Lentiviral transduction of the murine lung provides efficient pseudotype and developmental stage-dependent cell-specific transgene expression

Abstract

Gene transfer for cystic fibrosis (CF) airway disease has been hampered by the lung's innate refractivity to pathogen infection. We hypothesized that early intervention with an integrating gene transfer vector capable of transducing the lung via the lumen may be a successful therapeutic approach. An HIV-based lentiviral vector pseudotyped with the baculovirus gp64 envelope was applied to the fetal, neonatal or adult airways. Fetal intra-amniotic administration resulted in transduction of approximately 14% of airway epithelial cells, including both ciliated and non-ciliated epithelia of the upper, mid and lower airways; there was negligible alveolar or nasal transduction. Following neonatal intra-nasal administration we observed significant transduction of the airway epithelium (approximately 11%), although mainly in the distal lung, and substantial alveolar transduction. This expression was still detectable at 1 year after application. In the adult, the majority of transduction was restricted to the alveoli. In contrast, vesicular stomatitis virus glycoprotein pseudotyped virus transduced only alveoli after adult and neonatal application and no transduction was observed after fetal administration. Repeat administration did not increase transduction levels of the conducting airway epithelia. These data demonstrate that application at early developmental stages in conjunction with an appropriately pseudotyped virus provides efficient, high-level transgene expression in the murine lung. This may provide a modality for treatment for lung disease in CF.

Introduction

Progress towards an effective gene therapy strategy for cystic fibrosis (CF) is complicated by the lung's natural defences to invading pathogens. In humans, pathogenesis of the CF lung proceeds from early childhood, therefore, intervention should ideally precede these events.1, 2 A number of groups have attempted in utero gene therapy approaches to CF in the mouse3, 4, 5 and higher mammals6, 7, 8, 9 although the resultant expression was either at a low level or transient. Larson et al.10 reported controversial evidence of alleviation of meconium ileus in cftr-null mice following intra-amniotic injection of adenovirus expressing the CF transmembrane regulator (CFTR) cDNA.10 However, these results have not been repeatable by other groups.11 The fetal/neonatal period is characterized by reduced immune reactivity and stem-cell niches may also be more accessible to transduction. For example, Waddington et al.12 recently showed that in utero gene delivery of a lentiviral vector carrying human factor IX resulted in lifelong correction of the bleeding diathesis, permanent expression of and immune tolerance to the xenoprotein.

Although a variety of vector systems have been administered to the airways in adult patients, there has been limited clinical success due to inefficient transduction and/or local inflammatory responses.2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 Lentiviral vectors have the potential to integrate transgenic material into the host cell genome providing long-term expression.24, 25, 26, 27 However, airway epithelial gene transfer efficacy with this vector system has previously been restricted by a paucity of pseudotypes, which can be produced at high titres and which can infect lung epithelia from the apical surface. In recent years, apical transduction by lentivirus has been achieved both in vitro and in vivo using a number of viral envelopes from diverse origins, including filovirus,28, 29 baculovirus,30 influenza31 and parainfluenza32 viruses. For example, the Ebola virus envelope glycoprotein has been used successfully to achieve efficient transduction of the murine lung epithelium and human explants33, 34 although generation of consistently high viral titres has been problematic. Sinn et al.30 also showed that transgene expression after lung application of feline immunodeficiency virus pseudotyped with the baculoviral gp64 envelope applied in a viscoelastic gel formulation was significantly higher than observed with a vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped construct. However, Kremer et al.35 did not see increased apical uptake of gp64/HIV when compared to VSV-G.

In this study we have investigated the efficacy of airway epithelial transduction using application of high-titre pseudotyped lentivirus during the adult, neonatal and fetal periods of mouse development. HIV vectors pseudotyped with the gp64 envelope glycoprotein were compared with VSV-G pseudotyped vector. VSV-G pseudotyped lentivirus is known to only transduce polarized cells via the basolateral membrane, whereas the gp64 envelope has been shown to produce significant expression in the nasal epithelia of adult mice up to 1 year after application.

