markedly inhibited cationic liposome-mediated gene transfer, (iv) dipalmitoyl phosphatidylcholine was responsible for the inhibitory effect of Exosurf, and (v) inhibition of cationic liposome-mediated gene transfer by Exosurf was not due to inhibition of plasmid DNA-cationic liposome complex uptake or interference with the promoter and enhancer. The observed inhibition of cationic liposome-mediated gene transfer by surfactant may in part explain our previous observation that tracheal insufflation of plasmid DNA and plasmid-cationic liposome complex results in equal lung gene transfer.

Thérapie génique des hémophilies — Potentialités thérapeutiques et limitations technologiques

Defects in the genes encoding the human coagulation factor VIII (hFVIII) and IX (hFIX) result in life-threatening haemorrhages and severe arthropathies. While haemophiliacs are currently treated by blood-derived factors or recombinant hFVIII and hFIX, a number of recent technical advances make the prospect of using gene therapy to treat such genetic diseases a realistic goal. Several gene therapy strategies have therefore been developed and evaluated in recent years. Most of the initial protocols were ex vivo gene transfer approaches in which the target cells (fibroblasts, keratinocytes, myoblasts, ...) were expanded and genetically-engineered in the laboratory and then implanted in the host. However, the complexity of most ex vivo gene therapy strategies, together with the disappointing results obtained in various animal models stimulated the development of more direct in vivo gene therapy protocols. In aiming to establish such an in vivo gene transfer protocol for haemophilia B, we constructed and tested in vitro and in vivo various recombinant adenovirus vectors expressing human FIX. Intravenous administration of this vector into various strains of immunocompetent and immunodeficient mice led to an efficient hFIX gene transfer in liver and lung. As a consequence, the hFIX protein was correctly produced and secreted at high levels in the blood of the treated animals. However, expression was transient in all immunocompetent mice, except surprisingly in C57B1/6 animals. A systematic molecular and immunological analysis allowed us to identify the parameters that prevent the long-term in vivo expression of the human molecule and to improve the current adenovirus vectors.

Capital Appreciation of World Equity Markets 1971–1994

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air–liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and ‘benchmarked’ against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90–100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.

Gene therapy for cystic fibrosis: challenges and future directions.

for gene therapy when the gene responsible for the disease was isolated in 1989 (3-5). The wide distribution of possible cellular targets for gene transfer in the CF lung and the absence of a known lung epithelial stem cell suggested that an ex vivo approach to gene therapy would not be feasible (6, 7). Research immediately focused on in vivo approaches for gene transfer that could conveniently be delivered into the airway via aerosols. The ensuing research effort has literally dominated the field ofhuman gene therapy of genetic diseases while establishing the basic principles of in vivo gene delivery. This review summarizes critical scientific issues that have emerged in the development of in vivo gene therapy for CF using adenoviral vectors as a model.