Results

In this study, we have investigated the efficacy of airway epithelial transduction using application of high-titre pseudotyped lentivirus during the adult, neonatal and fetal periods of mouse development. Second-generation HIV-based lentiviral vectors expressing enhanced green fluorescent protein (GFP) from the SFFV (spleen focus-forming virus) LTR promoter were pseudotyped with the baculoviral gp64 envelope glycoprotein (gp64/HIV) or VSV-G (VSV-G/HIV) and concentrated by ultracentrifugation. Fresh preparations of lentivirus were administered by fetal intra-amniotic injection (50 μl) at 16 days post coitum (pcm), (normal gestation 20–21 days), or by instillation into the nostrils of either 1-day-old neonatal (20 μl) or adult mice (50 μl). Variations from preparation to preparation and the fact that intra-amniotic administration to the foetus is not lung specific inevitably leads to an unavoidable disparity in dose per lung surface area. Titres of multiple viral preparations generated during this investigation varied by 1- to 5-fold at most; nevertheless gp64/HIV production was reliable and reproducible. The initial viral preparations for both pseudotypes were quantified by biological titre on 293T cells by GFP fluorescence-activated cell sorting (FACS) (Table 1). Subsequent to this study we determined physical titres using both p24 and reverse transcriptase enzyme-linked immunosorbent assay (ELISA) for both pseudotypes. A comparison of all three titration methods showed that the consistent 5-fold higher biological titres for VSV-G/HIV over gp64/HIV were not apparent in physical titres (Table 2).

Table 1 Detail of viral titres for all experimental applications
Table 2 Comparison of biological and physical titres with VSV-G and gp64 pseudotypes

Macroscopic and quantitative analyses of lung lentiviral transduction

Mice were killed 2 weeks after vector administration and the lung were analysed by macroscopic observation, immunohistochemical analysis and GFP ELISA. Macroscopic observation for both pseudotypes revealed widespread and punctate GFP expression in the lung after both the adult and neonatal vector delivery but significantly lower levels of expression after fetal application, probably due to the above mentioned fluid dilution factor (Figure 1a). Consistent with the macroscopic observations, GFP ELISA on lung homogenates also showed significantly less expression after fetal versus neonatal and adult transduction (P<0.0005 in each case) (Figure 1b). Although there was no apparent difference between the two pseudotypes after adult or neonatal application, fetal administration of gp64/HIV resulted in significantly higher GFP expression than VSV-G/HIV application (P<0.0005).

Figure 1
figure1

Comparison of overt green fluorescent protein (GFP) expression in murine lung transduced at developmentally defined time points. Mice were administered with either gp64 or vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus by fetal intra-amniotic injection (50 μl), neonatal intra-nasal administration (2 × 10 μl) or adult intra-nasal administration (50 μl). Mice were killed at day 14 post-administration and macroscopic images recorded (magnification × 5) (a). Tissue lysates were analysed for GFP expression by enzyme-linked immunosorbent assay (ELISA) (b). Individual mice are represented by () and (-) represents the mean value. gp64/HIV administration provides significantly greater expression in neonates versus foetuses (P<0.0005). Expression after fetal administration of gp64/HIV is significantly greater than fetal VSV-G/HIV (P<0.0005). Scale bars represent 500 μm.

Microscopic analysis of lung lentiviral transduction

Immunohistochemical analysis of lung sections revealed a more complex expression profile (Figure 2a). VSV-G/HIV consistently transduced only alveoli and resident macrophages after both adult and neonatal nasal administrations. There was only rare transduction, of single cells, in the upper, middle or distal conducting airways in both developmental models. Intra-amniotic administration of the VSV-G/HIV resulted in negligible transduction of any pulmonary tissues. Adult intra-nasal administration of gp64/HIV resulted in sporadic transduction of the airway epithelium at low efficiency and less abundant alveolar transduction than with the VSV-G pseudotype. However, neonatal application of gp64/HIV provided more abundant transduction of alveoli and conducting airway epithelia. Interestingly, the majority of expression in the conducting airway epithelium was seen within the small, distal airways, close to the alveoli. Most notably, after intra-amniotic administration of gp64/HIV widespread and abundant expression within the airway epithelium was observed. A more detailed analysis after fetal gp64/HIV administration demonstrated widespread transduction from the trachea and upper airways to the bronchioles and mid airways to the distal airways (Figure 2b).

Figure 2
figure2

Immunohistological analyses of transduction after fetal, neonatal and adult vector administration. Mice were dosed as described in Figure 1 and killed on day 14 post-administration. Sections of lung were analysed for green fluorescent protein (GFP) expression by fluorescence immunohistochemistry. Co-immunolocalization was carried out with a mouse monoclonal β-tubulin IV antibody as a marker of ciliated cells. After fetal gp64/HIV-GFP application, extensive GFP reporter gene expression was observed in the conducting airway epithelium (a, magnification × 20 apart from fetal gp64 magnification × 40) including the upper, mid and the distal airways (b, magnification × 40). GFP-expressing cells were counted on at least 10 stained sections per animal (neonatal vesicular stomatitis virus glycoprotein (VSV-G) n=5, neonatal gp64 n=6, fetal gp64 n=8) and expressed as a percentage of the total transduced cells. Cell-type status was assigned by histology. All values are represented as means±standard error of mean (c). Experimental groups where >90% of cells were of one cell type or where less than 10 cells were counted per section are not represented as pie charts. Representative micrographs (magnification × 100) show that gp64/HIV-GFP transduced airway epithelia are equally distributed between ciliated and non-ciliated epithelia (di, ii) and evidence GFP-expressing clusters, which may represent evidence of clonal expansion (diii). Scale bars represent 100 μm.

Quantification of GFP-positive cells showed that fetal application of gp64/HIV resulted in GFP expression in 14±5% of airway epithelial cells and this was consistent throughout both proximal and distal pulmonary airways. Neonatal administration of the same vector achieved total transduction of 11±6% of all airway epithelia analysed although the majority of transduction was found in the distal airways. In contrast, adult intra-nasal administration of gp64/HIV or application of VSV-G/HIV resulted in little GFP expression (<1%) in airway epithelia but mainly transduced alveolar cells. We quantified the proportion of transduced cells in each tissue compartment for the three most promising pseudotype/developmental model combinations (Figure 2c). This figure clearly demonstrates that both the pseudotype and age at administration is crucial in targeting transduction to the airway epithelial cells.

Transduction of airway epithelia following fetal administration of gp64/HIV was assessed qualitatively using the ciliated cell-specific marker β-tubulin IV and showed no significant propensity for transduction of ciliated or non-ciliated airway epithelium (Figures 2di and dii). Apically presented sialic acid, a potential receptor for gp64, was analysed on transduced cells using fluorescently labelled lectins specific to either α2,3 (Sambucus nigra -SNA-I) or α2,6 (Maackia amurensis-MAA). This revealed no propensity for transduction in cells presenting either sialic acid conformation (data not shown).

Following fetal or neonatal transduction we observed that gp64/HIV resulted in GFP expression in contiguous groups of cells (Figure 2diii). This was consistently observed in many lung samples across several experiments and virus batches, suggesting expression in the progeny of originally transduced precursor cells. Similar observations were not evident after adult gp64/HIV administration or with VSV-G/HIV administration to any developmental model.

Repeat administration of lentivirus to the developing mouse lung

In order to maximize transduction we hypothesized that multiple administrations during the fetal and early neonatal period may result in transduction of an even greater proportion of the airway epithelium. We compared a single fetal administration at 16 days pcm (n=3) with the same administration followed by two neonatal administrations on days 0 and 1 after birth (n=2) or the two neonatal administrations alone (n=3). Figure 3 shows that the fetal/neonatal repeat administration (A) resulted in a similar level of airway epithelial transduction compared to a single fetal application (B) but a large increase in the number of transduced macrophages. Furthermore, the neonatal repeat administrations alone resulted in a similar level of macrophage transduction, but a reduced level of airway epithelial transduction (C) consistent with the comparative levels of transduction was seen with single neonatal administrations. This could be the result of macrophages infiltrating after the initial dose of gp64 pseudotyped lentivirus and being transduced by the second and third doses, but the animal groups were too small, therefore further investigations are required. In order to confirm the morphological identification of macrophages we probed sections from each experimental protocol with the F4/80 mouse macrophage marker (D).

Figure 3
figure3

Repeat administration of gp64/HIV. gp64/HIV was repeat dosed by a single intra-amniotic 50 μl injection at 16 days post coitum (pcm) followed by two subsequent intra-nasal administrations of 20 μl at 0 and 1 days after birth (a, magnification × 20) as compared to a single 50 μl intra-amniotic injection (b, magnification × 20). Alternatively, two neonatal administrations of 20 μl were performed on days 0 and 1 after birth (c, magnification × 20). Animals were killed at day 14 post-administration and green fluorescent protein (GFP) expression evaluated. Multiple administrations result in increased transduction of macrophages (d, magnification × 40). Airway epithelia are indicated by red arrows, transduced macrophages with white arrows and untransduced immunostained macrophages with hatched white arrows. Scale bars represent 100 μm.

Long-term expression in the mouse lung after a single neonatal application of gp64 pseudotyped lentivirus

In order to assess long-term transgene expression, a single intra-amniotic dose of gp64/HIV-luciferase (3 × 107 IU per mouse) was administered to neonatal mice at day 1 (n=5). Mice were subjected to bioimaging over the course of 1 year and beyond and luciferase bioluminescence compared to controls (n=2). Luciferase expression was substantially above background and persisted throughout this study (Figure 4).

Figure 4
figure4

Long-term transgene expression in the lung after neonatal administration. A single-dose intra-amniotic administration of gp64/HIV-luciferase (3 × 107 IU) was applied to day 1 neonatal mice (n=5). These animals, along with uninjected controls (n=2), were imaged after intra-nasal administration of 50 μl of 15 mg ml−1 luciferin. Luciferase expression in the lung is shown after removal of background (control) values and is detectable for the length of the study (390 days) (a). Graphic representation of luciferase expression in the lung and noses of the above mice. Images were taken 384 days of age (b). Scale bars represent 100 μm.

Discussion

Gene therapy providing long-term and persistent expression of the CFTR cDNA could result in persistent phenotypic correction of CF airway disease. This would require efficient transduction of affected tissues and prolonged expression or safe and efficacious repeat dosing. The use of vectors based on adenovirus serotype 5, which initially showed promise, has dwindled due to the significant immunogenicity against viral capsid proteins seen in clinical trials.14, 18 Recent interest has converged on pseudotyping; be it of adeno-associated virus (AAV)-2 genomes with novel AAV serotypes21, 36 or of integrating lentiviral vectors with viral envelopes from lung pathogens.28, 29, 31, 32, 33, 37 The Ebola virus envelope glycoprotein has been successfully used to pseudotype lentivirus and has achieved efficient transduction of the murine lung epithelium and human explants33, 34 although generation of consistently high viral titres has been problematic. Recently the baculovirus gp64 envelope has been shown to produce significant expression in the nasal epithelia of adult mice up to 1 year after application.30 Sinn et al.30 showed that expression after gp64-lentivirus application was significantly higher than observed with a VSV-G pseudotyped construct. However, the same phenomenon was not observed by Kremer et al.35 when carrying out similar experiments. Almost exclusively among these studies, vector has been applied to adult mammalian models.

In this study, we have combined the benefits of apical transduction and genomic integration seen with a gp64 pseudotyped lentivirus with an in utero/neonatal administration strategy. We hypothesized that this would avoid immune elimination of the transgenic protein12 and increase lung stem-cell transduction to result in long-term gene expression. Transduction of the murine lung was assessed at three developmental stages by the most amenable administration routes; adult (6 weeks), neonatal (1 day after birth) and fetal (16 days pcm). In utero, the lung is primarily a secretory organ although amniotic fluid is drawn into the lung by simulated breathing movements prior to birth.38 These breathing movements are of sufficient intensity and frequency at 16 days pcm to allow transduction of the fetal mouse airway after intra-amniotic injection.3 Immediately postpartum, the lung epithelium switches to fluid hyperabsorption in order to clear the lung of fluid and facilitate gaseous exchange. After this stage the only viable routes of administration are nasal or tracheal instillation or inhalation of aerosols.22, 36, 39

Each developmentally timed instillation was carried out using either gp64 pseudotyped lentivirus, or VSV-G pseudotyped lentivirus, which is only able to transduce polarized cells via the basolateral membrane.31, 40 Titres of multiple viral preparations were comparable and not subject to substantial intra-prep variation with either pseudotype. Mice were dosed with the maximum dose permissible in each developmental model. It was not possible to accurately quantify the viral dose per lung area or weight as intra-amniotic administration is not lung specific, so some viral dose will be reduced by non-specific transduction and fetal swallowing. The skin and amniotic sac are the major contact organs during intra-amniotic administration although we have previously shown that the skin is keratinized and refractory to transduction at day 16 pcm contrary to 14 days pcm when the periderm is easily transduced.3, 41 We also observed a higher degree of intra-experimental variation in fetal transduction likely due to variation in the uptake of vector-containing amniotic fluid into the developing lung and there is an inevitable dilution effect in the amniotic fluid (the amniotic fluid volume at 16 days pcm is approximately 300 μl). In a therapeutic context these considerations are of minimal relevance as dosing would most likely be performed by ultrasound-guided intra-tracheal administration or intra-luminal injection.7, 8

Macroscopic analysis of GFP expression in post-transduction lung, independent of pseudotype, suggests that adult and neonatal administrations are of equivalent efficiency and both show better overall transduction than in utero delivery owing to the fluid dilution factor as discussed above. The lack of significant transduction in the nasal epithelium may be due to minimal contact time, as the neonate rapidly inhales the virus and the majority of virus passes to the distal airways. Profiling of sialic acid presentation, a potential receptor for gp64 binding, does not alter markedly between the neonate and adult nose or lung but there may be other gp64 or VSV-G receptors, which are as yet uncharacterized. There was an equal distribution of transduced epithelial cells throughout the airways after fetal administration, whereas neonatal administration leads to transduction prevalent in the distal airways. We conclude that this is due to the difference in vector/host contact time rather than to differences in receptor distribution.42 Immunohistochemistry revealed that the majority of transduction after fetal administration was found in cell clusters, whereas neonatal and adult administration resulted in expression from homogenously scattered, non-contiguous cells. This was consistently observed in many lung samples across several experiments and virus batches. Such observations have also been made on the livers of mice after in utero intra-vascular injection of VSV-G pseudotyped HIV and equine infections anemia virus (EIAV) vectors.12, 43 We speculate that this clustering of transduced cells may be evidence of infection of progenitor cells and the result of clonal expansion.

We investigated methods of maximizing transduction efficiency by multiple dosing. We hypothesized that either a single fetal dose followed by a double dose at neonatal days 0 and 1, or a double neonatal doses, would be additive in effect. Surprisingly, repeat dosing in both instances appeared to have no additive effect on the transduction efficiency of airway epithelia but caused a substantial increase in macrophage transduction in both cases when compared to control single doses. This result could be explained by the initial dose of gp64 pseudotyped lentivirus causing an infiltration of macrophages to the lung and the vector having a tropism toward this cell type so the second-round dose results in transduction of these cells. We believe that these transduced are not phagocytosing cells as GFP fluorescence is intense. However, as we did not see a higher level of untransduced macrophages present in the lung after a single dose this phenomenon requires further investigation.

Finally, we carried out a long-term experiment of transgene expression in the lung after a single neonatal application of gp64/HIV expressing the luciferase transgene and compared the luciferase bioluminescent output to that of non-transduced controls over a period of over 1 year. Luciferase expression was significantly above background throughout the analysis period although it decreased up to 190 days after administration before increasing again up to the conclusion of the present data set at 390 days. This persistent length of expression is significant in a therapeutic context and warrants further investigation. It is notable that expression persisted through adulthood in these immune-competent mice. Whether this exemplifies immune tolerance or some form of immune ignorance was not examined in this study. Adult animals were not challenged with protein or repeated vector administration to look for cellular and humoral responses. Parallels may be drawn with a study by Zepeda and colleagues who administered adenovirus to deliver lacZ to neonatal and adult cotton rats.44 Unlike adult administration, neonatal delivery resulted in long-term expression although the apparent immune tolerance was broken by a second administration of adenovirus in adulthood (as evidenced by a full T-cell response and rapid extinction of transgene expression).

We conclude that gp64 pseudotyped lentivirus efficiently transduces airway epithelial cells after both fetal and neonatal administration, whereas adult administration resulted in low level transduction in this tissue but efficient transduction of alveoli. Intra-amniotic administration of gp64 pseudotyped lentivirus appears to be the most efficient mode of airway epithelial transduction in the murine model, resulting in expression from approximately 14% of airway epithelia. Interestingly, there is evidence that only approximately 5% of endogenous CFTR expression is required to correct the chloride ion transport defect.45, 46 The levels of airway epithelial transduction obtained after fetal or neonatal application might be sufficient to provide a significant phenotypic effect. However, the mouse pulmonary development at 16 days pcm is equivalent to human gestation at 16–17 weeks. Intra-tracheal access at this developmental stage has not yet been shown to be feasible in the human foetus and, therefore, interpretations based on the mouse model must be made cautiously. Previous studies have shown transduction of the fetal lung with adenoviral and lentiviral vectors3, 7, 47, 48 but to our knowledge this is the first example of fetal and/or neonatal transduction of the murine airways using a lentivirus. Furthermore, we have presented preliminary evidence of possible stem-cell transduction with the presence of clusters of transduced cells in lung after fetal and neonatal vector administration and transgene expression beyond 1 year. These data warrant further in-depth investigation.

Early intervention gene therapy for CF lung disease is ethically complex but so far no viable therapeutic strategy exists, that could lead to a potentially permanent correction. These data provide proof of concept that gp64 pseudotyped lentivirus could provide prolonged expression throughout adolescent lung development. Provided that the same pseudotype selectivity applies also to human fetal airway epithelia and that safe techniques are developed to administer the vector to the airways in utero, it may become possible in the future to offer early intervention gene therapy in conjunction with prenatal diagnosis as an alternative preventative therapy to individuals with a family history of CF.

Materials and methods

Vector production and validation

For the production of pseudotyped lentivector expressing GFP under the control of the SFFV LTR promoter, we used second-generation cassettes based on those previously described by Demaison et al.49 The gp64 pseudotyped cytomegalovirus (CMV) luciferase vector used for long-term analysis was produced as previously described by Seppen et al.50 The plasmid expressing the VSV-G pVSV-G has been previously described.51 The baculovirus envelope protein plasmid pHCMVwhvGP64 was kindly provided by Dr David Parsons and Dr Don Anson, Department of Pulmonary Medicine, Adelaide Women's and Children's Hospital, Australia and is based on that first described by Sinn et al.29 Lentivectors were prepared as follows: producer 293T cells were seeded at 2 × 107 cells per T-150 flask. After 18 h plasmid DNA was mixed in the following amounts per T-150 flask; vector construct (pHR.SINcpptSEW) 40 μg, pMDG.2/pHCMVwhvGP64 10 μg, pCMVΔ8.74 30 μg to a final volume of 5 ml in OptiMEM (Invitrogen, Paisley, UK). Polyethylenimine (PEI, 25 kDa) (Sigma, Poole, UK) was added to 5 ml of OptiMEM to a final concentration of 2 μM and filtered through a 0.22 μm filter. The DNA was added dropwise to the PEI solution and incubated at room temperature for 20 min. The DNA/PEI solution was added to the 293T cells and incubated for 4 h at 37 °C 5% CO2 before being replaced by complete Dulbecco's modified Eagle's medium (Invitrogen). Supernatant was harvested after a further 48 h and replaced with growth medium for a second collection after 72 h. Viral supernatant was initially centrifuged at 4000 g using a desktop centrifuge (MSE, Berg, Germany) for 10 min and then filtered through a 0.22 μm filter prior to ultracentrifugation (Sorvall, UK) at 23 000 g (100 000 g), 4 °C for 2 h. Medium was carefully decanted and viral pellets resuspended in 300 μl of phosphate-buffered saline (PBS) medium. Finally, viral suspensions were centrifuged at 4000 g for 10 min using a desktop microfuge to remove any remaining debris. All viral preparations were used fresh and titred on 293T cells for biological titre by limiting dilution and fluorescence activated cell sorting (FACS) analysis for GFP as well as physical titre by reverse transcriptase (Roche Applied Science, Mannheim, Germany) and p24 (Perkin Elmer, Beaconsfield, Bucks, England) ELISA assay as previously described.52 Therefore doses of each preparation were not necessarily identical. VSV-G titres were consistently 3–5 times higher than those of gp64 (Tables 1 and 2).

Animal studies

Male and female MF1 mice (Harlan, UK) were used in this study. For in utero administration, time-mated pregnant mice were anaesthetized by inhalation of isofluorane (Abbott Laboratories, UK). A midline laparotomy was performed and both horns of the gravid uterus exposed. Each amniotic cavity was injected (50 μl volume) by penetration of the uterus wall, the yolk sac and amniotic membranes with a 33-gauge Hamilton Microliter Syringe. Care was taken to ensure that injections were performed into the amniotic cavity and not into the extra-embryonic coelom.53 Following injection, the uterus was returned to the abdominal cavity and the abdominal wall closed in two layers with 5/0 Mersilk sutures (Ethicon, Brussels, Belgium). Animals were kept in a warmed cage in an undisturbed environment until awake and active. For neonatal administration, 1-day-old mice were manually restrained while they inhaled two 10 μl doses of vector separated by a 1 min interval. For adult administration, mice were anaesthetized by inhaled isofluorane and 50 μl was inhaled after direct application to the nostrils. Repeated administration protocols were carried out as follows; fetal/neonatal repeat administration consisted of a single fetal dose of 50 μl virus delivered into the amniotic fluid on 16 days pcm followed by two doses of 10 μl on days 0 and 1 after birth. Neonatal repeat dosing alone was as above without the fetal intra-amniotic administration.

All animal work was carried out under United Kingdom Home Office regulations and was compliant with the guidelines of the Imperial College London ethical review committee.

Detection of total GFP by ELISA

Briefly, 96-well plates were coated with monoclonal mouse anti-GFP antibody (Abcam, Cambridge, UK) and incubated at 4 °C overnight. Wells were washed and then blocked for 1 h at 37 °C with 1% bovine serum albumin (BSA; Sigma). Samples and standards were then added and incubated for 1 h at 37 °C. After washing, biotinylated secondary antibody (Abcam) was added in 1% BSA and incubated for 1 h at 37 °C. Plates were again washed and streptavidin-HRP (Dako, Cambridge, UK) added as to manufacturer's instructions. After a final incubation for 1 h at 37 °C, TMB chromagen (Dako) was added and plates were incubated in the dark for 20–30 min at room temperature. The enzymatic reaction was terminated by the application of 100 μl 3 M H2SO4 and samples were analysed on a microplate reader at a wavelength of 450 nm (SOFTMAX; Molecular Devices Corporation, Downington, PA, USA). A standard curve was produced ranging from 2000 to 31.25 pg ml−1 using recombinant GFP (Abcam).

Macroscopic observation

Prior to fixation, lung were observed and photographed using a Leica MZ16 Fluorescence Stereomicroscope. Images were autolevelled using Adobe Photoshop.

Fluorescence Immunohistochemistry

Murine lung were perfused in vivo with ice-cold 4% paraformaldehyde for 20 min prior to exsanguinations to preserve lung architecture. Excised lung were further fixed in 4% paraformaldeyhde for 16 h at 4 °C and then paraffin embedded and 4 μm-thick sections cut at 50 μm intervals. Sections were histologically stained with haematoxylin & eosin to verify the quality of sectioning prior to immunohistochemistry. Sections were immunolabelled with the following antibodies; rabbit anti-GFP polyclonal antibody (Abcam), mouse anti-β-tubulin IV monoclonal (Sigma). Antibody was detected using fluorescently labelled second antibodies; either FITC labelled goat anti-rabbit IgG or Cy3 labelled rabbit anti-mouse IgG (both Sigma). The F4/80 rat monoclonal anti-mouse antibody was used for immunostaining macrophages (Abcam). Briefly, sections were de-waxed and incubated in 4% BSA/PBS overnight. Primary antibody was added in 2% BSA/PBS for 4 h and then sections washed serially in PBS before fluorescent secondary antibody was added in 2% BSA/PBS and incubated in the dark for 1 hour. Sections were then washed serially in PBS before being coverslip mounted (Vectashield) and imaged using an Olympus BX51 fluorescence microscope.

In vivo luciferase bioimaging

Mice were anaesthetized with isofluorane (Abbott Laboratories, Maidenhead, Berkshire, UK) and 50 μl of 15 mg ml−1 D-luciferin (Gold Bio, St Louis, MO, USA) was administered intra-nasally and imaged 5 min later with a CCD camera (IVIS, Xenogen; MA, USA), After acquiring a grey scale photograph, a 5 min bioluminescent image was obtained using 12 cm field-of-view, binning (resolution) factor of 8, 1/f stop and open filter. Regions of interest (ROIs) were defined manually (using a standard area in each case), signal intensities were calculated using the Living Image software (Xenogen) and expressed as photons per second. Background photon flux was defined from an ROI drawn over the control mice where no vector had been administered.

Statistics

All values are represented as means±standard error of mean. Analysis of GFP ELISAs was performed by log transformation of values followed by general linear model analysis of variance with Tukey simultaneous tests.

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Acknowledgements

We thank Dr David Parsons and Associate Professor Don Anson, Women and Children's Children's Hospital, Adelaide, Australia for the kind gift of the gp64 plasmid. Plasmid was generated by Plasmid Factory (Bielefeld, Germany). TRM was funded by combined MRC/BBSRC/EPSRC as part of the UKCTE, University of Manchester. SNW is recipient of the Philip Gray Memorial Fellowship, Katharine Dormandy Trust. SMKB is a senior research fellow of the Clive Knight Laboratories and is part-funded by an FP6 grant from the European Union (LSHB-CT-2004-00213). SJH is supported by a European Commission Contract (CONSERT 005242) and AJT is a Wellcome Trust Senior Clinical Fellow.

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Correspondence to T R McKay.

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Buckley, S., Howe, S., Sheard, V. et al. Lentiviral transduction of the murine lung provides efficient pseudotype and developmental stage-dependent cell-specific transgene expression. Gene Ther 15, 1167–1175 (2008). https://doi.org/10.1038/gt.2008.74

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